Regional differences in arrhythmogenesis

JGP study shows that the subendocardium is more susceptible to spontaneous Ca²⁺ release events that can initiate arrhythmias, and this may be reduced by local CaMKII inhibition.

Calcium release and uptake must be carefully controlled in cardiomyocytes to ensure that the heart maintains a regular beat, and spontaneous Ca²⁺ release (SCR) from the sarcoplasmic reticulum—due to leaky ryanodine receptors, for example—can trigger lethal ventricular arrhythmias. In this issue of JGP, Dries et al. demonstrate that the subendocardial layer of the ventricular wall is particularly susceptible to arrhythmogenic SCR, and that this could potentially be treated by local inhibition of calcium/calmodulin-dependent kinase II (CaMKII; 1).Using living myocardial slices, Eef Dries (left), Cesare Terracciano (center), and colleagues show that, following injury, the subendocardial layer of the rat ventricular wall is more susceptible than the subepicardial layer to arrhythmogenic SCR events. High-resolution Ca²⁺ imaging of the subendocardium shows the increased number of SCRs (green dots) in the region bordering the injured tissue. The frequency of SCRs and ectopic contractions can be reduced by CaMKII inhibition.SCRs have been extensively studied in isolated cardiomyocytes, but arrhythmias are multicellular events (2) in which the behavior of individual cells is influenced by their interactions with neighboring cells and the extracellular matrix. “In addition, myocardial electrophysiology changes at different depths of the ventricular wall, and the vast majority of studies do not account for this transmurality,” explains Cesare Terracciano, a professor at the National Heart and Lung Institute, Imperial College London.Terracciano’s group has pioneered the use of living myocardial slices prepared from different layers of the ventricular wall to study regional differences in the electrical and mechanical properties of healthy hearts (3,4). However, it is unclear how these differences are impacted by injury or disease and whether this leaves some layers of the heart wall more susceptible to SCRs and arrhythmogenesis.Terracciano and colleagues, including first author Eef Dries, therefore prepared myocardial slices from different layers of the rat ventricular wall and subjected them to cryoinjury (1). Structural remodeling—in the form of reduced T-tubule density—was similar in both subendocardial and subepicardial slices after injury, but only subendocardial slices showed an increase in spontaneous, arrhythmic contractions.Dries et al. used a fluorescent Ca²⁺ indicator and high-resolution imaging to examine Ca²⁺ signaling in the “border zone” surrounding the cryoinjury, as this region has been implicated in triggering arrhythmias following myocardial infarction. “Intriguingly, and only in subendocardial slices after injury, we observed a reduction in the amplitude of calcium transients that also became slower to decline, changes that are hallmarks of heart failure,” Terracciano says. “SCR events were more frequent and more closely distributed when we cryoinjured the slices but, again, only in the subendocardium.”The clustering of multiple SCRs in both space and time makes them more likely to trigger an ectopic contraction. One possibility is that the open probability of ryanodine receptors is increased in subendocardial slices. This could be caused by enhanced CaMKII-mediated phosphorylation of ryanodine receptors and, indeed, Dries et al. found that, after cryoinjury, receptor phosphorylation is increased in subendocardial, but not subepicardial, slices (1).Accordingly, Terracciano and colleagues found that the CaMKII inhibitor AIP reduced the frequency of SCRs and spontaneous contractions in cryoinjured subendocardial slices. In contrast, AIP had no effect on injured subepicardial slices or on normal, healthy cardiac tissue. CaMKII inhibitors have been proposed as potential therapies for cardiac arrhythmias, but their use has so far been limited by off-target effects. Dries et al.’s results suggest that targeting CaMKII inhibitors to specific regions of the ventricular wall (using localized gene therapy, for example) could greatly improve their efficacy.“A picture is emerging that subendocardial slices are more susceptible to arrhythmogenic stimuli, and this can be important for understanding and treating arrhythmias,” Terracciano says. He now plans to study injured myocardial slices over longer time periods and investigate the molecular changes underlying the enhanced arrhythmogenic susceptibility of the subendocardium, as well as testing localized gene therapy approaches in animal models of disease.     

S2 domain gives myosin filaments some flexibility

JGP microscopy study supports the idea that the region linking myosin head and tail domains can be peeled away from filament backbone to prevent actin-attached heads from impeding filament movement.

Myosin II motors move along actin filaments by coupling cycles of ATP binding and hydrolysis to a repetitive process in which the myosin head domains attach to actin, undergo a conformational shift/powerstroke, and then detach. In muscle cells, myosin II molecules assemble into thick filaments containing hundreds of head domains, and any heads that remain attached to actin after completing their power stroke may impede the ability of other heads to move the filament and drive muscle contraction. In this issue of JGP, Brizendine et al. provide direct evidence that this potential drag on filament movement is limited by the flexibility of myosin II’s S2 subdomain (1).(Left to right) Richard Brizendine, Christine Cremo, and Murali Anuganti provide direct evidence that the S2 domain of myosin II is a flexible structure, which would allow it to prevent actin-attached heads from impeding the movement of myosin filaments. Quantum dots labeling a head domain (black) and the filament backbone (red) mostly follow the same trajectory as a filament moves in vitro. But, in rare instances (insets), an actin-attached head briefly lags the backbone’s trajectory before catching up, an event facilitated by the flexibility of the S2 region that connects the motor protein’s head and tail domains.For the past few years, Christine Cremo and colleagues at the University of Nevada, Reno, have been studying the kinetics of filament movement using fluorescently labeled myosin and actin filaments in vitro (2). Based on their data, Cremo’s team, in collaboration with Josh Baker, developed a mixed kinetic model that predicted a key mechanical function for the S2 subdomain of myosin II, which links the motor protein’s head domains to the C-terminal light meromyosin (LMM) domains that mediate filament assembly (3,4). According to the model, the flexibility of the S2 subdomain, and its ability to be peeled away from the filament backbone, provides some slack to actin-attached heads as the filament moves forward, giving them more time to detach before they impede the filament’s progress.“So now we wanted to see if we could directly observe this flexibility,” Cremo explains. To do this, two postdocs in Cremo’s laboratory, Richard Brizendine and Murali Anuganti, assembled smooth muscle myosin filaments labeled with two differently colored quantum dots, one attached to the LMM domain and the other attached to the head domain. Most of the time, these two labels should follow the same trajectory along actin filaments in vitro. If the S2 domain is flexible, however, it should be possible to occasionally observe an actin-attached head remain in place while the LMM domain continues moving forward. This brief “dwell” should then be followed by a “jump” as the head domain detaches from actin and catches up with the trajectory of the filament backbone.“We were looking for rare events in a sea of noise,” Cremo says, yet the researchers were able to identify dwells and jumps in the quantum dot trajectories consistent with the predicted flexibility of the S2 domain. The frequency and duration of these events fit the known kinetics of actomyosin motility.Based on their data, Brizendine et al. (1) estimate that, in smooth muscle, a myosin filament can move up to ∼52 nm without being impeded by an actin-attached head, a figure close to that predicted by the mixed kinetic model. To provide this flexibility, the researchers calculate that as much as 26 nm of the S2 domain can be unzipped from the filament backbone. Intriguingly, this matches the maximum length that S2 can be seen to project from thick filaments in tomograms of Drosophila flight muscle (5), and the forces generated by working myosin heads should be more than sufficient to achieve this unzipping.Many cardiomyopathy-associated mutations are located in the S2 region of myosin II. However, the mixed kinetic model predicts that, compared with smooth muscle, myosin filaments in cardiac and skeletal muscle cannot move quite as far without being impeded by actin-attached heads. “What leads to these differences?” Cremo wonders. “Are there differences in the biophysical behavior of the S2 domain in different muscle types?”     

cMyBPC phosphorylation alters response to heart failure drug

JGP study shows that the phosphorylation state of cMyBPC modulates the ability of omecamtiv mecarbil to enhance myocardial force generation.

The small molecule omecamtiv mecarbil (OM) is a cardiac-specific myosin activator that is currently undergoing clinical trials for the treatment of heart failure with reduced ejection fraction. In this issue of JGP, Mamidi et al. demonstrate that OM’s ability to increase cardiac force production is altered by the phosphorylation state of cardiac myosin-binding protein C (cMyBPC), a target of β-adrenergic signaling that is often dysregulated in late-stage heart failure patients (1).(Left to right) Ranganath Mamidi, Joshua Holmes, Julian Stelzer, and colleagues reveal that the effects of the heart failure drug OM are modulated by the phosphorylation state of the contractile protein cMyBPC. For example, OM’s ability to increase force generation is significantly blunted in mouse myocardial preparations expressing phosphoablated (SA) rather than WT cMyBPC due to changes in myosin cross-bridge kinetics.OM enhances myocardial force generation by increasing the number of strongly bound myosin cross-bridges (2), partly by slowing ADP release and cross-bridge detachment (3). Though the drug has progressed to phase 3 clinical trials, little is known about how its effects may be influenced by pathophysiological changes in other sarcomeric proteins, such as cMyBPC, that regulate myosin cross-bridges and force production.During exercise or other physiological stresses, adrenaline stimulates the phosphorylation of cMyBPC by PKA, thereby accelerating cross-bridge kinetics and myocardial contractility to meet the increased demand for cardiac output (4). In late-stage heart failure patients, however, β-adrenergic signaling is dysregulated and cMyBPC phosphorylation is greatly reduced. “We wanted to test how the phosphorylation state of cMyBPC would effect OM treatment,” explains Julian Stelzer, a professor at Case Western Reserve University.Stelzer’s team, including cofirst authors Ranganath Mamidi and Joshua Holmes, prepared myocardial tissue from both WT mice and mice expressing a cMyBPC mutant that lacks the three main PKA phosphorylation sites. The researchers treated the preparations with OM and found that the ablation of cMyBPC phosphorylation significantly blunted OM’s ability to increase force production (1).Dephosphorylated cMyBPC is thought to stabilize the super-relaxed state of myosin, in which the head domains are folded back toward the filament backbone and are less available to form active cross-bridges (5). Stelzer and colleagues have previously shown that ablating cMyBPC phosphorylation slows cross-bridge kinetics (6).“This is exacerbated by the addition of OM,” Stelzer says. “It creates an even slower system that limits cross-bridge recruitment, and those that are recruited can’t really be detached.” This may reduce the effectiveness of OM in end-stage heart failure patients with low levels of cMyBPC phosphorylation.In contrast, phosphorylation of cMyBPC by PKA usually accelerates myosin cross-bridge kinetics. However, when Stelzer and colleagues treated their myocardial preparations with both PKA and OM, mimicking the scenario of an early-stage heart failure patient exercising or experiencing stress, the effects of the drug dominated the effects of the kinase.“OM did not allow any acceleration and, in fact, slowed cross-bridge kinetics even further, completely negating the effect of PKA on contractility,” Stelzer says. This could mean that early-stage patients on OM are unable to increase their cardiac output during exercise, elevating the risk of ischemia.New iterations of OM are already being explored as potential next-generation treatments for heart failure. Stelzer says that it will be important to investigate how these drugs interact with cMyBPC and other components of the contractile machinery. In the meantime, Stelzer’s laboratory is focused on developing novel therapeutic approaches involving the direct manipulation of cMyBPC phosphorylation.     

Anti-science kills: From Soviet embrace of pseudoscience to accelerated attacks on US biomedicine

The United States witnessed an unprecedented politicization of biomedical science starting in 2015 that has exploded into a complex, multimodal anti-science empire operating through mass media, political elections, legislation, and even health systems. Anti-science activities now pervade the daily lives of many Americans, and threaten to infect other parts of the world. We can attribute the deaths of tens of thousands of Americans from COVID-19, measles, and other vaccine-preventable diseases to anti-science. The acceleration of anti-science activities demands not only new responses and approaches but also international coordination. Vaccines and other biomedical advances will not be sufficient to halt COVID-19 or future potentially catastrophic illnesses, unless we simultaneously counter anti-science aggression.

This Essay argues that COVID-19 exposed how a rising tide of anti-science rhetoric and activities can dramatically exploit society''s vulnerabilities to an infectious disease, suggesting that anti-science extremism has become as big a threat as the virus itself.

“Without science, democracy has no future.”—Maxim Gorky, April 1917

The newest (October 2020) projections from the University of Washington Institute of Health Metrics and Evaluation (IHME) Coronavirus Disease 2019 (COVID-19) forecasting team reveal a grim reality. Their estimates indicate that more than 510,000 Americans could lose their lives by February 28, 2021 [1], representing more than a doubling of the current estimates of 220,000 deaths (although not all groups agree with these estimates). For most of 2020, the US has been the epicenter of the COVID-19 pandemic, leading the world in new cases and deaths. This dire situation is a consequence of our government’s failure to launch a coordinated national response and roadmap and refusing to aggressively promote nonpharmaceutical interventions (NPIs), especially face masks, social distancing mandates, school closures, testing, and contact tracing [2]. In its place, and as the cases and deaths mounted, the White House and its coronavirus task force, and famously the President himself, organized a campaign of disinformation [3].Central to White House anti-science promotion efforts were attempts by key officials to downplay the severity of COVID-19 and its long-haul consequences, inflate the curative properties of certain medicines such as hydroxychloroquine, falsely attribute COVID-19 deaths to comorbidities in order to artificially reduce actual disease mortality rates, and make scientifically unsubstantiated claims about herd immunity (or its links to the Great Barrington Declaration, which argued without evidence that restrictions cause more harm than the virus). There were also efforts to discredit the effectiveness of face masks to prevent COVID-19 or to refuse implementing mask mandates, invoking at times new political terms or slogans that gained popularity in recent years such as “health freedom” or “medical freedom” [4]. This is exemplified by a recent October 22 tweet from the Republican Governor Kristi Noem of South Dakota [5]:

If folks want to wear a mask, they are free to do so. Those who don’t want to wear a mask shouldn’t be shamed into it, and govt should not mandate it. We need to respect each other’s decisions. In SD, we know a little common courtesy can go a long way.

The open questioning of face masks or refusal to enforce mandates will likely continue to have tragic consequences for the American people. According to the IHME COVID-19 forecasting team, 95% public mask use would save almost 130,000 lives from September 22, 2020, through February 28, 2021 [1]. Thus, anti-science disinformation that advocates shunning masks could inflict a mass casualty event in the US. Its occurrence should not surprise us. Instead, our tragic loss of American lives would reflect the handiwork of an evolving anti-science movement that aggressively accelerated in the last 5 years beginning in California and Texas. In this Essay, I argue that to understand how a nation state might seek to attack and dissolve modern biomedicine, it is helpful to revisit a tragic period in 20th century Russia (see Box 1). The relentless attacks on science and scientists during Stalin’s Great Purge and the ascendancy of Lysokenkoism and other pseudoscientific theories provide a useful framework for addressing some stark reminders about the politicization of science occurring now in America, even if they play out at a far lesser scale.Box 1. Lessons from a dark chapter in historyOne of the darkest chapters in the history of the Soviet Union, the Great Purge, or the Great Terror (Большой террор), saw the widespread imprisonment, execution, and persecution of millions considered an enemy of Joseph Stalin’s government. It began following the assassination of Sergei Kirov in 1934, a Soviet leader and revolutionary, before halting in 1938, although significant elements of the purge remained throughout the 1940s. The intelligentsia was a Great Purge target, as were entire fields of science, including astrophysics, which was ultimately deemed as a “political platform” running counter to Marxism [6]. Another was the field of mendelian genetics, then led in the USSR by Nikolai Vavilov in his role as head of the Lenin All Union Academy of Agricultural Sciences, the scientific branch of the Commissariat of Agriculture. Vavilov was a botanist and a scientific pioneer in using genetic approaches to improve cereal crops for the USSR [6–8]. Ultimately, Vavilov came under attack by Trofim Lysenko, a peasant with no doctoral degree who popularized and laid claims to the concept of “vernalization” [6]. Lysenko and his colleagues proposed moistening and chilling winter wheat and allowing it to germinate in order to sense these conditions in time for the spring when it would supposedly flourish [6]. Through vernalization—which bore some resemblance to Lamarckian evolutionary theories by claiming that acquired traits could be inherited—Lysenko aspired to adapt wheat to the harsh Russian climate. As a sort of proof of concept, he had his father soak his winter wheat in water before burying it in a snowbank to keep it cold prior to spring planting [6].Initially, Vavilov took on a mentoring role for Lysenko, even touting his accomplishments at the Sixth International Congress of Genetics held at Cornell University in Ithaca, New York, in the summer of 1932 [8]. See, for example, Vavilov’s praise of Lysenko in a special news “flash,” as it was called by R. C. Cook, the editor of the Journal of Heredity during the 1940s [8]:

The remarkable discovery recently made by T. D. Lysenko of Odessa opens enormous new possibilities to plant breeders and plant geneticists of mastering individual variation.. . .The essence of these methods, which are specific for different plants and different variety groups, consists in the action upon the seeds of definite combinations of darkness (photo-periodism), temperature and humidity. This discovery enables us to utilize in our climate for breeding and genetic work tropical and sub-tropical varieties.... This creates the possibility of widening the scope of breeding. . . to an unprecedented extent, allowing the crossing of varieties requiring entirely different periods of vegetation.

Lysenko’s vernalization technology would theoretically make it possible, argues Simon Ings in his book, Stalin and the Scientists, “to grow alligator pears and Bananas in New York and lemons in New England” [6]. Its extraordinary claims aside, vernalization was seen as a form of Soviet homegrown science and a source of national pride. In contrast, Lysenko was able to convince Stalin that genetics was an evil science, much like relativity. Political expediency became the rationale for promoting pseudoscience even if it meant that millions of rural peasants would die of starvation in the USSR when Lysenko’s cold-resistant crops failed to materialize. Ultimately, Lysenko became the President of the Lenin Academy of Agricultural Science in 1939, whereas Vavilov was arrested in 1940 and rounded up with other intellectuals, including the founder of the Marx-Lenin Institute of World Literature. He was interrogated and sent to a Soviet prison in Saratov where he perished, possibly by starvation in January 1943, despite repeated appeals from international leaders including British Prime Minister, Winston Churchill (Fig 1) [6].Open in a separate windowFig 1Photo of the prisoner Nikolai Vavilov.Official photo from the file of the investigation. The People''s Commissariat for Internal Affairs (Народный комиссариат внутренних дел), Central Archive of the Federal Security Service of the Russian Federation (Moscow) (Центральный архив ФСБ РФ (Москва)) Institute of Plant Industry (Всероссийский институт растениеводства имени Н. И. Вавилова), created January 1, 1942. https://en.wikipedia.org/wiki/Nikolai_Vavilov#/media/File:Vavilov_in_prison.jpg.Vavilov received a posthumous pardon by Nikita Khrushchev during the 1950s, and in 2008, a book about his life, The Murder of Nikolai Vavilov: The Story of Stalin''s Persecution of One of the Great Scientists of the Twentieth Century was published in English [9]. It remains a great irony that Vavilov devoted his scientific career to the humanitarian cause of feeding the population of the Soviet Union only to die by starvation.Following the death of Stalin in 1953, the USSR began reopening to international science, ushering in a new era in vaccine development. Throughout the 1950s, both the US and Soviet Union suffered from severe polio epidemics prompting the 2 nations to embark on an unprecedented scientific collaboration [10]. Dr. Albert Sabin sent his polio strains to the USSR where they were manufactured at large scale to produce a trivalent vaccine. During the “Khrushchev Thaw,” it was tested in tens of millions of Soviet citizens and shown to be both safe and effective at preventing polio. A decade later, the US and USSR collaborated to improve a vaccine leading to the eradication of smallpox [10]. Nonetheless, state oppression of Soviet scientists continued, and Krushchev supported Lysenko’s work. Moreover, the physicist and father of the Soviet hydrogen bomb, Andrei Sakharov, won the Nobel Prize in 1975 advocating for human rights, but was subsequently arrested and exiled to Gorky [11]. The mathematician and chess champion, Natan Sharansky, was arrested on treason charges in 1977 and kept in solitary confinement before he was released through a prisoner exchange, later emigrating to Israel in 1980. The American physicist Robert Oppenheimer also endured persecution during the red scare in the 1950s, though on a lesser scale, having had his national security clearance revoked.     

Understanding Ca2+ alternans

JGP study reveals that insufficient reuptake of calcium into the sarcoplasmic reticulum underlies arrhythmogenic variations in cardiac calcium transients.

Ca²⁺ alternans (Ca-Alts) are beat-to-beat changes in the amplitude of the Ca²⁺ transients evoked in cardiomyocytes, which can lead to arrhythmias and sudden cardiac death. Ca-Alts can be induced by an elevated heart rate (tachycardia) or metabolic impairments such as ischemia or hypothermia, but the molecular mechanisms underlying the phenomenon are unclear. In this issue of JGP, Millet et al. reveal that Ca-Alts arise when SERCA pumps are unable to fully replenish Ca²⁺ levels in the SR (1).Jose Millet, Yuriana Aguilar-Sanchez, Ariel L. Escobar (left to right), and colleagues investigate the mechanisms underlying arrhythmogenic Ca-Alts. The FLOM technique shows how these beat-to-beat changes in Ca²⁺ transients can be induced in intact hearts by increased heart rate and local reductions in temperature produced by a cold finger. The researchers find that Ca-Alts result from insufficient replenishment of SR Ca²⁺ levels by SERCA pumps.“Ca-Alts are very arrhythmogenic,” says Ariel L. Escobar, a professor at the University of California, Merced. “If you develop these alternans, you have a very high chance of suffering ventricular fibrillation.”Yet the mechanisms underlying Ca-Alts remain unclear. Though they appear to involve changes in the amount of Ca²⁺ released from the SR (2,3,4), Ca-Alts could be triggered by variations in the duration of action potentials (APD-Alts) that stimulate calcium-induced calcium release, an incomplete recovery of the ryanodine receptor that releases Ca²⁺ from the SR, or incomplete refilling of the SR by SERCA ATPases.To investigate the phenomenon in more detail, Escobar and colleagues, including co-first authors Jose Millet and Yuriana Aguilar-Sanchez, developed a new technique called fluorescence local field optical mapping (FLOM), which uses optical conduits containing >70,000 optical fibers to map the fluorescence of calcium-sensitive or potentiometric dyes in the epicardium of intact mouse hearts. “This approach allows us to study the spatiotemporal dynamics of calcium and membrane potential changes in a functional heart,” Escobar explains.FLOM imaging confirmed that Ca-Alts can be induced by increased heart rate and/or global reductions in temperature, two conditions that also induce APD-Alts. More crucially, however, Escobar and colleagues used a small, crescent-shaped cold finger to show that local reductions in tissue temperature also induce Ca-Alts but do not cause APD-Alts, demonstrating that the two phenomena can be uncoupled and that Ca-Alts are not driven by changes in action potential duration.Because the crescent-shaped cold finger creates a temperature gradient within the epicardium, Escobar and colleagues were able to carefully analyze the temperature dependence of Ca²⁺ dynamics. The relaxation of Ca²⁺ transients becomes gradually slower at lower temperatures, and a thermodynamic analysis of this process suggested that it involves not only active mechanisms—such as the ATPases that pump Ca²⁺ into the SR—but also passive mechanisms such as diffusion and binding to cytosolic buffers.In contrast, the relatively steep temperature dependence of Ca-Alts indicated that they exclusively depend on an active process like SERCA-mediated Ca²⁺ reuptake into the SR. Indeed, Escobar and colleagues found that the Q₁₀ temperature coefficient of Ca-Alts is remarkably similar to the Q₁₀ of SERCA-mediated Ca²⁺ transport in vitro.To confirm the importance of Ca²⁺ reuptake in Ca-Alts, Escobar and colleagues treated hearts with the SERCA inhibitor Thapsigargin. Partial blockade of SERCA-mediated reuptake enhanced the level of Ca-Alts, the researchers found, indicating that Ca-Alts are induced when SERCA pumps fail to fully replenish SR Ca²⁺ stores between heart beats. This could occur when the heart is beating particularly fast or when the metabolic activity of cardiomyocytes is impaired by, for example, low temperatures.Escobar’s team is now developing a needle-shaped optical conduit that can be used to probe any layer within the ventricular wall. “We hope to measure Ca-Alts in each layer, including the endocardium where SERCA levels are lower and Ca-Alts tend to be initiated,” Escobar says.     

Dissecting neurotransmission with artificial synapses

JGP study demonstrates how recordings from neuron–HEK cell cocultures provide a clearer picture of the factors involved in synaptic transmission.

Resolving the rapid series of steps involved in synaptic transmission and assessing the contributions of different molecules to each of them is an enormous challenge. In this issue of JGP, Chiang et al. show that the process can be studied with greater resolution at the artificial synapses formed between neurons and cocultured human embryonic kidney (HEK) cells (1).Chung-Wei Chiang (left), Meyer Jackson (right), and colleagues studied synaptic transmission between hippocampal neurons and cocultured HEK cells. Mutations in the SNARE protein synaptobrevin 2 alter the shape of mEPSCs generated in HEK cells, an effect made clearer by the absence of dendritic filtering in this artificial system.Meyer Jackson and colleagues at the University of Wisconsin School of Medicine and Public Health are particularly interested in exocytosis. Though this process can be measured directly in endocrine cells, its role in controlling the dynamics of synaptic transmission can be difficult to separate from all the downstream steps required to elicit a response in the postsynaptic neuron. “We wanted to study a surrogate synapse with a simplified response to neurotransmitter that would allow us to focus on vesicle release with greater resolution,” Jackson explains.For years, researchers have studied synaptogenesis by transfecting HEK cells with a handful of postsynaptic factors that enable them to assemble functional synapses when cocultured with neurons (2, 3). Jackson realized that these artificial synapses lack two key sources of variability that can obscure the contribution of vesicle release to synaptic transmission. First, the postsynaptic apparatus of neuron–HEK synapses is consistent and can be precisely controlled (in contrast to regular synapses, where the molecular composition may vary from synapse to synapse). Second, the compact shape of HEK cells greatly reduces the influence of dendritic filtering, the phenomenon by which synaptic inputs take varying lengths of time to reach the cell body, depending on the location of the synapse within the dendritic arbor.Jackson and colleagues, including first author Chung-Wei Chiang, transfected HEK cells with four postsynaptic proteins—the AMPA receptor subunit GluA2, the adhesion molecule neuroligin 1, the scaffold protein PSD-95, and the accessory factor stargazin—and cocultured them with hippocampal neurons (1). The researchers then measured the miniature excitatory postsynaptic currents (mEPSCs) evoked in the HEK cells by the spontaneous release of single synaptic vesicles from neighboring neurons.The mEPSCs generated at these artificial synapses were larger and faster than mEPSCs produced by regular, neuron–neuron synapses (though they involved comparable amounts of charge, suggesting that the vesicle populations are similar at both types of synapse).Notably, the rise rate of mEPSCs in HEK cells was faster and less variable, in keeping with the absence of dendritic filtering and the consistent, shorter distance between the artificial synapses and the HEK cell body. This allowed Chiang et al. (1) to resolve the contribution of vesicle release to mEPSC dynamics using mutant versions of the SNARE protein synaptobrevin 2 that impede the flux of neurotransmitters through synaptic fusion pores (4). These mutant proteins decreased the rise rate and decay rate of mEPSCs at artificial synapses. “However, the effect was much clearer in HEK cells than we’d previously seen in neurons,” Jackson says.Chiang et al. (1) were also able to resolve the contribution of postsynaptic receptors to mEPSC shape, but Jackson is most interested in using the neuron–HEK coculture system to investigate synaptic vesicle release in more detail. “It opens up a new approach that will allow us to study synaptic exocytosis more precisely and look for much subtler effects,” Jackson says. For example, Jackson hopes to explain why mutations in some exocytotic proteins have major effects on endocrine cells but little to no effect at synapses.     

Policy changes and physicians opting out from Medicare in Quebec: an interrupted time-series analysis

BACKGROUND:In all Canadian provinces, physicians can decide to either bill the provincial public system (opt in) or work privately and bill patients directly (opt out). We hypothesized that 2 policy events were associated with an increase in physicians opting out in Quebec.METHODS:The 2 policy events of interest were the 2005 Supreme Court of Canada ruling on Chaoulli v. Quebec and a regulatory clampdown forbidding double billing that was implemented by Quebec’s government in 2017. We used interrupted time-series analyses of the Quebec government’s yearly list of physicians who chose to opt out from 1994 to 2019 to analyze the relation between these events and physician billing status.RESULTS:The number of family physicians who opted out increased from 9 in 1994 to 347 in 2019. Opting out increased after the Chaoulli ruling, and our analysis suggested that between 2005 and 2019, 284 more family physicians opted out than if pre-Chaoulli trends had continued. The number of specialist physicians who opted out rose from 23 in 1994 to 150 in 2019. Our analysis suggested that an additional 69 specialist physicians opted out after the 2017 clampdown on double billing than previous trends would have predicted.INTERPRETATION:We found that the number of physicians who opted out increased in Quebec, and increases after 2 policy actions suggest an association with these policy interventions. Opting out decisions are likely important inputs into decision-making by physicians, which, in turn may influence the provision of publicly funded health care.

In all Canadian provinces, most physicians work as part of a private business. Whether they are solo practitioners or part of larger group practices, physicians can bill the provincial public Medicare system, or work privately and bill patients directly. Engaging in both is referred to as “dual practice,” and evidence exists to suggest that it can negatively affect the accessibility of care in the public system where it is permitted.¹ As a result, most provinces have enacted legal barriers designed to prohibit or discourage dual practice.² In Quebec, British Columbia, Alberta, Saskatchewan and New Brunswick, physicians have to opt out formally from the public system to be able to bill patients for publicly covered services. In Ontario, Manitoba and Nova Scotia, physicians who decide to rely on private billing are not permitted to bill their patients more than the public fee schedule. Given these regulations, it is widely thought that only a small proportion of Canadian physicians choose to work outside of the public funding scheme for medical care. Nevertheless, private out-of-pocket payment to physicians still accounts for hundreds of millions of dollars per year in Canada³ and, in Quebec, the number of physicians who opt out has been steadily growing for the past decade. However, our understanding of how policies and legal events have affected these rates in Quebec or other Canadian provinces is limited. Over the past 20 years, 2 important policy events directly related to dual practice have occurred in Quebec.First, in 2005, in its ruling for Chaoulli v. Quebec, the Supreme Court of Canada concluded that Quebec’s prohibition of private insurance for publicly insured medical services violated Section 1 of the Quebec Charter of Human Rights and Freedoms.^4–6 Quebec’s government was granted 1 year to adjust its laws to the ruling, during which time there were intense policy debates about the private system.^7–9 Many argued that there was a demand for out-of-pocket privately financed medical services, pushing many private investors, physicians and patients to debate the role of private delivery of health services.^7,10–12 Second, in the years following this ruling, Quebec witnessed substantial interest and investments in private facilities for elective medical interventions. In many specialties with a high volume of outpatient elective interventions (e.g., ophthalmology, dermatology and orthopedic surgery), a certain level of double billing became the norm. Patients were routinely asked to pay out-of-pocket to cover things like eye drops, anesthetics, use of the intervention room and record management — so-called “frais accessoires” [“incidental expenses”]. Faced with increased public and media scrutiny of the legality of those fees and strong pressures from Ottawa, Quebec started, in January 2017, to enforce a new regulation that clearly outlawed double billing for publicly funded medical services.¹³ Because many clinics had come to rely on these added fees, this clampdown threatened their business model, which may have pushed some physicians to opt out of the public system altogether.As physicians who have opted out are not available to deliver services for publicly insured patients, any trend toward more privately delivered care will have obvious implications for delivery of publicly funded health care in Canada. Furthermore, international evidence suggests that dual practice is associated with challenges to equity and efficiency.^14–16 Therefore, we analyzed the association of these 2 policy events with physicians’ decisions to opt out in Quebec.     

A Gs-RhoGEF interaction: An old G protein finds a new job

The heterotrimeric G proteins are known to have a variety of downstream effectors, but G_s was long thought to be specifically coupled to adenylyl cyclases. A new study indicates that activated G_s can also directly interact with a guanine nucleotide exchange factor for Rho family small GTPases, PDZ-RhoGEF. This novel interaction mediates activation of the small G protein Cdc42 by G_s-coupled GPCRs, inducing cytoskeletal rearrangements and formation of filopodia-like structures. Furthermore, overexpression of a minimal PDZ-RhoGEF fragment can down-regulate cAMP signaling, suggesting that this effector competes with canonical signaling. This first demonstration that the Gα_s subfamily regulates activity of Rho GTPases extends our understanding of Gα_s activity and establishes RhoGEF coupling as a universal Gα function.

The canonical G protein pathway consists of a cell surface receptor, a heterotrimeric G protein, and an effector protein that controls signaling within the cells. This fundamental paradigm, familiar to every biologist, is rooted in discoveries by the laboratories of Sutherland, Rodbell, and Gilman, which in the 1970s and 1980s dissected biochemical mechanisms of adenylyl cyclase activation by hormones. Their breakthrough came after experiments showing that the G protein G_s is essential to transfer agonist stimulation from the receptor to adenylyl cyclase (1). This G protein consists of the ∼42-kDa α subunit, which binds and hydrolyzes GTP, and the permanently associated dimer of 35-kDa β and ∼10-kDa γ subunits (Gβγ). Their findings helped establish a canonical model in which the agonist-bound receptor causes the G protein to release GDP, and the heterotrimer dissociates into Gα-GTP and free Gβγ; in this state, the G protein can activate its effector (i.e. Gα_s will activate adenylyl cyclase until GTP is hydrolyzed). Although the rod photoreceptor G protein, transducin, was discovered by that time (2), the ubiquitously expressed G_s can be considered the founding member of the G protein family.The subsequent cloning and identification of the other three families (G_i, G_q, and G₁₂) completed the rough map of G protein–mediated transduction. These initial studies suggested that the α subunits were responsible for activation of one type of effector (e.g. Gα_s for adenylyl cyclase and cAMP; Gα_q for phospholipase C, phosphoinositides, and Ca²⁺; and Gα_i for ion channels and inhibition of adenylyl cyclase), whereas the free Gβγ complexes interact with a remarkably large number of binding partners, including some effector enzymes and ion channels (3). Later, Gα₁₂ and Gα₁₃ were found to regulate a distinct type of effectors, the RhoGEFs (4, 5). These multidomain proteins contain pleckstrin homology (PH) domains, which facilitate their membrane localization, and Dbl homology (DH) domains, which catalyze GDP-for-GTP exchange (guanine nucleotide exchange factor; GEF) in the Rho family of small (∼20-kDa) G proteins. At the time, the G₁₂-RhoGEF pathway seemed odd as it contained two G proteins: the receptor-activated “large” G₁₂ class protein and the “small” Rho G protein, which is activated by RhoGEF. However, it was then discovered that Gα_q could activate a RhoGEF called Trio (6), and that Gβγ complexes activate other RhoGEFs, indicating that this pathway, if unusual, is at least popular. Gα_s, however, mostly appeared to be faithful to its originally determined role—to stimulate adenylyl cyclase(s)—possibly contributing to the enduring perception that regulation of a second messenger–generating enzyme is the “real” function of a heterotrimeric G protein.In the current issue of JBC, Castillo-Kauil et al. (7) force a reexamination of the existing canon, presenting data that show Gα_s can also interact with a specific RhoGEF, in this case PDZ-RhoGEF (PRG). The authors made this discovery as part of an examination of the regulation of cell shape by the Rho family. They began by expressing a series of short constructs of three RhoGEF proteins, p115RhoGEF, PRG, and LARG, all of which activated RhoA as expected, promoting cell contraction. However, they noticed that the DH/PH domain of PRG also activated Cdc42 and induced filopodia-like cell protrusions. To investigate which G protein is responsible for activation of this Cdc42-mediated pathway, they overexpressed constitutively active mutants of different Gα subunits. These mutants are stabilized in the active GTP-bound state due to substitution of the glutamine residue crucial for GTP hydrolysis. Surprisingly, the PRG-Cdc42 pathway was stimulated by Gα_sQ227L, the one Gα subtype not known for interaction with RhoGEFs. Furthermore, they showed that binding of PRG to Cdc42 was promoted only by G_s-coupled receptors, and not by G_q- or G_i-coupled GPCRs. The authors then investigated the PRG site responsible for the interaction with Gα_s, narrowing it down to the isolated PRG DH and PH domains and their linker region. A construct encompassing these domains was able to inhibit (i) GPCR-mediated activation of Cdc42, (ii) the Gα_sQ227L-promoted interaction of PRG with Cdc42, and (iii) some protein phosphorylation events downstream of the canonical cAMP pathway. Taken together, their work identifies PRG as a novel effector for G_s; the Gα_s-PRG interaction mediates activation of Rho family protein Cdc42, leading to cytoskeletal remodeling.The unexpected results of Castillo-Kauil et al. open up new opportunities to explore this mechanism at different levels of biology. The experiments described in the paper were performed in vitro using cultured cells, imaging, and pulldown of protein complexes containing the overexpressed Gα_s Q227L mutant. Considering the multitude of G_s-coupled receptors and RhoGEFs in the body (8, 9), it will be important to understand the physiological context where the new G_s-mediated pathway plays a significant role. This will require experimentation in vivo and possibly reevaluation of the phenotypes associated with known pathogenic mutations in Gα_s (GNAS) and other relevant genes. At the molecular level, it would be important to delineate the biochemical mechanisms of Gα_s interaction with PRG. For example, at what stage of the GTP/GDP cycle does Gα_s bind to PRG: in the GTP-bound state, which also activates adenylate cyclase, or in the transition state (i.e. just before the terminal phosphate of GTP is removed)? Indeed, there is precedent for proteins that bind preferentially with the transition state—specifically RGS proteins, which accelerate the GTPase reaction. Another possibility is that, by analogy with p115RhoGEF, which stimulates GTPase activity of Gα₁₂ and Gα₁₃, PRG (and other RhoGEFs with similar DH-PH sequences) can influence interaction of Gα_s with nucleotides, Gβγ, and other partners.Since defining the receptor, G protein, and effector as the three essential members of the G protein pathway, researchers have discovered many additional proteins that regulate the amplitude and duration of the stimulus and/or participate in cross-talk with other signaling circuits. These “new” proteins include arrestins, receptor kinases, nonreceptor exchange factors, GTPase-activating proteins, special chaperones, etc. Thus, in a way, discovering a novel binding partner for a signaling molecule is not as surprising as it would have been 20 years ago. However, the new partner identified by Castillo-Kauil et al. makes the result of extra significance; until now, we knew that three of four G protein subfamilies could regulate Rho GTPases by activating RhoGEFs: G₁₂ and G_q via their α subunits and G_i via the Gβγ subunits (10). The demonstration that the G_s subfamily is no exception shows that activation of RhoGEFs by heterotrimeric G proteins may be a truly universal mechanism (Fig. 1). The significance of this insight is that the multitude of biological processes regulated by Rho-GTPase networks can potentially respond to the entire repertoire of GPCR-mediated stimuli.Open in a separate windowFigure 1.Activation of the Rho family by heterotrimeric G proteins. The Rho family of small GTPases is activated by RhoGEF proteins, some of which can be stimulated by heterotrimeric G proteins. Of four families of heterotrimeric G proteins, three (G₁₂, G_q, and G_i, shown in shades of gray) were known to activate certain RhoGEFs. The new results (highlighted in orange) (7) show that G_s, the G protein known to stimulate production of cAMP, can also stimulate a particular RhoGEF; this suggests that the Rho GTPases can potentially be stimulated by the multitude of signals from the entire class of GPCRs, including those coupled to G_s. IP₃, inositol 1,4,5-trisphosphate.

Funding and additional information—This work was supported in part by National Institutes of Health Grant R56DK119262 (to V. Z. S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

PH

pleckstrin homology

DH

Dbl homology

GEF

guanine nucleotide exchange factor

PRG

PDZ-RhoGEF

GPCR

G protein–coupled receptor.

     

Rickettsial infections: A blind spot in our view of neglected tropical diseases

Rickettsial diseases are a group of vector-borne bacterial infections that cause acute febrile illness with potentially severe or fatal complications. These vector-borne diseases are prevalent in tropical and subtropical regions worldwide and disproportionately affect poorer communities but are scientifically underrecognized. Despite this, they are not included in the World Health Organization’s list of neglected tropical diseases nor were they mentioned in Peter Hotez’s recent reflections on “What constitutes a neglected tropical disease?” in PLOS Neglected Tropical Diseases [1]. Here we present the case that rickettsial infections, as an overlooked cause of morbidity, mortality, and economic losses in marginalized populations, should be recognized as neglected tropical diseases. We describe how this oversight is the result of a number of factors and how it negatively impacts patient outcomes. We then propose measures to address the neglect of rickettsial infections in both scientific research and public health interventions.     

Unexpectedly high mutation rate of a deep-sea hyperthermophilic anaerobic archaeon

Deep-sea hydrothermal vents resemble the early Earth, and thus the dominant Thermococcaceae inhabitants, which occupy an evolutionarily basal position of the archaeal tree and take an obligate anaerobic hyperthermophilic free-living lifestyle, are likely excellent models to study the evolution of early life. Here, we determined that unbiased mutation rate of a representative species, Thermococcus eurythermalis, exceeded that of all known free-living prokaryotes by 1-2 orders of magnitude, and thus rejected the long-standing hypothesis that low mutation rates were selectively favored in hyperthermophiles. We further sequenced multiple and diverse isolates of this species and calculated that T. eurythermalis has a lower effective population size than other free-living prokaryotes by 1-2 orders of magnitude. These data collectively indicate that the high mutation rate of this species is not selectively favored but instead driven by random genetic drift. The availability of these unusual data also helps explore mechanisms underlying microbial genome size evolution. We showed that genome size is negatively correlated with mutation rate and positively correlated with effective population size across 30 bacterial and archaeal lineages, suggesting that increased mutation rate and random genetic drift are likely two important mechanisms driving microbial genome reduction. Future determinations of the unbiased mutation rate of more representative lineages with highly reduced genomes such as Prochlorococcus and Pelagibacterales that dominate marine microbial communities are essential to test these hypotheses.Subject terms: Archaea, Population genetics

One theory for the origin of life is that the last universal common ancestor was an anaerobic hyperthermophilic organism inhabiting the deep-sea hydrothermal vents, as these environments display a few characteristics paralleling the early Earth [1]. While hydrothermal vents vary with chemical parameters, they all share a high temperature zone near the black chimney with anaerobic fluid from it. In the past decades, great efforts were made to understand the metabolic strategies deep-sea hyperthermophiles use to conserve energy and cope with physicochemical stresses, and to appreciate the molecular mechanisms leading to the stabilization of nucleic acids and proteins at exceedingly high temperatures [2, 3]. However, little is known whether they have a low or high intrinsic (i.e., not selected by environmental pressure) rate to change their genetic background information and whether this intrinsic potential itself is a result of selection shaped by these unique habitats.A previous population genomic analysis showed that protein sequences are under greater functional constraints in thermophiles than in mesophiles, suggesting that mutations are functionally more deleterious in thermophiles than in mesophiles [4]. This explanation is also supported by experimental assays showing nearly neutral mutations in temperate conditions become strongly deleterious at high temperature [5]. Furthermore, fluctuation tests on a hyperthermophilic archeaon Sulfolobus acidocaldarius [6] and a hyperthermophilic bacterium Thermus thermophilus [7] consistently showed that hyperthermophiles have much lower mutation rate compared to mesophiles. This appears to support the hypothesis that selection favors high replication fidelity at high temperature [5].Nevertheless, mutation rates measured using fluctuation experiments based on reporter loci are known to be biased, since the mutation rate of the organism is extrapolated from a few specific nonsynonymous mutations enabling survival in an appropriate selective medium, which renders the results susceptible to uncertainties associated with the representativeness of these loci and to inaccuracies of the assumptions made in extrapolation methods [8–10]. These limitations are avoided by the mutation accumulation (MA) experiment followed by whole-genome sequencing (WGS) of derived lines. In the MA part, multiple independent MA lines initiated from a single progenitor cell each regularly pass through a single-cell bottleneck, usually by transferring on solid medium. As the effective population size (N_e) becomes one, selection is unable to eliminate all but the lethal mutations, rendering the MA/WGS an approximately unbiased method to measure the spontaneous mutation rate [11].Members of the free-living anaerobic hyperthermophilic archaeal family Thermococcaceae are among the dominant microbial lineages in the black-smoker chimney at Guaymas Basin [12] and other deep-sea hydrothermal vents [13, 14]. This family only contains three genera: Thermococcus, Pyrococcus and Palaeococcus. In this study, the MA/WGS procedure was applied to determine the unbiased spontaneous mutation rate of a representative member Thermococcus eurythermalis A501, a conditional pizeophilic archaeon which can grow equally well from 0.1 MPa to 30 MPa at 85 °C [15, 16]. The MA lines were propagated at this optimal temperature on plates with gelrite which tolerates high temperature, and the experiment was performed under normal air pressure and in strictly anaerobic condition (Fig. 1A–D). To the best of our knowledge, this is the first report of unbiased mutation rate of a hyperthermophile and an obligate anaerobe.Open in a separate windowFig. 1Experimental determination of the unbiased mutation rate of the Thermococcus eurythermalis A501 is challenging because this archaeon has unusual physiology (i.e., obligate anaerobic and obligate hyperthermophilic).A The preparation of anaerobic high temperature tolerant gelrite plate. After sterilization and polysulfide addition via syringe, the plates are made in an anaerobic chamber. B The incubation of the strain T. eurythermalis A501 at 85 °C in liquid medium. C The initiation of mutation accumulation (MA) by spreading cells from a single founding colony to 100 lines. Plates are placed in an anaerobic jar for incubation in strictly anaerobic condition at 85 °C. D The MA process followed by whole-genome sequencing and data analysis. Single colony of each line is transferred to a new plate for N times (here N = 20). E Base-substitution mutations and insertion/deletion mutations across the whole genome of T. eurythermalis. The dashed vertical line separates the chromosome and plasmid. The height of each bar represents the number of base-substitution mutations across all MA lines within 10 kbp window. Green and red triangles denote insertion and deletion, respectively. The locus tags of the 14 genes with statistical enrichment of mutations are shown.Our MA experiment allowed accumulation of mutations over 314 cell divisions (after correcting the death rate (Table S1) [17]) in 100 independent lines initiated from a single founder colony and passed through a single cell bottleneck every day. By sequencing genomes of 96 survived lines at the end of the MA experiment, we identified 544 base-substitution mutations over these lines (Table S2), which translates to an average mutation rate (µ) of 85.01 × 10⁻¹⁰ per cell division per nucleotide site (see Supplementary information). The ratio of accumulated nonsynonymous to synonymous mutations (371 vs 107) did not differ from the ratio of nonsynonymous to synonymous sites (1,485,280 vs 403,070) in the A501 genome (χ² test; p > 0.05). Likewise, there was no difference of the accumulated mutations between intergenic (65) and protein-coding sites (478) (χ² test; p > 0.05). These are evidence for minimal selective elimination of deleterious mutations during the MA process. In general, the mutations were randomly distributed along the chromosome and the plasmid, though 86 base-substitution mutations fell into 14 genes which showed significant enrichment of mutations (bootstrap test; p < 0.05 for each gene) and 52 out of the 86 base-substitution mutations were found in five genes (TEU_RS04685 and TEU_RS08625-08640 gene cluster) (Fig. 1E, Table S3). A majority of mutations in these five genes may have inactivated these genes (38 out of 71 in the former gene and 33 out of 43 in the latter gene cluster) either by nonsense mutation or insertion-deletion (INDEL) mutation. The phenomenon of mutation clustering is not unique to this organism; it was reported in another MA study with the yeast Schizosaccharomyces pombe, and these genomic regions may represent either mutational hotspots or that mutations confer selective advantages under experimental conditions [18]. The TEU_RS04685 encodes the beta subunit of the sodium ion-translocating decarboxylase which is an auxiliary pathway for ATP synthesis by generating sodium motive force via decarboxylation [19], and the TEU_RS08625-08640 encodes a putative nucleoside ABC transporter. These genes appear to be important for energy conservation in the highly fluctuating deep-sea hydrothermal fluids. Under the culture conditions in which peptides and amino acids were stably and sufficiently supplied (see the TRM medium recipe in Supplementary information), however, these genes may be dispensable because peptides and amino acids are the preferred carbon and energy sources for T. eurythermalis [15]. On the other hand, some of these genes (e.g., TEU_RS08625) were shown to be upregulated under alkaline stress [16], and thus may be similarly induced under the culture condition in which pH is elevated compared to the vents. Besides, the laboratory condition differed from the vents in a number of other physicochemical features including hydrostatic pressure (0.1 MPa during the MA process versus 20 MPa in situ), temperature and salinity, which likely imposed additional selective pressures on the mutation accumulation processes. Taken together, deleting these genes were likely translated to a net fitness gain and were thus driven by selection. Removing these mutations led to a spontaneous mutation rate of 71.57 × 10⁻¹⁰ per cell division per site for T. eurythermalis A501. After removing the mutations in these 14 genes, both the accumulated mutations at nonsynonymous sites (288) relative to those (104) at synonymous sites (χ² test; p = 0.014) and the accumulated mutations at intergenic regions (65) relative to protein-coding regions (392) (χ² test; p = 0.013) showed marginally significant differences.To date, over 20 phylogenetically diverse free-living bacterial species and two archaeal species isolated from various environments have been assayed with MA/WGS, and their mutation rates vary from 0.79 × 10⁻¹⁰ to 97.80 × 10⁻¹⁰ per cell division per site [20]. The only prokaryote that displays a mutation rate (97.80 × 10⁻¹⁰ per cell division per site) comparable to A501 is Mesoplasma florum L1 [21], a host-dependent wall-less bacterium with highly reduced genome (~700 genes). Our PCR validation of randomly chosen 20 base-substitution mutations from two MA lines displaying highest mutation rates and of all nine INDEL mutations involving >10 bp changes across all lines (Table S2) indicates that the calculated high mutation rate did not result from false bioinformatics predictions.The extremely high mutation rate of T. eurythermalis is unexpected. One potential explanation in line with the “mutator theory” [22–24] is that high mutation rate may allow the organisms to gain beneficial mutations more rapidly and thus is selectively favored in deep-sea hydrothermal vents where physicochemical parameters are highly fluctuating. Alternatively, high mutation rate is the result of random genetic drift according to the “drift-barrier model” [21]. In this model, increased mutation rates are associated with increased load of deleterious mutations, so natural selection favors lower mutation rates. On the other hand, increased improvements of replication fidelity come at an increased cost of investments in DNA repair activities. Therefore, natural selection pushes the replication fidelity to a level that is set by genetic drift, and further improvements are expected to reduce the fitness advantages [11, 21]. These two explanations for the high mutation rate of T. eurythermalis are mutually exclusive, and resolving them requires the calculation of the power of genetic drift, which is inversely proportional to N_e.A common way to calculate N_e for a prokaryotic population is derived from the equation π_S = 2 × N_e × µ, where π_S represents the nucleotide diversity at synonymous (silent) sites among randomly sampled members of a panmictic population [25]. We therefore sequenced genomes of another eight T. eurythermalis isolates available in our culture collections. Like T. eurythermalis A501, these additional isolates were collected from the same cruise but varying at the water depth from 1987 m to 2009 m at Guaymas Basin. They differ by only up to 0.135% in the 16S rRNA gene sequence and share a minimum whole-genome average nucleotide identity (ANI) of 95.39% (Table S4), and thus fall within an operationally defined prokaryotic species typically delineated at 95% ANI [26]. Population structure analysis with PopCOGenT [27] showed that these isolates formed a panmictic population and that two of them were repetitive as a result of clonal descent (see Supplementary information). Using the median value of π_S = 0.083 across 1628 single-copy orthologous genes shared by the seven non-repetitive genomes, we calculated the N_e of T. eurythermalis to be 5.83 × 10⁶.Next, we collected the unbiased mutation rate of other prokaryotic species determined with the MA/WGS strategy from the literature [11, 28–30]. While the N_e data were also provided from those studies, the isolates used to calculate the N_e were identified based on their membership of either an operationally defined species (e.g., ANI at 95% cutoff) or a phenotypically characterized species (e.g., many pathogens), which often create a bias in calculating N_e [25]. We therefore again employed PopCOGenT to delineate panmictic populations from those datasets and re-calculated N_e accordingly. There was a significant negative linear relationship between µ and N_e on a logarithmic scale (dashed gray line in Fig. 2A [r² = 0.83, slope = −0.85, s.e.m. = 0.09, p < 0.001]) according to a generalized linear model (GLM) regression. This relationship cannot be explained by shared ancestry, as confirmed by phylogenetic generalized least square (PGLS) regression analysis (solid blue line in Fig. 2A [r² = 0.81, slope = −0.81, s.e.m. = 0.09, p < 0.001]). The nice fit of T. eurythermalis to the regression line validated the drift-barrier hypothesis. This is evidence that the high mutation rate of T. eurythermalis is driven by genetic drift rather than by natural selection.Open in a separate windowFig. 2The scaling relationship involving the base-substitution mutation rate per cell division per site (µ), the estimated effective population size (N_e), and genome size across 28 bacterial and two archaeal species.All three traits’ values were logarithmically transformed. The mutation rates of these species are all determined with the mutation accumulation experiment followed by whole-genome sequencing of the mutant lines. The mutation rate of species numbered 1–29 (blue) is collected from literature and that of the species 30 (red) is determined in the present study. Among the numbered species shown in the figure, the species #6 Haloferax volcanii is facultative anaerobic halophilic archaeon, and the species #30 is an obligate anaerobic hyperthermophilic archaeon. A The scaling relationship between µ and N_e. B The scaling relationship between µ and genome size. C The scaling relationship between genome size and N_e. Numbered data points 21–29 are not shown in A and C because of the lack of population dataset for estimation of N_e. The dashed gray lines and blue lines represent the generalized linear model (GLM) regression and the phylogenetic generalized least square (PGLS) regression, respectively. The Bonferroni adjusted outlier test for the GLM regression show that #7 Janthinobacterium lividum is an outlier in the scaling relationship between µ and N_e, and #9 Mesoplasma florum is an outlier in the scaling relationship between genome size and N_e. No outlier was identified in the PGLS regression results.As stated in the drift-barrier theory, high mutation rate is associated with a high load of deleterious mutations. In the absence of back mutations, recombination becomes an essential mechanism in eliminating deleterious mutations [31]. In support of this argument, the ClonalFrameML analysis [32] shows that members of the T. eurythermalis population recombine frequently, with a high ratio of the frequency of recombination to mutation (ρ/θ = 0.59) and a high ratio of the effect of recombination to mutation (r/m = 5.76). In fact, efficient DNA incorporation to Thermococcaceae genomes from external sources has been well documented experimentally [33, 34]. A second potentially important mechanism facilitating T. eurythermalis adaptation at high temperature is strong purifying selection at the protein sequence level, as protein sequences in thermophiles are generally subjected to stronger functional constraints compared to those in mesophiles [4, 35].Our result of the exceptionally high mutation rate of a free-living archaeon is a significant addition to the available collection of the MA/WGS data (Table S5), in which prokaryotic organisms with very high mutation rate have only been known for a host-dependent bacterium (Mesoplasma florum L1) with unusual biology (e.g., cell wall lacking). The availability of these two deeply branching (one archaeal versus the other bacterial) organisms adopting opposite lifestyles (one free-living versus the other host-restricted; one hyperthermophilic versus the other mesophilic; one obligate anaerobic versus the other facultative anaerobe), along with other phylogenetically and ecologically diverse prokaryotic organisms displaying low and intermediate mutation rates, provides an opportunity to help illustrate mechanisms potentially driving genome size evolution across prokaryotes. We found a negative linear relationship (dashed gray line in Fig. 2B [r² = 0.49, slope = −1.66, s.e.m. = 0.32, p < 0.001]) between genome size and base-substitution mutation rate, which is consistent with the hypothesis that increased mutation rate drives microbial genome reduction. We also showed a positive linear relationship (dashed gray line in Fig. 2C [r² = 0.47, slope = 0.24, s.e.m. = 0.06, p < 0.001]) between genome size and N_e, which suggests that random genetic drift drives genome reduction across prokaryotes. These correlations remain robust when the data were analyzed as phylogenetically independent contrasts (blue solid lines in Fig. 2B [r² = 0.47, slope = −1.75, s.e.m. = 0.34, p < 0.001] and in Fig. 2C [r² = 0.45, slope = 0.25, s.e.m. = 0.06, p < 0.001]). Our results are consistent with recent studies which employed mathematical modeling and/or comparative sequence analyses to show random genetic drift [36] and increased mutation rate [37] driving genome reduction across diverse bacterial lineages including both free-living and host-dependent bacteria. One benefit of the present study is that it directly measures µ and N_e, as compared to those recent advances which relied on proxies for these metrics (e.g., using the ratio of nonsynonymous substitution rate to synonymous substitution rate to represent N_e) to infer mechanisms of genome reduction.Despite this advantage, there are important caveats to our conclusions related to the mechanisms of genome reduction. The correlation analyses performed here are inspired by Lynch and colleagues’ work, who had great success explaining eukaryotic genome expansion with genetic drift [11, 38]. However, there are a few key differences of genomic features between prokaryotes and eukaryotes, which makes it more difficult to explain the correlation observed in prokaryotes. Importantly, genome sizes of eukaryotes can vary over several orders of magnitude, whereas those of free-living prokaryotes differ by only an order of magnitude [11], so there is much less variability to explain in prokaryotes. Moreover, eukaryotic genomes experience dramatic expansions of transposable elements which are often considered as genomic parasites, whereas prokaryotic genomes including those large ones are usually depleted with transposable elements and their genome size variations are largely driven by gene content [39]. Aside from these conceptual difficulties, the plots (Fig. 2B, C) are poorly populated with typical free-living species carrying small genomes such as the Prochlorococcus (mostly 1.6–8 Mb) and Pelagibacterales (1.3–1.5 Mb), which dominate the photosynthetic and heterotrophic microbial communities, respectively, in the ocean [40]. It has been generally postulated that bacterial species in these lineages have very large N_e [39–41], though there has been little direct evidence for it [42, 43]. If confirmed through the measurement of the unbiased mutation rate (µ) followed by the calculation of N_e based on µ, it might compromise the linear relationship between genome size and N_e observed here (Fig. 2C). It is also not necessarily appropriate to translate correlations to causal relationships. For example, the correlation between increased mutation rates and decreased genome sizes (Fig. 2B) does not necessarily mean that increased mutation rate drives genome reduction. This is because high mutation rates are observed in species with small N_e. Given that deletion bias is commonly found in prokaryotes [44, 45], genome reduction can be easily explained by increased fixation of deletional mutations in species with smaller N_e. High mutation rates in these species are simply the result of random genetic drift as explained by the drift-barrier theory, and they may have a limited role in driving genome reduction.Whereas our analysis based on the available data did not support natural selection as a universal mechanism driving genome reduction across prokaryotes (Fig. 2B, C), it does not mean that selection has no role in genome reduction of a particular taxon. In the case of thermophiles, proponents for selection acting to reduce genomes explained that genome size, due to its positive correlation with cell volume, may be an indirect target of selection which strongly favors smaller cell volume [35]. The underlying principle is that high temperature requires cells to increase the lipid content and change the lipid composition of the cell membranes, which consumes a large part of the cellular energy, and thus lower cell volume is selectively favored at high temperature [35]. Our calculations of a relatively small N_e in T. eurythermalis does not necessarily contradict with this selective argument, given that the fitness gained by decreasing cell volume and thus reducing genome size is large enough to overcome the power of random genetic drift. On the other hand, our data strongly indicate that neutral forces dictate the genome evolution of T. eurythermalis, and they are not negligible with regard to its genome reduction process. The significantly more deletion over insertion events (t test; 95 versus 37 events with p < 0.001 and 48 versus 20 events with p < 0.05 before and after removing the 14 genes enriched in mutations, respectively) and the significantly more nucleotides involved in deletions over insertions (t test; 433 versus 138 bp with p < 0.05 and 386 versus 121 bp with p < 0.001 before and after removing the 14 genes enriched in mutations, respectively) suggest that the deletion bias, combined with increased chance fixation of deletion mutants due to low N_e, is a potentially important neutral mechanism giving rise to the small genomes of T. eurythermalis (2.12 Mbp).The globally distributed deep-sea hydrothermal vents are microbe-driven ecosystems, with no known macroorganisms surviving at the vent fluids. Sample collections, microbial isolations, and laboratory propagations of mutation lines at hot and anoxic conditions are challenging. In the present study, we determined that T. eurythermalis, and perhaps Thermococcaceae in general, has a highly increased mutation rate and a highly decreased effective population size compared to all other known free-living prokaryotic lineages. While it remains to be tested whether this is a common feature among the vents’ microbes, the present study nevertheless opens a new avenue for investigating the hyperthemophile ecology and evolution in the deep sea.     

Hypertension and atrial fibrillation: Closing a virtuous circle

Ying Gue and Gregory Lip discuss the accompanying study by Ana-Catarina Pinho-Gomes and co-workers on blood pressure lowering treatment in patients with atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with an increased risk of major adverse cardiovascular events (MACE) [1]. Patients with AF typically have other concomitant cardiovascular risk factors—hypertension being one of the commonly associated conditions with a prevalence of up to 90% in major clinical trials of AF [2]. Not only is hypertension common in AF, but it is also an independent risk factor for ischaemic and haemorrhagic strokes, thus bearing important implications for patient prognosis [3]. Therefore, optimal management of hypertension in patients with AF is vital to prevent future MACE. In the accompanying individual-participant data (IPD) meta-analysis [4], the authors from the Blood Pressure Lowering Treatment Trialists’ Collaboration (BPLTTC) aimed to investigate the effects of blood pressure (BP) lowering treatment on MACE when comparing patients with and without AF at baseline. They aimed to address 4 main questions: firstly, whether AF at baseline modifies BP treatment effects; secondly, whether associations between intensity of BP reduction and outcomes are similar with or without AF; thirdly, whether treatment effect is dependent on baseline systolic BP; and lastly, whether classes of antihypertensives have different treatment effect in AF.A total of 22 trials were included with a total of 188,570 participants and 13,266 patients with a history AF at baseline. Baseline characteristics were different between the 2 groups, with AF patients being older (mean age 70 years versus 65 years), had lower baseline BP (mean 143/84 mmHG versus 155/88 mmHg), and were more commonly prescribed diuretics (50.5% versus 23.8%), angiotensin converting enzyme inhibitors (59.6% versus 44%), beta-blockers (51.3% versus 36%), and alpha-blockers (10.7% versus 4.4%) at baseline. This reflects the more commonly associated cardiovascular comorbidities (hypertension, heart failure, and older age) in patients with AF at baseline [3,5,6].Among the authors’ findings, firstly, the mean difference in Systolic blood pressure (SBP) reduction was 7.2 mmHg in placebo-controlled studies (8 studies), 2.3 mmHg in drug–drug comparisons (12 studies), and 10.9 mmHg in more-versus-less intensive treatment trials (2 studies) with an overall difference of 3.7 mmHg. When comparing differences in SBP reduction, the authors reported no difference between patients with or without AF (3.3 mmHg versus 3.7 mmHg).Secondly, meta-regression showed that each 5 mmHg reduction in BP equated to a 10% reduction in MACE in patients with and without AF. Thirdly, authors found no evidence of difference in treatment effects at different baseline systolic BP. And lastly, there was no difference between classes of antihypertensives (renin-angiotensin-aldosterone system inhibitor (RAAS-I) and calcium channel blockers (CCB)), although this conclusion was limited by small numbers of participants with AF in these studies.The authors conclude that due to the higher risk of MACE in AF patients compared to those without AF, the same relative risk reduction with BP control translates to greater absolute risk reduction in AF patients and, therefore, more focus should be placed on addressing the associated cardiovascular risk factor such as hypertension to better improve the outcomes in patients with AF.We congratulate the authors for performing this highly relevant IPD meta-analysis to highlight the importance of the holistic management of patients with AF and the need for more evidence in this area. This thought process is echoed in the most recent European Society of Cardiology (ESC) guideline on the management of AF with a shift from managing AF, from the CC (Confirm AF and Characterise AF) to ABC (“A” Anticoagulation/Avoid stroke, “B” Better symptom control, and “C” Comorbidities/Cardiovascular risk factor management) approaches of managing AF [7].The association of BP control and reduction in MACE in patients with AF does not come as a surprise as hypertension has been linked not only with adverse cardiovascular outcomes but also with an increased risk of AF [8]. The importance of BP control has previously been shown in a large meta-analysis of 61 prospective observational studies involving 12.7 million person-years, i.e., that there is a linear relation between BP and vascular (and overall) mortality, starting from values of 115/75 mmHg [9]. BP control reduces mortality from ischaemic vascular events and haemorrhagic complications from anticoagulation treatment in patients with AF [3]. The reduction in mortality was reflected in the present study [4], although there was no differentiation between haemorrhagic and ischaemic stroke in the outcomes.One limitation of this work is the inclusion of trials involving only patients with AF. AF status being the inclusion or exclusion criteria prior to randomisation could add to the risk of selection bias within the analysis. In addition, the majority of AF participants are from the Atrial Fibrillation Clopidogrel Trial With Irbesartan for Prevention of Vascular Events (ACTIVE-I) trial dataset [10], which can bias the effect seen given the difference in patient characteristic at baseline. Similarly, including trials with only patients without AF may dilute the effects of BP lowering that could be seen in patients with AF. The authors have addressed this by performing sensitivity analyses excluding these studies which have shown comparable results, reassuring us that the impact of selection bias is not significant on the study conclusions.In the new 2020 ESC guidelines on the “ABC” approach to the management of AF [7], the management of other associated cardiovascular risk factors has become an integral component of optimal management of AF. The shift in the management of AF towards a more holistic approach is one step in the right direction as has been shown by the improvement in outcomes [11,12] and reduction in healthcare-associated costs [13]. Compliance with the “ABC” management approach requires clear, evidence-based guidelines in terms of treatment targets. With regard to hypertension, the currently recommended BP target (≤130/80) is based upon the current ESC hypertension guidelines [14] and observational data showing greatest benefit of BP between 120 and 129 systolic [15–17]. Whether this target is optimal for the reduction of future MACE in patients with AF is unknown.This IPD meta-analysis by the BPLTTC has shown that the presence of AF does not alter the treatment effects of antihypertensives. BP lowering in patients with and without AF shows a corresponding reduction in MACE to a similar extent. Owing to the higher absolute risk of MACE in patients with AF, BP lowering in these patients would result in greater absolute risk reduction. This should provide sufficient evidence to convince clinicians regarding the benefits of strict BP control in patients with AF, and the consultation for patients with AF should always involve a conversation about managing hypertension, be it lifestyle modification or pharmacological treatment. However, the potential benefits (or harms) of a much lower BP target (below the recommended 130/80 mmHg) and ideal choice or combination of antihypertensives remain unanswered and would require future studies to provide further insight.     

A Novel Single-Domain Na+-Selective Voltage-Gated Channel in Photosynthetic Eukaryotes

The evolution of Na⁺-selective four-domain voltage-gated channels (4D-Na_vs) in animals allowed rapid Na⁺-dependent electrical excitability, and enabled the development of sophisticated systems for rapid and long-range signaling. While bacteria encode single-domain Na⁺-selective voltage-gated channels (BacNa_v), they typically exhibit much slower kinetics than 4D-Na_vs, and are not thought to have crossed the prokaryote–eukaryote boundary. As such, the capacity for rapid Na⁺-selective signaling is considered to be confined to certain animal taxa, and absent from photosynthetic eukaryotes. Certainly, in land plants, such as the Venus flytrap (Dionaea muscipula) where fast electrical excitability has been described, this is most likely based on fast anion channels. Here, we report a unique class of eukaryotic Na⁺-selective, single-domain channels (EukCatBs) that are present primarily in haptophyte algae, including the ecologically important calcifying coccolithophores, Emiliania huxleyi and Scyphosphaera apsteinii. The EukCatB channels exhibit very rapid voltage-dependent activation and inactivation kinetics, and isoform-specific sensitivity to the highly selective 4D-Na_v blocker tetrodotoxin. The results demonstrate that the capacity for rapid Na⁺-based signaling in eukaryotes is not restricted to animals or to the presence of 4D-Na_vs. The EukCatB channels therefore represent an independent evolution of fast Na⁺-based electrical signaling in eukaryotes that likely contribute to sophisticated cellular control mechanisms operating on very short time scales in unicellular algae.

Electrical signals trigger rapid physiological events that underpin an array of fundamental processes in eukaryotes, from contractile amoeboid locomotion (Bingley and Thompson, 1962), to the action potentials of mammalian nerve and muscle cells (Hodgkin and Huxley, 1952). These events are mediated by voltage-gated ion channels (Brunet and Arendt, 2015). In excitable animal cells, Ca²⁺- or Na⁺-selective members of the four-domain voltage-gated cation channel family (4D-Ca_v/Na_v) underpin well-characterized signaling processes (Catterall et al., 2017). The 4D-Ca_v/Na_v family is broadly distributed across eukaryotes, contributing to signaling processes associated with motility in some unicellular protist and microalgal species (Fujiu et al., 2009; Lodh et al., 2016), although these channels are absent from land plants (Edel et al., 2017). It is likely that the ancestral 4D-Ca_v/Na_v channel was Ca²⁺-permeable, with Na⁺-selective channels arising later within the animal lineage (Moran et al., 2015). This shift in ion selectivity represented an important innovation in animals, allowing rapid voltage-driven electrical excitability to be decoupled from intracellular Ca²⁺ signaling processes (Moran et al., 2015).Na⁺-selective voltage-gated channels have not been described in other eukaryotes, although a large family of Na⁺-selective channels (BacNa_v) is present in prokaryotes (Ren et al., 2001; Koishi et al., 2004). BacNa_v are single-domain channels that form homotetramers, resembling the four-domain architecture of 4D-Ca_v/Na_v. Studies of BacNa_v channels have provided considerable insight into the mechanisms of gating and selectivity in voltage-dependent ion channels (Payandeh et al., 2012; Zhang et al., 2012). The range of activation and inactivation kinetics of native BacNa_v are generally slower than observed for 4D-Na_v, suggesting that the concatenation and subsequent differentiation of individual pore-forming subunits may have enabled 4D-Na_v to develop specific properties such as fast inactivation, which is mediated by the conserved intracellular Ile–Phe–Met linker between domains III and IV (Fig. 1A; Irie et al., 2010; Catterall et al., 2017).Open in a separate windowFigure 1.EukCatBs represent a novel class of single-domain channels. A, Schematic diagram of a single-domain EukCatB channel. The voltage-sensing module (S1–S4, blue), including conserved positively charged (++) residues of segment (S4) that responds to changes in membrane potential, is shown. The pore module (S5–S6, red) is also indicated, including the SF motif (Ren et al., 2001). The structure of a 4D-Na_v (showing the SF of rat 4D-Na_v1.4 with canonical “DEKA” locus of Na⁺-selective 4D-Na_v1s) is also displayed (right). The Ile–Phe–Met motif of the fast inactivation gate is indicated (West et al., 1992) B, Maximum likelihood phylogenetic tree of single-domain, voltage-gated channels including BacNa_v and the three distinct classes of EukCat channels (EukCatA–C). Representatives of the specialized family of single-domain Ca²⁺ channels identified in mammalian sperm (CatSpers) are also included. SF for each sequence is shown (right). “Position 0” of the high-field–strength site that is known to be important in determining Na⁺ selectivity (Payandeh et al., 2011), is colored red. Channel sequences selected for functional characterization in this study are shown in bold. EukCatA sequences previously characterized (Helliwell et al., 2019) are also indicated, as is NaChBac channel from B. halodurans (Ren et al., 2001). Maximum likelihood bootstrap values (>70) and Bayesian posterior probabilities (>0.95) are above and below nodes, respectively. Scanning electron micrographs of coccolithophores E. huxleyi (scale bar = 2 μm) and S. apsteinii, (scale bar = 10 μm) are shown.We recently identified several classes of ion channel (EukCats) bearing similarity to BacNa_v in the genomes of eukaryotic phytoplankton. Characterization of EukCatAs found in marine diatoms demonstrated that these voltage-gated channels are nonselective (exhibiting permeability to both Na⁺ and Ca²⁺) and play a role in depolarization-activated Ca²⁺ signaling (Helliwell et al., 2019). Two other distinct classes of single-domain channels (EukCatBs and EukCatCs) were also identified that remain uncharacterized. These channels are present in haptophytes, pelagophytes, and cryptophytes (EukCatBs), as well as dinoflagellates (EukCatCs; Helliwell et al., 2019). Although there is a degree of sequence similarity between the distinct EukCat clades, the relationships between clades are not well resolved, and there is not clear support for a monophyletic origin of EukCats. The diverse classes of EukCats may therefore exhibit significant functional differences. Characterization of these different classes of eukaryote single-domain channels is thus vital to our understanding of eukaryote ion channel structure, function, and evolution, and to our gaining insight into eukaryote membrane physiology more broadly.Notably, EukCatB channels were found in ecologically important coccolithophores, a group of unicellular haptophyte algae that represent major primary producers in marine ecosystems. Coccolithophores are characterized by their ability to produce a cell covering of ornate calcium carbonate platelets (coccoliths; Fig. 1B; Taylor et al., 2017). The calcification process plays an important role in global carbon cycling, with the sinking of coccoliths representing a major flux of carbon to the deep ocean. Patch-clamp studies of coccolithophores indicate several unusual aspects of membrane physiology, such as an inwardly rectifying Cl⁻ conductance and a large outward H⁺ conductance at positive membrane potentials, which may relate to the increased requirement for pH homeostasis associated with intracellular calcification. Here we report that EukCatB channels from two coccolithophore species (Emiliania huxleyi and Scyphosphaera apsteinii) act as very fast Na⁺-selective voltage-gated channels that exhibit many similarities to the 4D-Na_vs, which underpin neuronal signaling in animals. Thus, our findings demonstrate that the capacity for rapid Na⁺-based signaling has evolved in certain photosynthetic eukaryotes, contrary to previous widely held thinking.     

Drugs that target early stages of Onchocerca volvulus: A revisited means to facilitate the elimination goals for onchocerciasis

Several issues have been identified with the current programs for the elimination of onchocerciasis that target only transmission by using mass drug administration (MDA) of the drug ivermectin. Alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed. We posit that the addition of “prophylactic” drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may hasten the time to elimination and also will support achieving a sustained elimination of onchocerciasis. These “prophylactic” drugs will target the infective third- (L3) and fourth-stage (L4) larvae of Onchocerca volvulus and consequently prevent the establishment of new infections not only in uninfected individuals but also in already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin), and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development. Prophylaxis of onchocerciasis is not a new concept. We present new data showing that these drugs can inhibit L3 molting and/or inhibit motility of L4 at IC₅₀ and IC₉₀ that are covered by the concentration of these drugs in plasma based on the corresponding pharmacological profiles obtained in human clinical trials when these drugs were tested using various doses for the therapeutic treatments of various helminth infections.

Onchocerca volvulus is an obligate human parasite and the causative agent for onchocerciasis, which is a chronic neglected tropical disease prevalent mostly in the sub-Saharan Africa. In 2017, 20.9 million people were infected, with 14.6 million having skin pathologies and 1.15 million having vision loss [1]. The socioeconomic impact of onchocerciasis and the debilitating morbidity caused by the disease prompted the World Health Organization (WHO) to initiate control programs that were first focused on reducing onchocerciasis as a public health problem, and since 2012, the ultimate goal is to eliminate it by 2030 [2]. Over the years, WHO sponsored and coordinated 3 major programs: The Onchocerciasis Control Programme (OCP), the African Programme for Onchocerciasis Control (APOC), and the Onchocerciasis Elimination Program of the Americas (OEPA). Since 1989, the control measures depended on mass drug administration (MDA) annually or biannually with ivermectin, which targets the transmitting stage of parasite, the microfilariae [3–5]. However, several issues have been identified with the current MDA programs including the need to expand the treatment to more populations depending on baseline endemicity and transmission rates [2,6]. Moreover, it became apparent that alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed [2]. Ivermectin has only mild to moderate effects on the adult stages of the parasite [7–9], and there are communities in Africa where the effects of ivermectin are suboptimal [10]. It is also contraindicated in areas of Loa loa co-endemicity [11], as well as in children under the age of 5 and in pregnant women. By relying only on MDA with ivermectin, the most optimistic mathematical modeling predicts that elimination will occur only in 2045 [12].To support the elimination agenda, much of the recent focus has been on improving efficacy outcomes through improved microfilariae control with moxidectin and the discovery of macrofilaricidal drugs that target the adult O. volvulus parasites [13–18]. We posit that the addition of “prophylactic” drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may also hasten the time for elimination and support achieving a sustained elimination of onchocerciasis. These “prophylactic” drugs will target the infective third- (L3) and fourth-stage (L4) larvae of O. volvulus and consequently prevent the establishment of new infections not only in the uninfected individuals but also in the already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin) [19,20], and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development [21].Prophylaxis of onchocerciasis is not a new concept. In the 1980s, once ivermectin was introduced as a “prophylactic” drug against the filarial dog heartworm, Dirofilaria immitis [22], its prophylactic effects were also examined in Onchocerca spp. In chimpanzees, a single dose of ivermectin (200 μg/kg) was highly protective (83% reduction in patent infections) when given at the time of the experimental infection and tracked for development of patency over 30 months. It was, however, much less effective (33% reduction in patent infections) when given 1 month postinfection with the L3s, at which time the L4s had already developed [23]. Moreover, monthly treatment with ivermectin at either 200 μg/kg or 500 μg/kg for 21 months completely protected naïve calves against the development of O. ochengi infection as compared to untreated controls, which were 83% positive for nodules and 100% positive for patency [24]. When naïve calves exposed to natural infection were treated with either ivermectin (150 μg/kg) or with moxidectin (200 μg/kg) monthly or quarterly, none of the animals developed detectable infections after 22 months of exposure, except 2 animals in the quarterly ivermectin treated group which had 1 nodule each; in the non-treated control group, the nodule prevalence was 78.6% [25]. These prophylactic studies in calves exposed to natural infections clearly demonstrated that monthly or quarterly treatments with ivermectin and/or moxidectin over 22 months were highly efficacious against the development of new infections. When ivermectin was administered in a highly endemic region of onchocerciasis in Cameroon every 3 months over a 4-year period, it resulted in reduced numbers of new nodules (17.7%) when compared to individuals who were treated annually. This recent study suggests that ivermectin may have also a better prophylactic effect in humans when administered quarterly [26].Importantly, moxidectin, a member of the macrocyclic lactone family of anthelmintic drugs, also used in veterinary medicine like ivermectin [20], was recently approved for the treatment of onchocerciasis as a microfilaricidal drug in individuals over the age of 12 [20]. In humans, a single dose of moxidectin (8 mg) appeared to be more efficacious than a single dose of ivermectin (150 μg/kg) in terms of lowering microfilarial loads [17]. Modeling has shown that an annual treatment with moxidectin and a biannual treatment with ivermectin would achieve similar reductions in the duration of the MDA programs when compared to an annual treatment with ivermectin [27].In our efforts to identify macrofilaricidal drugs, we tested a selection of drugs for their ability to inhibit the molting of O. volvulus L3 to L4 as part of the in vitro drug screening funnel [13,28–31]. With some being highly effective, we decided to also examine the effects of the known MDA drugs and those already in clinical development for macrofilaricidal effects on molting of L3 and the motility of L4 (S1 Text) as potential “prophylactic” drugs. When ivermectin and moxidectin were evaluated, we found that both drugs were highly effective as inhibitors of molting: IC₅₀ of 1.048 μM [918.86 ng/ml] and IC₉₀ of 3.73 μM [2,949.1 ng/ml] for ivermectin and IC₅₀ of 0.654 μM [418.43 ng/ml] and IC₉₀ of 1.535 μM [985.3 ng/ml] for moxidectin (Table 1 and S1 Fig), with moxidectin being more effective than ivermectin. When both drugs were tested against the L4, we found that both drugs inhibited the motility of L4s after 6 days of treatment: Ivermectin had an IC₅₀ of 1.38 μM [1,207.6 ng/ml] and IC₉₀ of 31.45 μM [27,521.9 ng/ml] (Table 1 and S1 Fig), while moxidectin had an IC₅₀ of 1.039 μM [665.4 ng/ml] and IC₉₀ of approximately 30 μM [approximately 19,194 ng/ml] (Table 1 and S1 Fig). Interestingly, when the treatment of L4 with both drugs was prolonged, the IC₅₀ values for the inhibition of L4 motility on day 11 with ivermectin and moxidectin were 0.444 μM and 0.380 μM, respectively. Significantly, from the prospect of employing both drugs for prophylaxis against new infections with O. volvulus, moxidectin (8 mg) has an advantage as it achieves a maximum plasma concentration of 77.2 ± 17.8 ng/ml, is metabolized minimally, and has a half-life time of 40.9 ± 18.25 days with an area under the curve (AUC) of 4,717 ± 1,494 ng*h/ml in healthy individuals [32], which covers the experimental IC₅₀ achieved by moxidectin for inhibiting both L3 molting and L4 motility, and the IC₉₀ for L3s. In comparison, ivermectin reaches a maximum plasma concentration of 54.4 ± 12.2 ng/ml with a half-life of 1.5 ± 0.43 days and an AUC of 3,180 ± 1,390 ng*h/ml in healthy humans [33], which only covers the IC₅₀ for inhibiting molting of L3 and motility of L4. We therefore reason that based on the significantly improved pharmacokinetic profile of moxidectin and its efficacy against both L3 and L4 larvae in vitro (Table 1), it might have a better “prophylactic” profile than ivermectin for its potential to interrupt the development of new O. volvulus infections, and thus ultimately affect transmission and further support the elimination of onchocerciasis. Adding to moxidectin’s significance, in dogs, it is a highly effective prophylactic drug against ivermectin-resistant D. immitis strains [19], an important attribute in the event that a suboptimal responsiveness to ivermectin treatment becomes more widespread in the onchocerciasis endemic regions of Africa. Testing the potential effect of moxidectin on the viability or development of transmitted L3 larvae was already recommended by Awadzi and colleagues in 2014 [34], when the excellent half-life of moxidectin in patients with onchocerciasis was realized. We have to acknowledge, however, that the key parameters that can predict the potency of a drug is actually a combination of exposure (drug concentrations) at the site of action and the duration of that exposure that is above the determined IC₅₀/IC₉₀. As we have access to only the AUC, half-life, and Cmax data for each of the in vitro–tested drugs, the use of plasma concentrations for predicting the anticipated potency of these putative “prophylactic” drugs in vivo has to be further assessed with care during clinical trials.Table 1Inhibition of O. volvulus L3 molting and L4 motility in vitro by the prospective prophylactic drugs and their essential pharmacokinetic parameters at doses currently used or deemed safe for use in humans.

Collaboration between NDH and KEA3 Allows Maximally Efficient Photosynthesis after a Long Dark Adaptation

In angiosperms, the NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around PSI (CET). K⁺ Efflux Antiporter3 (KEA3) is a putative thylakoid H⁺/K⁺ antiporter and allows an increase in membrane potential at the expense of the ∆pH component of the proton motive force. In this study, we discovered that the chlororespiratory reduction2-1 (crr2-1) mutation, which abolished NDH-dependent CET, enhanced the kea3-1 mutant phenotypes in Arabidopsis (Arabidopsis thaliana). The NDH complex pumps protons during CET, further enhancing ∆pH, but its physiological function has not been fully clarified. The observed effect only took place upon exposure to light of 110 µmol photons m⁻² s⁻¹ after overnight dark adaptation. We propose two distinct modes of NDH action. In the initial phase, within 1 min after the onset of actinic light, the NDH-dependent CET engages with KEA3 to enhance electron transport efficiency. In the subsequent phase, in which the ∆pH-dependent down-regulation of the electron transport is relaxed, the NDH complex engages with KEA3 to relax the large ∆pH formed during the initial phase. We observed a similar impact of the crr2-1 mutation in the genetic background of the PROTON GRADIENT REGULATION5 overexpression line, in which the size of ∆pH was enhanced. When photosynthesis was induced at 300 µmol photons m⁻² s⁻¹, the contribution of KEA3 was negligible in the initial phase and the ∆pH-dependent down-regulation was not relaxed in the second phase. In the crr2-1 kea3-1 double mutant, the induction of CO₂ fixation was delayed after overnight dark adaptation.

Photosynthesis consists of two sets of reactions, the light reactions and the Calvin-Benson cycle. It takes place in the chloroplast and fixes CO₂ into organic compounds using solar energy. In the light reactions, the absorption of photons activates electron transport in two photosystems. In linear electron transport (LET), PSII catalyzes the light-dependent oxidation of water, resulting in the release of oxygen and protons (H⁺) in the thylakoid lumen. The water-derived excised electrons are transferred to PSI through the cytochrome (Cyt) b₆f complex and ultimately to NADP⁺, producing NADPH. This electron transport is coupled with the translocation of H⁺ from the stroma to the thylakoid lumen via the quinone cycle at the Cyt b₆f complex, resulting in the formation of a proton concentration gradient across the thylakoid membrane. This ∆pH contributes to the formation of proton motive force (pmf) in addition to the membrane potential formed across the thylakoid membrane (∆ψ) that results from the uneven distribution of ions across the membrane. The pmf energizes ATP synthesis via F_oF₁-ATP synthase in chloroplasts (Kramer et al., 2003; Soga et al., 2017) and thus influences the efficiency of the light reactions.The Calvin-Benson cycle depends on NADPH and ATP produced by the light reactions. To fix a molecule of CO₂ into a carbohydrate, three molecules of ATP and two molecules of NADPH are needed. However, this ratio of ATP to NADPH (1.5) is not satisfied by LET (Shikanai, 2007). Photorespiration, which takes place due to the low specificity of Rubisco, the CO₂-fixing enzyme for CO₂, increases the energetic requirements in terms of ATP, raising the above ratio to 1.67. The additional ATP is thought to be supplied by cyclic electron transport around PSI (CET; Yamori and Shikanai, 2016). In contrast to LET, CET is driven solely by PSI and does not contribute to the net production of reducing power. CET recycles electrons from ferredoxin (Fd) to the plastoquinone (PQ) pool and contributes to the additional generation of ∆pH via the quinone cycle. As a result, CET balances the production ratio of ATP and NADPH. In angiosperms, CET has been proposed to consist of two pathways: the PROTON GRADIENT REGULATION5 (PGR5)/PGR5-like Photosynthetic Phenotype1 (PGRL1) protein-dependent, antimycin A-sensitive pathway and the NADH dehydrogenase-like (NDH) complex-dependent antimycin A-insensitive pathway (Munekage et al., 2004). The NDH complex pumps four protons, coupled with the movement of two electrons, from Fd to PQ, further increasing the efficiency of ∆pH formation (Strand et al., 2017).In addition to ATP synthesis, the ∆pH component of pmf also contributes to the down-regulation of electron transport (Shikanai, 2014). Acidification of the thylakoid lumen triggers the thermal dissipation of excessively absorbed light energy from the PSII antennae, a process that is monitored by nonphotochemical quenching (NPQ) of chlorophyll fluorescence (Müller et al., 2001). Low lumenal pH also down-regulates the activity of the Cyt b₆f complex, slowing down the rate of electron transport toward PSI (Stiehl and Witt, 1969). CET-dependent ∆pH formation is also necessary to induce the down-regulation of electron transport, as indicated by the phenotype of the pgr5 mutant. The Arabidopsis (Arabidopsis thaliana) pgr5 mutant cannot induce thermal dissipation under excessive light conditions (Munekage et al., 2002), suggesting that CET-generated ∆pH plays an important role in providing a sufficiently acidic lumen pH that can trigger NPQ. The pgr5 mutant is also defective in the down-regulation of Cyt b₆f activity, resulting in hypersensitivity of PSI to fluctuating light intensity (Tikkanen et al., 2010). Compared with the physiological function of the PGR5/PGRL1-dependent CET, the contribution of the NDH-dependent CET to photoprotection is somewhat minor, although clear phenotypes have been observed in these mutants at low light intensities and fluctuating light levels (Ueda et al., 2012; Yamori et al., 2015, 2016). Furthermore, the physiological function of the NDH complex has not been fully clarified.Both ∆pH and ∆ψ contribute to pmf, but only ∆pH down-regulates electron transport. To optimize the operation of the accelerator (ATP synthesis) and the brake on electron transport, it is necessary to precisely regulate the ratio of the two pmf components as well as the total size of pmf (Cruz et al., 2001; Kramer et al., 2003). Several channels and antiporters localized to the thylakoid membrane regulate the partitioning of the pmf components (Spetea et al., 2017). K⁺ Efflux Antiporter3 (KEA3) is thought to be an H⁺/K⁺ antiporter localized to the thylakoid membrane (Armbruster et al., 2014; Kunz et al., 2014), although its antiport activity has not been experimentally demonstrated (Tsujii et al., 2019). Based on its structure, topology, and the mutant phenotypes, KEA3 most likely moves H⁺ from the thylakoid lumen while taking up K⁺ as a counter ion. Consequently, KEA3 transforms ∆pH to ∆ψ and is necessary to rapidly relax the down-regulation of electron transport by raising the luminal pH (i.e. by alkalinizing the lumen). The C-terminal domain of KEA3, KTN (K⁺ transport/nucleotide binding), is exposed to the stroma (Wang et al., 2017) and is thought to regulate its activity by monitoring ATP or NADPH levels (Schlosser et al., 1993; Roosild et al., 2002). However, information on the regulation of KEA3 is limited. Armbruster et al. (2014) demonstrated that KEA3 contributes to efficient photosynthesis under fluctuating light conditions. The disturbed proton gradient regulation is a dominant mutant allele of KEA3, and its mutant phenotype is evident after a long period of dark adaptation (overnight; Wang et al., 2017). KEA3 is likely important during the induction of photosynthesis as well as under fluctuating light intensities. The similarity between the two conditions suggests that KEA3 is required for readjusting the ∆pH-dependent regulation immediately after any drastic change in light conditions.In this study, we characterized double mutants defective in the CET pathways and KEA3 to understand whether and how the synergy between CET and KEA3 in the regulatory network of photosynthesis affects this process. We focused on the contribution of NDH-dependent CET during the induction of photosynthesis after overnight dark adaptation in the kea3-1 mutant context. Based on our results, we propose a novel physiological function of the NDH complex: that of allowing flexibility of the regulatory network during the induction of photosynthesis.     

A call for diversity,equity, and inclusion: Highlights from the Consortium of Universities for Global Health 2021 conference

Beryne Odeny reports from the CUGH 2021 virtual conference.

The first virtual Consortium of Universities for Global Health (CUGH) 2021 conference was held in March, 2021 [1]. Two weeks of satellite symposia culminated in this highly prestigious conference, which drew an eclectic group of renowned speakers, global health leaders, program implementers, researchers, and students from across the globe. There were more than 5000 delegates from diverse disciplines including public health, politics, education, medicine, planetary health, and finance. Top of the agenda was addressing critical gaps in global health and development against the backdrop of the COVID-19 pandemic.CUGH is an organization of over 170 academic institutions and organizations throughout the world, engaged in addressing global health challenges [1]. The 2021 conference was meticulously and creatively planned as was evidenced by the dynamic virtual platform, which hosted several global leader interviews, general sessions, 40 concurrent sessions, 7 plenary sessions, over 700 poster programs, and the Pulitzer Center Film festivals–yes, movies were on the menu [2]. Best of all, the platform held up, with minimal technical difficulties. The conference agenda had curated sessions carefully customized to varying attendee interests and expertise. Participants could seamlessly and discreetly shuttle between sessions.The inaugural interviews, with Dr. Anthony Fauci of the United States and Dr. Hugo Lopez-Gatell of Mexico, set the tone with emphasis on a much-needed global response to the ongoing pandemic. “2020 was a watershed moment in Global Health,” said Dr. Fauci. The COVID-19 pandemic indiscriminately unveiled the fragility of health systems in high income countries (HIC) and low- and middle-income countries (LMICs) alike. He unpacked the origins, evolution, and contention around current public health mandates such as mask wearing. He discussed vaccines–exploring vaccine manufacturing in LMICs, open patents, implications of emerging COVID-19 variants, and advice on curbing the prevailing vaccine infodemic (i.e., pandemic of misinformation) [2–4]. Dr. Lopez-Gatell described the pandemic as a “massive social event” fueled by deficits in health systems, politics, and governance, and by the growing tide of non-communicable diseases (NCDs) [5]. In a brief video recording, Dr. Tedros Adhanom Ghebreyesus, WHO’s Director-General, implored global partners to sign the COVID-19 Declaration on vaccine equity which he termed “the defining challenge of 2021” [6].The post-pandemic forecast for global health was dire. COVID-19 has disrupted decades of progress toward attainment of Universal Health Care (UHC) and it will be doubly difficult to restore, by 2035, health indicators to their levels prior to the pandemic [7–9]. A modelling study by Dr. Wenhui Mao of Duke University showed that, even in the most optimistic scenario, it may not be possible to achieve UHC in the next decade without breakthrough technologies and exceptional political commitment. Among four critical indicators of TB mortality rate, HIV mortality rate, under 5 mortality ratio, and maternal mortality ratio, Dr. Mao found that only the HIV indicator had potential for recovery by 2035.The metaphorical elephant in the room, and now its opposite, “the elephant not in the room”, respectively encapsulate two themes: neocolonialism and equity, especially for marginalized groups. Neocolonialism–a progeny of colonialism–resulting from sustained global North-South power imbalances, manifests in low prioritization of the most pressing challenges and diseases in LMICs. Equity was a poignant theme across the CUGH sessions and satellite symposia. Sessions were dedicated to exploring the hegemonic structures and institutional systems that underpin adverse health system performance and outcomes. A sampling of wide-ranging topics on global challenges exacerbated by neocolonialism and inequities comprised: a) elevating the visibility and power of researchers in LMICs, including fragile and conflict affected settings, through equitable access to funding, research autonomy and leadership, access to scholarly publishing, and senior authorship of research articles [10]; b) training next-generation global health professionals and building capacity for resource-challenged settings to address NCDs, including cancer care [5]; c) the Latin American and Caribbean health crises drawn by social gradients and inequities; d) navigating conflicting interests between public health and the corporate food industry; e) the dearth and role of women leaders in global health and in the COVID-19 response; f) the disproportionate incidence of HIV in adolescent girls and young women in sub-Saharan Africa (SSA) [11]; g) the disparate burden of neonatal mortality in LMICs and marginalized communities within HIC; and h) leveraging the power of film to evoke emotion and induce a consolidated response to global challenges. In addition, various facets of the human ecosystem were unpacked including climate change, biodiversity preservation, political climate, and the global kleptocracy, with attention to their implications for the health of the most marginalized populations.Despite the highlighted issues, there is, potentially, a panacea for these inequities and challenges. One speaker, Dr. Lisa Adams of Dartmouth College, proposed a paradigm shift that summarized a wide range of deliberations–“moving global health out of the realm of charity into global citizenship, security, human rights, equal partnership, and interdisciplinary collaboration between LMICs and HICs.” Moving forward, more deliberate effort should be given to some elements. First, rethinking governance and funding at a global level while promoting the autonomy of LMICs and conflict-affected settings to drive their health agenda–independent from HIC interests. Bringing the elephant into the room by making equal space for LMICs to set the agenda at global tables of discussion around funding, research, and development will be pivotal to dismantling neocolonialism. Furthermore, funders and partners should work with in-country systems in LMICs as opposed to bypassing them. This is essential to building resilient health systems unified at national levels to allow for cross-discipline collaborations and swift responses to health threats. Rwanda is a laudable example, having swiftly remodeled its existing health systems including routine electronic information systems for nationwide COVID-19 surveillance, testing, contact tracing, and vaccination. Second, investing time to build trusting relationships between researchers or implementers and policy makers by upholding a participatory approach to research and implementation of evidence-based practices. This is essential globally, to support development of global public goods such as vaccines, free from market dynamics and aimed at universal and equitable access. Third, introduce policies that engage economies to produce with less fragmentation of nature and reduced pollution. These include protected area management, financing of nature-positive projects, and conservationist work for natural capital preservation. Global and public health practitioners need to educate and empower citizens to choose healthy and ecologically sustainable consumption practices. Fourth, promoting development of novel technologies for preventing HIV infection, such as broadly neutralizing antibodies, could overturn the unequal burden of HIV in adolescents and young women in SSA. Finally, HIC have a lot they can learn from LMICs. COVID-19 evidently demonstrated that a country’s Global Health Security Index ranking is not necessarily commensurate to its degree of success in handling pandemics, among other public health threats [8,12,13].Throughout the conference, it was apparent that equity and collectivity in global health are necessary–not optional. Dr. Elvin Geng of Washington University, St. Louis remarked that the path to equity should be measurable with routinely incorporated metrics that track interventions to redress inequity and foster accountability. To achieve this, the tools of implementation science can be employed at both regional and global levels [14]. Overall, the remarkable interlacing of diverse disciplinary sessions at CUGH 2021 not only brought to light pressing world problems but equipped participants with a wellspring of potential remedies and collaborative opportunities. The panelists and speakers effectively portrayed the layered and multidimensional nature of global challenges underscoring the need for similarly multifaceted solutions. CUGH 2021 sparked thought-provoking discourse around global health strategies and re-invigorated the collective passion of global health experts, novices, and everyone in between, to build forward better.     

Training matters! Narrative from a Black scientist

As STEM (Science, Technology, Engineering, and Math) professionals, we are tasked with increasing our understanding of the universe and generating discoveries that advance our society. An essential aspect is the training of the next generation of scientists, including concerted efforts to increase diversity within the scientific field. Despite these efforts, there remains disproportional underrepresentation of Black scientists in STEM. Further, efforts to recruit and hire Black faculty and researchers have been largely unsuccessful, in part due to a lack of minority candidates. Several factors contribute to this including access to opportunities, negative training experiences, lack of effective mentoring, and other more lucrative career options. This is a narrative of a Black male scientist to illustrate some of the issues in retaining Black students in STEM and to highlight the impact of toxic training environments that exists at many institutions. To increase Black participation in STEM careers, we must first acknowledge, then address, the problems that exist within our STEM training environments in hopes to inspire and retain Black students at every level of training.

I write this today as the curtain of systemic racism and oppression has lifted on our nation. I write this today knowing that difficult conversations about race are happening all across America. As a result of tremendous sacrifices and lives lost, there have been demonstrations and rallies internationally demanding change, prompting governments, organizations, and companies to issue statements claiming that Black Lives Matter (Asmelash, 2020). While the rage has sparked the demand for equity in our society, what does this mean for science?My heart is heavy with these discussions as I have reflected on my own journey in science and revisit the toxic environment that often makes up our science culture. The journey has been long and brutal. It has taken me from first realizing that I wanted to become a scientist, to having this dream deferred by racism, to adopting a persona of persistence and resilience, and finally becoming a professor and cell biologist. This trek through science is one that is not traversed by many Black people (Graf et al., 2018).When confronted by the pervasiveness of racism in science, I remember surviving the assault by learning about the resilience story of Carl Brashear (Robbins, 2000). In 1970, Master Chief Petty Officer Brashear became the first African American master diver in the Navy, and he showed unwavering strength and persistence in the face of racism. Brashear faced an onslaught of racism during his training that endangered his life countless times, but he persisted and eventually won the admiration of his fellow divers. Upon reflection, his story has many signs of an abusive hazing relationship. However, at the time, I thought emulating his behaviors of persistence was the answer to success in science. I thought, “All you have to do is not give up.” I focused on what I thought I could control and kept the Japanese proverb, “Fall down seven, stand up eight” above my bench. I worked long hours, made many mistakes, but always got right back up to the bench to try again. I never saw myself as the brightest or smartest, but I would tell myself “I will be the one who does not give up.” When I recall these stories and talk to students about my journey, I would always say I wanted to be like the cockroach. Because, as is commonly known, you can never get rid of the cockroach. What I never realized with this persistence or “grit” mentality was that it never addressed the problems of systemic racism within the culture of science (Das, 2020). This message of persistence is akin to blaming the victim and not dealing with the root problems in science, including the lack of mentoring, implicit bias, and hostile teaching and training environments (Barber et al., 2020; Team, 2020).In her book, We Want to Do More Than Survive, Bettina Love talks about the idea of teaching persistence or “grit” to African American students as the educational equivalent to the Hunger Games, a fictional competition where participants battle to the death until there is only one victor (Love, 2019). Instead of addressing institutional barriers to success for African Americans in science (i.e., dismantling the Hunger Games arena), we prepare them to survive in a toxic environment. We tell African American students at a young age that the system is structured against them and that they have to be twice as good and work twice as hard as white students (Thomas and Wetlaufer, 1997; Cavounidis and Lang, 2015; Danielle, 2015). We heap a tremendous amount of pressure and responsibility on their shoulders without ever addressing the question, why is it like this? We are in effect training them for the Hunger Games. As they enter college as science majors, they are pitted against each other, and the few victors move into science careers.This Hunger Games analogy (Love, 2019) is reflective of my thinking early on in my science career. As a freshman marine biology major, I imagined myself, like Brashear, a soldier during basic training. I was a member of the “people of color” (POC) squad that was given the least amount of resources and the most dangerous duties. As part of the POC squad, we moved forward through our college years. I saw many fellow soldiers drop from science, and there were only a handful of us left when I reached my junior year (Koenig, 2009).Recently, Michael Eisen, Editor-in-Chief of eLife, authored an opinion article entitled “Racism in Science: We need to act now” (Eisen, 2020). In this article, he reflected on the current racial climate in science and examined his role as both a principle investigator (PI) of a research laboratory and an editor of a prestigious journal. Of note, he highlighted the dire lack of African Americans he had worked with over his career, including the number of researchers he trained in his laboratory, senior editors, and even reviewers for the articles sent for publication to eLife. I appreciated his honesty in shedding light on the issue that so many people whisper about in department hallways or during coffee breaks at national conferences. Based on my journey, I truly understand this lack of diversity, as so few of us are victors in the scientific Hunger Games.As we struggle as a nation with the role of policing within our society, I find similarities between aggressive policing in the Black community and training of Black and Brown students (North, 2020). There are strong implicit biases that we hold within our training environment, and Black students usually find themselves very quickly judged (or prejudged) for a perceived lack of commitment, motivation, or focus (Park et al., 2020). They are also stereotyped as lacking in quantitative abilities (especially the ability to do math) (McClain, 2014). Taken together, these biased judgements result in a lack of trust regarding their data (Steele, 1997). In other words, research supervisors may implicitly expect Black students to be untrustworthy. This is extremely problematic because educational research shows that one of the greatest determinants of students’ success is their teachers’ expectations (Boser et al., 2014). Consequently, it is predictable that if research supervisors expect Black students to be untrustworthy, they will fail.As PIs, we must trust our research students because they are extensions of ourselves in the laboratory. Due to our inability to spend significant amounts of time at the bench, we must trust our students to figure it out and get the work done. Inevitably, experimental approaches will fail; however, based on my experiences in science, Black students are often not given the benefit of the doubt. Instead, I have seen mis/distrust of their commitment, values, and abilities that creates the narrative that they are not motivated, do not care about science, and/or are unable to get the work done, resulting in a broken trainer/trainee relationship. I have witnessed too many Black students fall victim to a “one strike” policy. This was true of me in my early training in marine biology, where I was asked to leave after only 6 months of working in a laboratory. The professor suggested that I had a lack of commitment to my project and was told by other lab members that they collected “my” data, thus providing justification to ask me not to continue. However, what the professor did not know (or care to ask about) was that the other lab members deemed me as someone who did not belong. Consequently, without my knowledge, they collected data on my project and sent it to the PI, thereby working to reinforce the narrative of my lack of commitment. This experience significantly hindered my access to research opportunities and blacklisted me from any other marine biology labs at my university because I was labeled as uncommitted to science. This ended my career in marine biology. I lost the Hunger Games.As a graduate student, I found another opportunity in a cell biology laboratory, and I tried to apply lessons learned from my earlier participation in the Games. I overcommitted to lab work, blocking out any activities related to my culture or personal life. Instead, I dedicated myself completely to the lab. Working 12-h days, I found that my research was progressing, but I was burning out and losing any desire toward a research career. In particular, my burnout was connected to the perception that any interest in my culture and community would not be allowed or accepted or would signal a lack of adequate commitment to science. In effect, I was learning that being a scientist meant that I could not be Black. This, coupled with the constant microaggressions that I faced from professors in classes, among my graduate cohort, and my laboratory colleagues, broadcasted the message that I was an intruder in science. Luckily, I received good mentoring and advice on how to succeed in my graduate program, learning that it was not a sprint, but a marathon. I learned how to balance my personal and professional life, and I always kept them separate. Additionally, the mental image of the resilient cockroach helped me repeatedly during my graduate training, from failing my qualifying exams and failed experiments at the bench to rejections of papers and fellowship applications. While all scientists know that being a scientist means accepting significant amounts of failure, I could not help but feel that the failures I experienced were more frequent, more recognized by others, and even expected by some. This culture of expected failure for people of color (i.e., presumed incompetence), combined with implicit biases and microaggressions, can establish significant barriers for entering and staying in STEM training environments (Smith et al., 2007).To overcome barriers to success in STEM, I worked hard to become a professor in cell biology. I believed that as a professor, I could make a difference, change the environment, and contribute to the change that is so desperately needed. However, I have discovered that the current science culture is just as toxic as when I was a student. Yes, there are programs targeting the inclusion of historically underrepresented groups. There are also a growing number of institutions that are adopting inclusive teaching strategies. Further, we are seeing hiring committees require diversity statements from their applicants as well as receiving implicit bias trainings (Wood, 2019). However, there remains nearly a complete lack of Black faculty members at universities and colleges (Jayakumar et al., 2009; Garrison, 2013; Li and Koedel, 2017). This is, in part, because we have not changed the systemic racism that exists within our training environments. In fact, this racism comes from our noninclusive faculty bodies (Hardy, 2020). In essence, we have nearly a complete absence of Black faculty in STEM because so few Black trainees survive the Hunger Games. More troubling, if they survive, they may be found otherwise unacceptable.Changing the system starts with the belief that Black students can be scientists, followed by acting to proactively encourage and support Black students in STEM. As Eisen states, “This is a solvable problem, we have chosen not to solve it” (Eisen, 2020). Recruiting Black students and scientists at every level is a good start, but without changing the scientific environment to be more welcoming and affirming, those recruited to science will continue to be traumatized. In other words, while increasing access to science is required, it is not sufficient. The dominant majority in science also needs to identify and address their own biases to create antiracist environments. This will only happen when scientists from all groups recognize our convergent interests to advance our universal missions, which is to increase our understanding of the world around us and to solve research questions that will benefit our communities. This is best achieved by a diverse and inclusive scientific workforce for greater knowledge, discovery, and innovation.     

The impact of COVID-19 on pregnancy outcomes: a systematic review and meta-analysis

Background:The impact of coronavirus disease 2019 (COVID-19) on maternal and newborn health is unclear. We aimed to evaluate the association between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during pregnancy and adverse pregnancy outcomes.METHODS:We conducted a systematic review and meta-analysis of observational studies with comparison data on SARS-CoV-2 infection and severity of COVID-19 during pregnancy. We searched for eligible studies in MEDLINE, Embase, ClinicalTrials.gov, medRxiv and Cochrane databases up to Jan. 29, 2021, using Medical Subject Headings terms and keywords for “severe acute respiratory syndrome coronavirus 2 OR SARS-CoV-2 OR coronavirus disease 2019 OR COVID-19” AND “pregnancy.” We evaluated the methodologic quality of all included studies using the Newcastle–Ottawa Scale. Our primary outcomes were preeclampsia and preterm birth. Secondary outcomes included stillbirth, gestational diabetes and other pregnancy outcomes. We calculated summary odds ratios (ORs) or weighted mean differences with 95% confidence intervals (CI) using random-effects meta-analysis.RESULTS:We included 42 studies involving 438 548 people who were pregnant. Compared with no SARS-CoV-2 infection in pregnancy, COVID-19 was associated with preeclampsia (OR 1.33, 95% CI 1.03 to 1.73), preterm birth (OR 1.82, 95% CI 1.38 to 2.39) and stillbirth (OR 2.11, 95% CI 1.14 to 3.90). Compared with mild COVID-19, severe COVID-19 was strongly associated with preeclampsia (OR 4.16, 95% CI 1.55 to 11.15), preterm birth (OR 4.29, 95% CI 2.41 to 7.63), gestational diabetes (OR 1.99, 95% CI 1.09 to 3.64) and low birth weight (OR 1.89, 95% CI 1.14 to 3.12).INTERPRETATION:COVID-19 may be associated with increased risks of preeclampsia, preterm birth and other adverse pregnancy outcomes.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was declared a global pandemic in March 2020.¹ Pregnant people and infants may be particularly susceptible to COVID-19 because the physiologic changes of pregnancy involve cardiorespiratory and immune systems, which may result in an altered response to SARS-CoV-2 infection in pregnancy. ² Fetuses may be exposed to SARS-CoV-2 during critical periods of fetal development.³ The nature of the association between COVID-19 and pregnancy outcomes remains unclear, and meta-analyses involving patients with COVID-19 who are pregnant are limited. Previous reviews have focused mostly on prevalence estimates from case reports or case series that are difficult to interpret and potentially biased.^4,5 A 2020 systematic review suggested that people who are pregnant did not have an increased risk of SARS-CoV-2 infection or symptomatic COVID-19, but they were at risk of severe COVID-19 compared with those who were not pregnant.⁵ However, this review included suspected COVID-19 cases in addition to confirmed cases.⁵ Although some recent observational studies have suggested that people with confirmed asymptomatic and symptomatic COVID-19,^6–15 as well as mild and severe infections,^{6,8,9,15–22} may be at risk of adverse pregnancy outcomes, we are unaware of any systematic reviews that have comprehensively evaluated these data.We performed a systematic review and meta-analysis of maternal, fetal and neonatal outcomes among pregnant patients with COVID-19. We aimed to determine the association between SARS-CoV-2 infection and adverse pregnancy outcomes, including preeclampsia, preterm birth and stillbirth.     

Ensuring the quality and specificity of preregistrations

Researchers face many, often seemingly arbitrary, choices in formulating hypotheses, designing protocols, collecting data, analyzing data, and reporting results. Opportunistic use of “researcher degrees of freedom” aimed at obtaining statistical significance increases the likelihood of obtaining and publishing false-positive results and overestimated effect sizes. Preregistration is a mechanism for reducing such degrees of freedom by specifying designs and analysis plans before observing the research outcomes. The effectiveness of preregistration may depend, in part, on whether the process facilitates sufficiently specific articulation of such plans. In this preregistered study, we compared 2 formats of preregistration available on the OSF: Standard Pre-Data Collection Registration and Prereg Challenge Registration (now called “OSF Preregistration,” http://osf.io/prereg/). The Prereg Challenge format was a “structured” workflow with detailed instructions and an independent review to confirm completeness; the “Standard” format was “unstructured” with minimal direct guidance to give researchers flexibility for what to prespecify. Results of comparing random samples of 53 preregistrations from each format indicate that the “structured” format restricted the opportunistic use of researcher degrees of freedom better (Cliff’s Delta = 0.49) than the “unstructured” format, but neither eliminated all researcher degrees of freedom. We also observed very low concordance among coders about the number of hypotheses (14%), indicating that they are often not clearly stated. We conclude that effective preregistration is challenging, and registration formats that provide effective guidance may improve the quality of research.

Researchers face many, often seemingly arbitrary choices in formulating hypotheses, designing protocols, collecting data, analyzing data, and reporting results. A study of two formats of preregistration available on the OSF reveals that the opportunistic use of researcher degrees of freedom aimed at obtaining statistical significance is restricted by using more extensive preregistration guidelines; however, these guidelines should be further improved.     

A Major Fraction of Glycosphingolipids in Model and Cellular Cholesterol-containing Membranes Is Undetectable by Their Binding Proteins

Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains and provide receptors for protein ligands. Lipid-based “aglycone” interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb₃), was analyzed in simple GSL/cholesterol, detergent-resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb_3/cholesterol-containing populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb anti-Gb₃, HIVgp120 or Bandeiraea simplicifolia lectin. Only IgM anti-Gb₃, more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryo-immuno-EM confirmed these results. This appears to be a general GSL-cholesterol property, because similar receptor-inactive vesicles were separated for other GSL-protein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer and GlcCer) rendered VT1-unreactive Gb₃/cholesterol vesicles, VT1-reactive. We found GalCer and GlcCer bind Gb₃, suggesting GSL-GSL interaction can counter cholesterol masking of Gb₃. The similar separation of Vero cell membrane-derived vesicles into minor “binding,” and major “non-binding” fractions when probed with VT1, CTx, or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol- and actin-dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of “available” and “cholesterol-masked” plasma membrane Gb₃ pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions.