A CRISPR response to pandemics?: Exploring the ethics of genetically engineering the human immune system

Ethical challenges should be addressed before gene editing is made available to improve the immune response against emerging viruses. Subject Categories: S&S: Economics & Business, Genetics, Gene Therapy & Genetic Disease, Immunology

In 1881, Louis Pasteur proved the “germ theory of disease”, namely that microorganisms are responsible for causing a range of diseases. Following Pasteur’s and Robert Koch’s groundbreaking work on pathogens, further research during the 20^th century elucidated how the immune system fends off disease‐causing microorganisms from a molecular perspective.The COVID‐19 pandemic has again focused scientific and public attention on immunology not the least owing to the race of employing vaccines to halt the spread of the virus. Although most countries have now started vaccination programs to immunize a large part of the world''s population, the process will take time, vaccines may not be available to everyone, and a number of unresolved issues remain including the potential contagiousness of vaccinated individuals and the duration of protection (Polack et al, 2020).It would therefore be extremely helpful from a public health perspective—and indeed lifesaving for those with elevated risk of developing severe course of the disease—if we could boost the human immune system by other means to better fight off SARS‐CoV‐2 and possibly other viruses. Recent studies showing that some individuals may be less susceptible to contract severe COVID‐19 depending on their genetic status support such visions (COVID‐19 Host Genetics Initiative, 2020). This could eventually inspire research projects on gene therapy with the aim of generally enhancing immunity against viral infections.

It would therefore be extremely helpful from a public health perspective […] if we could boost the human immune system by other means to better fight off SARS‐CoV‐2 …

The idea of genetically enhancing the human immune response is not new and spread from academic circles to policymakers and the general public even before the pandemic, when He Jiankui announced in November 2018 the birth of genetically edited twins who, he claimed, were resistant to HIV. The public outcry was massive, not only because He violated standards of methodological rigor and research ethics, but also because of fundamental doubts about the wisdom and legitimacy of human germline manipulation (Schleidgen et al, 2020).Somatic gene therapy has been met with a less categorical rejection, but it has also been confronted with skepticism when major setbacks or untoward events occurred, such as the death of Jesse Gelsinger during an early clinical trial for gene therapy in 1999. Nonetheless, given the drastic impact the current pandemic has on so many lives, there may be a motivation to put concerns aside. In fact, even if we managed to get rid of COVID‐19 owing to vaccines—or at least to keep its infectiousness and mortality low—another virus will appear sooner or later; an improved resistance to viral pathogens—including coronaviruses—would be an important asset.Interventions to boost the immune system could in fact make use of either germline gene editing, as has been the case of the Chinese twins, or through somatic gene editing. The first requires time and only the next generation would potentially benefit while the latter could be immediately applied and theoretically used to deal with the ongoing COVID‐19 pandemic.

Interventions to boost the immune system could in fact make use of either germline gene editing, as has been the case of the Chinese twins, or through somatic gene editing.

     

USP26: a genetic risk factor for sperm X‐Y aneuploidy

Segregation of the largely non‐homologous X and Y sex chromosomes during male meiosis is not a trivial task, because their pairing, synapsis, and crossover formation are restricted to a tiny region of homology, the pseudoautosomal region. In humans, meiotic X‐Y missegregation can lead to 47, XXY offspring, also known as Klinefelter syndrome, but to what extent genetic factors predispose to paternal sex chromosome aneuploidy has remained elusive. In this issue, Liu et al (2021) provide evidence that deleterious mutations in the USP26 gene constitute one such factor.Subject Categories: Cell Cycle, Development & Differentiation, Molecular Biology of Disease

Analyses of Klinefelter syndrome patients and Usp26‐deficient mice have revealed a genetic influence on age‐dependent sex chromosome missegregation during male meiosis.

Multilayered mechanisms have evolved to ensure successful X‐Y recombination, as a prerequisite for subsequent normal chromosome segregation. These include a distinct chromatin structure as well as specialized proteins on the pseudoautosomal region (Kauppi et al, 2011; Acquaviva et al, 2020). Even so, X‐Y recombination fails fairly often, especially in the face of even modest meiotic perturbations. It is perhaps not surprising then that X‐Y aneuploidy—but not autosomal aneuploidy—in sperm increases with age (Lowe et al, 2001; Arnedo et al, 2006), as does the risk of fathering sons with Klinefelter syndrome (De Souza & Morris, 2010).Klinefelter syndrome is one of the most common aneuploidies in liveborn individuals (Thomas & Hassold, 2003). While most human trisomies result from errors in maternal chromosome segregation, this is not the case for Klinefelter syndrome, where the extra X chromosome is equally likely to be of maternal or paternal origin (Thomas & Hassold, 2003; Arnedo et al, 2006). Little is known about genetic factors in humans that predispose to paternal XY aneuploidy, i.e., that increase the risk of fathering Klinefelter syndrome offspring. The general notion has been that paternally derived Klinefelter syndrome arises stochastically. However, fathers of Klinefelter syndrome patients have elevated rates of XY aneuploid sperm (Lowe et al, 2001; Arnedo et al, 2006), implying a persistent defect in spermatogenesis in these individuals rather than a one‐off meiotic error.To identify possible genetic factors contributing to Klinefelter syndrome risk, Liu et al (2021) performed whole‐exome sequencing in a discovery cohort of > 100 Klinefelter syndrome patients, followed by targeted sequencing in a much larger cohort of patients and controls, as well as Klinefelter syndrome family trios. The authors homed in on a mutational cluster (“mutated haplotype”) in ubiquitin‐specific protease 26 (USP26), a testis‐expressed gene located on the X chromosome. Effects of this gene’s loss of function (Usp26‐deficient mice) on spermatogenesis have recently been independently reported by several laboratories and ranged from no detectable fertility phenotype (Felipe‐Medina et al, 2019) to subfertility/sterility associated with both meiotic and spermiogenic defects (Sakai et al, 2019; Tian et al, 2019). With their Klinefelter syndrome cohort findings, Liu et al (2021) also turned to Usp26 null mice, paying particular attention to X‐Y chromosome behavior and—unlike earlier mouse studies—including older mice in their analyses. They found that Usp26‐deficient animals often failed to achieve stable pairing and synapsis of X‐Y chromosomes in spermatocytes, produced XY aneuploid sperm at an abnormally high frequency, and sometimes also sired XXY offspring. Importantly, these phenotypes only occurred at an advanced age: XY aneuploidy was seen in six‐month‐old, but not two‐month‐old Usp26‐deficient males. Moreover, levels of spindle assembly checkpoint (SAC) proteins also reduced in six‐month‐old males. Thus, in older Usp26 null mice, the combination of less efficient X‐Y pairing and less stringent SAC‐mediated surveillance of faithful chromosome segregation allows for sperm aneuploidy, providing another example of SAC leakiness in males (see Lane & Kauppi, 2019 for discussion).Liu et al’s analyses shed some light on what molecular mechanisms may be responsible for the reduced efficiency of X‐Y pairing and synapsis in Usp26‐deficient spermatocytes. USP26 codes for a deubiquitinating enzyme that has several substrates in the testis. Because USP26 prevents degradation of these substrates, their levels should be downregulated in Usp26 null testes. Liu et al (2021) show that USP26 interacts with TEX11, a protein required for stable pairing and normal segregation of the X and Y chromosomes in mouse meiosis (Adelman & Petrini, 2008). USP26 can de‐ubiquitinate TEX11 in vitro, and in Usp26 null testes, TEX11 was almost undetectable. It is worth noting that USP26 has several other known substrates, including the androgen receptor (AR), and therefore, USP26 disruption likely contributes to compromised spermatogenesis via multiple mechanisms. For example, AR signaling‐dependent hormone levels are misregulated in Usp26 null mice (Tian et al, 2019).The sex chromosome phenotypes observed in Usp26 null mice predict that men with USP26 mutations may be fertile, but producing XY aneuploid sperm at an abnormally high frequency, and that spermatogenic defects should increase with age (Fig 1). These predictions were testable, because the mutated USP26 haplotype, present in 13% of Klinefelter syndrome patients, was reasonably common also in fertile men (7–10%). Indeed, sperm XY aneuploidy was substantially higher in fertile men with the mutated USP26 haplotype than in those without USP26 mutations. Some mutation carriers produced > 4% aneuploid sperm. Moreover, age‐dependent oligospermia was also found associated with the mutated USP26 haplotype.Open in a separate windowFigure 1Mutated USP26 as genetic risk factor for age‐dependent X‐Y defects in spermatogenesisMouse genetics demonstrate that deleterious USP26 mutations lead to less‐efficient X‐Y pairing and recombination with advancing age. Concomitant decrease of spindle assembly checkpoint (SAC) protein levels leads to less‐efficient elimination of metaphase I spermatocytes that contain misaligned X and Y chromosomes. This allows for the formation of XY aneuploid sperm in older individuals and subsequently increased age‐dependent risk for fathering Klinefelter syndrome (KS) offspring, two correlates also observed in human USP26 mutation carriers. At the same time, oligospermia/subfertility also increases with advanced age in both Usp26‐deficient mice and USP26 mutation‐carrying men, tempering Klinefelter syndrome offspring risk but also decreasing fecundity.As indicated by its prevalence in the normal control population, the USP26 mutated haplotype is not selected against in the human population. With > 95% of sperm in USP26 mutation carriers having normal haploid chromosomal composition, the risk of producing (infertile) Klinefelter syndrome offspring remains modest, likely explaining why USP26 mutant alleles are not eliminated. Given that full Usp26 disruption barely affects fertility of male mice during their prime reproductive age (Felipe‐Medina et al, 2019; Tian et al, 2019; Liu et al, 2021), there is little reason to assume strong negative selection against USP26 variants in humans. USP26 as the first‐ever genetic risk factor predisposing to sperm X‐Y aneuploidy and paternal origin Klinefelter syndrome offspring in humans, as uncovered by Liu et al, may be just one of many. 90% of Liu et al’s Klinefelter syndrome cases were not associated with USP26 mutations. But even in the age of genomics, discovery of Klinefelter syndrome risk factors is not straightforward, since most sperm of risk mutation carriers will not be XY aneuploid and thus not give rise to Klinefelter syndrome offspring. In addition, as Usp26 null mice demonstrate, both genetic and non‐genetic modifiers impact on penetrance of the XY aneuploidy phenotype: Spermatogenesis in the absence of Usp26 was impaired in the DBA/2 but not the C57BL/6 mouse strain background (Sakai et al, 2019), and in older mice, there was substantial inter‐individual variation in the severity of the X‐Y defect (Liu et al, 2021). In human cohorts, genetic and non‐genetic modifiers are expected to blur the picture even more.Future identification of sex chromosome aneuploidy risk factors has human health implications beyond Klinefelter syndrome. Firstly, XXY incidence is not only relevant for Klinefelter syndrome livebirths—it also contributes to stillbirths and spontaneous abortions, at a 4‐fold higher rate than to livebirths (Thomas & Hassold, 2003). Secondly, persistent meiotic X‐Y defects can, over time, result in oligospermia and even infertility. Since the mean age of first‐time fathers is steadily rising and currently well over 30 years in many Western countries, age‐dependent spermatogenic defects will be of ever‐increasing clinical relevance.     

Reply to Bronstein and Vinogradov

The response by the author. Subject Categories: S&S: Economics & Business, S&S: Ethics

I thank Michael Bronstein and Sophia Vinogradov for their interest and comments. I would like to respond to a few of their points.First, I agree with the authors that empirical studies should be conducted to validate any approaches to prevent the spread of misinformation before their implementation. Nonetheless, I think that the ideas I have proposed may be worth further discussion and inspire empirical studies to test their effectiveness.Second, the authors warn that informing about the imperfections of scientific research may undermine trust in science and scientists, which could result in higher vulnerability to online health misinformation (Roozenbeek et al, 2020; Bronstein & Vinogradov, 2021). I believe that transparency about limitations and problems in research does not necessarily have to diminish trust in science and scientists. On the contrary, as Veit et al put it, “such honesty… is a prerequisite for maintaining a trusting relationship between medical institutions (and practitioners) and the public” (Veit et al, 2021). Importantly, to give an honest picture of scientific research, information about its limitations should be put in adequate context. In particular, the public also should be aware that “good science” is being done by many researchers; we do have solid evidence of effectiveness of many medical interventions; and efforts are being taken to address the problems related to quality of research.Third, Bronstein and Vinogradov suggest that false and dangerous information should be censored. I agree with the authors that “[c]ensorship can prevent individuals from being exposed to false and potentially dangerous ideas” (Bronstein & Vinogradov, 2021). I also recognize that some information is false beyond any doubt and its spread may be harmful. What I am concerned about are, among others, the challenges related to defining what is dangerous and false information and limiting censorship only to this kind of information. For example, on what sources should decisions to censor be based and who should make such decisions? Anyone, whether an individual or an organization, with a responsibility to censor information will likely not only be prone to mistakes, but also to abuses of power to foster their interests. Do the benefits we want to achieve by censorship outweigh the potential risks?Fourth, we need rigorous empirical studies examining the actual impact of medical misinformation. What exactly are the harms we try to protect against and what is their scale? This information is necessary to choose proportionte and effective measures to reduce the harms. Bronstein and Vinogradov give an example of a harm which may be caused by misinformation—an increase in methanol poisoning in Iran. Yet, as noticed by the authors, misinformation is not the sole factor in this case; there are also cultural and other contexts (Arasteh et al, 2020; Bronstein & Vinogradov, 2021). Importantly, the methods of studies exploring the effects of misinformation should be carefully elaborated, especially when study participants are asked to self‐report. A recent study suggests that some claims about the prevalence of dangerous behaviors, such as drinking bleach, which may have been caused by misinformation are largely exaggerated due to the presence of problematic respondents in surveys (preprint: Litman et al, 2021).Last but not least, I would like to call attention to the importance of how veracity of information is determined in empirical studies on misinformation. For example, in a study of Roozenbeek et al, cited by Bronstein and Vinogradov, the World Health Organization (WHO) was used as reliable source of information, which raises questions. For instance, Roozenbeek et al (2020) used a statement “the coronavirus was bioengineered in a military lab in Wuhan” as an example of false information, relying on the judgment of the WHO found on its “mythbusters” website (Roozenbeek et al, 2020). Yet, is there a solid evidence to claim that this statement is false? At present, at least some scientists declare that we cannot rule out that the virus was genetically manipulated in a laboratory (Relman, 2020; Segreto & Deigin, 2020). Interestingly, the WHO also no longer excludes such a possibility and has launched an investigation on this issue (https://www.who.int/health‐topics/coronavirus/origins‐of‐the‐virus, https://www.who.int/emergencies/diseases/novel‐coronavirus‐2019/media‐resources/science‐in‐5/episode‐21‐‐‐covid‐19‐‐‐origins‐of‐the‐sars‐cov‐2‐virus); the information about the laboratory origin of the virus being false is no longer present on the WHO “mythbusters” website (https://www.who.int/emergencies/diseases/novel‐coronavirus‐2019/advice‐for‐public/myth‐busters). Against this backdrop, some results of the study by Roozenbeek et al (2020) seem misleading. In particular, the perception of the reliability of the statement about bioengineered virus by study participants in Roozenbeek et al (2020) does not reflect the susceptibility to misinformation, as intended by the researchers, but rather how the respondents perceive reliability of uncertain information.I hope that discussion and research on these and related issues will continue.     

Membrane protein biogenesis by the EMC

Recent cryo‐EM‐based models reveal how the ER membrane protein complex may accomplish insertion of protein transmembrane domains with limited hydrophobicity.

Insertion of strongly hydrophobic TMDs into the ER membrane is mediated by the Sec61 complex for co‐translational insertion and the GET complex for post‐translational insertion of tail‐anchors (Volkmar & Christianson, 2020). By contrast, the EMC inserts TMDs of limited hydrophobicity, frequently located at the N‐ or C‐termini of proteins, and is involved in biogenesis of multi‐spanning membrane proteins (Volkmar & Christianson, 2020).The EMC is highly conserved (Wideman, 2015). In vertebrates, ten subunits have been identified (EMC1‐10), two of which, EMC8 and EMC9, are homologous and the result of a vertebrate‐specific gene duplication (Wideman, 2015). In Saccharomyces cerevisiae, EMC8 has been lost (Wideman, 2015). Only EMC3 displays clear homology to other membrane protein insertases, the Oxa1 family (Wideman, 2015; Volkmar & Christianson, 2020). This family includes YidC, which inserts TMDs into the bacterial cytoplasmic membrane, usually in cooperation with the Sec61‐homologous SecYEG channel (Volkmar & Christianson, 2020). Their association, along with the SecDF ancillary complex, forms a holo‐translocon capable of protein secretion and TMD insertion, with striking similarities to the EMC complex (Martin et al, 2019).Recent work by Pleiner et al (2020) presented a 3.4 Å cryo‐EM structure of the human EMC purified via a GFP‐tag on EMC2 and incorporated into a phospholipid nanodisc. The complex is formed by nine proteins (EMC1‐8, EMC10) (Pleiner et al, 2020). EMC8 and EMC9 are structurally similar, and their association with EMC2 is mutually exclusive (O''Donnell et al, 2020). Of the 12 TMDs, nine constitute the pseudosymmetric central ordered core, with a basket‐shaped cytosolic vestibule formed primarily by alpha‐helices of the EMC3 and EMC6 TMDs and cytosolic EMC2 (Fig 1A; Pleiner et al, 2020). The L‐shaped lumenal domain of the EMC consists mostly of beta‐sheets (Fig 1A; Pleiner et al, 2020), flanked by a conspicuous and conserved amphipathic alpha‐helix of EMC1 sealing the vestibule at the interface between the membrane and the ER lumen, together with another smaller amphipathic helix contributed by EMC3 (Fig 1A; Pleiner et al, 2020). In the ER lumen, the two 8‐bladed propellers of EMC1 contact six of the eight other subunits and stabilize the entire complex (Fig 1A; Pleiner et al, 2020). Beta‐sandwiches of EMC7 and EMC10 are anchored to the EMC1 lumenal domain (Fig 1A; Pleiner et al, 2020). In the cytosol, the tetratricopeptide repeat (TPR) spiral of EMC2 forms a cup underneath the partially hydrophilic vestibule in the membrane between the TMDs of EMC3 and EMC6, bridging the cytosolic ends of TMDs of EMC1, 3 and 5 (Fig 1A; Pleiner et al, 2020). Cytosolic EMC8 is bound to the opposite face of EMC2 (Fig 1A).Open in a separate windowFigure 1Comparison of the structures of human and yeast EMC(A) Cryo‐EM 3D map of the human (emdb‐21929) and yeast (emdb‐21587) EMC, showing front and back views with individual subunits coloured. Membrane position, obtained from the OPM database, is shown by grey discs. (B) Close‐up view of the EMC cavity formed by EMC3 and EMC6. Left, shown in a hydrophobicity surface pattern. Right, surface representation overlapped with the TMDs of EMC3 and EMC6. EMC4, flexible and with a gate function at the substrate‐binding place, is shown in pink in the yeast representation. EMC4 is not visible at the atomic EMC human structure, although is observed as a weak density at the human model, accompanied by TMs of EMC7 and EMC10 (Pleiner et al, 2020). (C) The yeast EMC following > 5 µs of CG‐MD simulation. The protein is shown as surface and coloured as per Pleiner et al (2020). The computed densities of waters and phospholipid tails and phosphates are shown as blue, yellow and lime green densities, sliced to bisect the cavity for clarity. Right, inset of the EMC cavity. Methods: CG‐MD simulations were built using PDB 6WB9 in a solvated symmetric POPC/POPE/cholesterol membrane and run in the Martini forcefield as described in Martin et al (2019). 3 µs unrestrained simulations were run, followed by 2.5 µs backbone restrained simulation for density calculation, done using VolMap in VMD (Humphrey et al, 1996).The 3.0 Å cryo‐EM structure of the yeast EMC presented by Bai and colleagues shows a very similar overall organization (Bai et al, 2020). Here, purification was via a 3xFLAG‐tag on EMC5, and the structure of the 8‐subunit complex (without EMC8/9) was visualized in detergent solution (Bai et al, 2020). The yeast complex has twelve TMDs like the human EMC, but unlike the human structure, EMC4 in yeast has three TMDs that are clearly visible (Bai et al, 2020). They are angled in the membrane pointing away from the complex at the cytosolic end (Fig 1A), and Bai et al (2020) propose that TMDs of EMC4, EMC3 and EMC6 form a substrate‐binding pocket similar to that of YidC. As in the human EMC, there are two amphipathic helices (EMC1 and EMC3) at the membrane/lumen interface (Fig 1A; Bai et al, 2020). In the ER lumen, yeast EMC1 only has one 8‐bladed beta‐propeller, to which the beta‐sandwiches of EMC7 and EMC10 are anchored (Fig 1A; Bai et al, 2020). In the cytosol, EMC2 bridges EMC3, 4 and 5, and its TPR repeats form a cup underneath the vestibule similar to human EMC2 (Fig 1A; Bai et al, 2020).The authors propose that insertion of a partially hydrophilic TMD by the yeast EMC is mechanistically similar to insertion by bacterial YidC (Bai et al, 2020). Yeast EMC is proposed to bind substrate between TMD2 of EMC3 and TMD2 of EMC4 in a pocket with polar and positively charged amino acids at either end and hydrophobic amino acids in the centre (Fig 1B; Bai et al, 2020). Much has been made of a conserved positive region within the EMC complex here, present in an equivalent position also in YidC (Kumazaki et al, 2014): It is claimed to be important for the incorporation of more‐hydrophilic TMDs and perhaps responsible for the “positive‐inside” orientation rule (von Heijne, 1992). Yeast and human EMC3 contain a specific R31 and R26 residue, respectively, conserved also in YidC and important for function of the EMC, as well as for YidC in Gram‐positive, but interestingly not Gram‐negative, bacteria (Chen et al, 2014; Pleiner et al, 2020; Bai et al, 2020). Another interesting feature, also conserved with YidC, is the flexibility of the TMDs flanking the substrate‐binding pocket, critical for EMC entry of substrates (Bai et al, 2020).In the human EMC, methionine residues in a cytosolic loop of EMC3 act as a substrate bait (Pleiner et al, 2020). Polar and charged residues within the substrate‐binding groove guide the lumenal domain across the membrane, facilitated by local membrane thinning (Pleiner et al, 2020; Fig 1B). The positive charges within the substrate‐binding site exclude signal peptides and enforce the “positive‐inside rule” (von Heijne, 1992; Pleiner et al, 2020). Flexible TMDs of EMC4, EMC7 and EMC10 forming a “lateral gate” of the substrate‐binding groove allow sampling of the bilayer by the substrate TMD (Pleiner et al, 2020). As the shortened TMDs of EMC3 and EMC6 cannot stably bind the substrate TMD, they favour its release into the bilayer (Pleiner et al, 2020). The EMC1 beta‐propeller(s) may recruit additional protein maturation factors in the ER lumen (Pleiner et al, 2020; Bai et al, 2020) or bind the Sec61 channel to allow cooperation between the two insertases (Bai et al, 2020).Arguably, the most interesting feature of the EMC complex is the location of a large interior cavity with distinctive hydrophilic character, which likely aids TMD insertion (Fig 1B). We ran a coarse‐grained molecular dynamics (CG‐MD) simulation of the yeast EMC structure, which highlights a profound perturbation of the phospholipid bilayer in the EMC interior cavity (Fig 1C). Here, a deep gorge forms in the cytoplasmic leaflet of the bilayer, allowing the cavity to become flooded with water (Fig 1C). Note the location of the lipid head groups here (lime green), which presumably define the site of amphipathic TMD insertion. The incursion of phospholipids into the centre of the EMC complex is a feature shared by the bacterial holo‐translocon (Martin et al, 2019) and perhaps by all membrane protein insertases. The shape and character of the EMC cavity presumably dictate its predisposition for less hydrophobic TMDs; it would be interesting to see whether the cavities of different insertases are similarly tailored to suit their substrates.     

Mycobiota dysbiosis: a new nexus in intestinal tumorigenesis

While there is growing evidence that perturbation of the gut microbiota can result in a variety of pathologies including gut tumorigenesis, the influence of commensal fungi remains less clear. In this issue, Zhu et al (2021) show that mycobiota dysbiosis stimulates energy metabolism changes in subepithelial macrophages promoting colon cancer via enhancing innate lymphoid cell activity. These findings provide insights into a role of the gut flora in intestinal carcinogenesis and suggest opportunities for adjunctive antifungal or immunotherapeutic strategies to prevent colorectal cancer.Subject Categories: Cancer, Immunology, Metabolism

Recent work reports a role for the commensal gut flora in driving aberrant host immunity and malignant cytokine signaling.

There is growing evidence for an important role for the microbiota in influencing tumorigenesis (Helmink et al, 2019). It is now well documented that gut microbiota represents a highly diverse polymicrobial population of bacteria, fungi, viruses, and protozoa. Recent evidence highlights involvement of the bacterial component of the gut microbiota in protection or enhancement of colorectal tumorigenesis. In contrast, the importance of the mycobiota is less well understood although recently suggested to promote pancreatic oncogenesis and colitis‐associated colon cancer (CAC) (Wang et al, 2018; Aykut et al, 2019). Therefore, gut fungi may play a role in the development of other gastro‐intestinal cancer types, such as CRC. Notably, there is emerging evidence suggesting that mycobiota imbalance modulates immune cells and can trigger inflammatory bowel disease (IBD) (Richard & Sokol, 2019).Here, Zhu et al (2021) provide new insight into the association between mycobiota dysbiosis, immunomodulation, and tumorigenesis in the mouse gut (Fig 1).Open in a separate windowFigure 1Dectin‐3 deficiency induces fungal dysbiosis and tumorigenesis in mice by orchestrating immune cell metabolism and cytokine signalingIn the gut of wild‐type mice, the natural population of the commensal yeast Candida albicans is detected by the Dectin‐3 receptor located on the subepithelial macrophage cell surface. This recognition allows macrophages to maintain gut homeostasis by exerting an antifungal activity. In Dectin‐3‐deficient mice, the mycobiota becomes disrupted and aberrantly increased populations of C. albicans emerge. Elevated C. albicans load triggers increased glycolysis in macrophages and interleukin‐7 (IL‐7) secretion. Macrophage‐derived IL‐7 finally induces IL‐22 secretion by group‐3 innate lymphoid cells that in turn promote tumor cell proliferation in the gut epithelium.The current study (Zhu et al, 2021) is based on previous observations suggesting that human pathogenic fungi are recognized by the C‐type lectin receptor Dectin‐3. This led Zhu et al (2021) to test whether the mycobiota influenced gut tumor formation and is linked to immune recognition mediated by Dectin‐3. First, the authors demonstrated that mice lacking the Dectin‐3 receptor had increased colonic tumorigenesis in response to the azoxymethane (AOM) and dextran sodium sulfate (DSS). This was evident histologically in marked differences in tumor number, size, and burden in Dectin‐3‐deficient mice. Of note, immunohistochemical staining revealed that the lack of Dectin‐3 induced gut tumor formation by triggering epithelial cell proliferation rather than preventing cell apoptosis. In fact, first insight into the impact of microbes in CAC was suggested by the observation that co‐housed WT and Dectin‐3‐deficient mice displayed no difference in tumorigenesis. The pivotal role of the microbiota was then underlined in fecal transplantation experiments. Chemically induced germ‐free mice that received feces from Dectin‐3 tumor‐bearing mice displayed exacerbated tumor development compared to wild‐type controls. In addition, the fungal burden was specifically increased in tumor‐bearing Dectin‐3‐deficient animals. Deep profiling of the mycobiota alterations demonstrated an increase in a single yeast species, i.e., Candida albicans, that normally behaves as commensal in the gut (Papon et al, 2013; Wilson, 2019). Preliminary experiments suggested that the increased burden of C. albicans in Dectin‐3‐deficient tumor‐bearing mice is due to impaired antifungal killing by macrophages. Consistently, elevated C. albicans populations triggered glycolysis and inflammatory IL‐7 secretion from lamina propria macrophages, suggesting that Dectin‐3 deficiency‐induced fungal dysbiosis resulted in modulation of gut macrophage metabolism, promoting tumorigenesis. Exploring the molecular and cellular mechanisms that linked macrophage‐derived IL‐7 secretion and CRC development, Zhu et al (2021) showed in vitro that IL‐7 produced by subepithelial macrophages induced IL‐22 secretion by group‐3 innate lymphoid cells (ILC3s). In turn, up‐regulation of IL‐22 in Dectin‐3‐deficient mice contributed to the oncogenesis seen in these animals. Finally, a detailed analysis of tumor tissues collected from 172 patients with CRC showed correlation and poorer clinical outcome in patients with decreased expression of Dectin‐3, but increased expression of IL‐22 and mycobiota burden, although they did not directly link this to the presence of C. albicans in these patients.Overall, Zhu et al (2021) define a new cell paradigm linking mycobiota dysbiosis, macrophage energy metabolism, and innate lymphoid cell function to tumor development in the mouse gut. In this context, this study also sheds additional light on a new role of ILC3s, a recently described type of lymphoid effectors (Serafini et al, 2015). Indeed, ILC3s have been shown in the present article to act as cornerstone cells orchestrating cytokine‐regulated tumorigenesis in the gut. Beyond these pathophysiological considerations, the study opens up new opportunities for developing adjunctive antifungal or immunotherapeutic strategies for the prevention of high morbidity in CRC. Importantly, this enlightening article provides firm evidence that colonic C. albicans populations promote metabolic reprogramming in lamina propria macrophages and tumor cell formation. Metabolic reprogramming has been observed with other fungi, such as Aspergillus fumigatus, which induces metabolic rewiring of alveolar macrophages in the lung epithelium (Gonçalves et al, 2020). In line, the report by Zhu et al (2021) adds to previous work suggesting that mycobiota promotes pancreatic oncogenesis via activation of mannose‐binding lectins (Aykut et al, 2019). Mycobiota dysbiosis therefore stands out as an important new field of investigation in cancer research that is ripe for future exploration.     

Restraining CDK1–cyclin B activation: PP2A on the cUSP(7)

USP7 inhibitors are gaining momentum as a therapeutic strategy to stabilize p53 through their ability to induce MDM2 degradation. However, these inhibitors come with an unexpected p53‐independent toxicity, via an unknown mechanism. In this issue of The EMBO Journal, Galarreta et al report how inhibition of USP7 leads to re‐distribution of PP2A from cytoplasm to nucleus and an increase of deleterious CDK1‐dependent phosphorylation throughout the cell cycle, revealing a new regulatory mechanism for the progression of S‐phase cells toward mitosis to maintain genomic integrity.Subject Categories: Cell Cycle, Post-translational Modifications, Proteolysis & Proteomics

Recent work reveals untimely activation of mitotic cyclin‐dependent kinase as a molecular basis for p53‐independent cell toxicity of USP7 deubiquitinase inhibitors.

The G2‐M transition in the eukaryotic cell cycle is a critical point to ensure that cells with damaged DNA are unable to enter the mitotic phase. This checkpoint is highly regulated by a number of kinases, including ATR, ATM and WEE1, and ends upon activation of the CDK1–cyclin B1 kinase complex (Visconti et al, 2016). Since premature activation of CDK1–cyclin B1 causes replication fork collapse, DNA damage, apoptosis, and mitotic catastrophe (Szmyd et al, 2019 and references therein), restricting CDK1–cyclin B1 activity prior to mitosis is key to maintaining genomic integrity.A body of recent work has suggested that the deubiquitinase USP7 is a master regulator of genomic integrity; it is required for DNA replication in numerous ways, including indirect regulation of cyclin A2 during the S‐phase, origin firing, and replication fork progression. USP7 also regulates mitotic entry by stabilizing PLK1, another kinase which is highly active in the M phase and ensures proper alignment of chromatids prior to segregation. Notably, USP7 inhibitors have become an attractive cancer therapeutic strategy based on their ability to trigger degradation of MDM2, and thereby stabilize p53 (Valles et al, 2020). However, there is growing evidence of USP7 inhibitor‐related toxicity that is not mediated through p53 (Lecona et al, 2016; Agathanggelou et al, 2017), indicating that USP7 inhibitors impact other cellular processes. Therefore, Galarreta et al (2021) investigated the potential functional relationship between USP7 and CDK1, given the role of both factors in regulating the cell cycle.Through a series of in vitro experiments, the authors confirmed that five USP7 inhibitors induce premature mitotic kinase activity, including increased MPM2 signal (indicative of mitosis‐specific phosphorylation events) and phosphorylation of histone H3 Ser10 (H3S10P) in all cells, regardless of where they are in the cell cycle. To determine whether USP7 affects CDK1 during the cell cycle, Galarreta et al (2021) demonstrate that cell lines treated with USP7 inhibitors exhibit reduced levels of inhibitory Tyr‐15 phosphorylation on CDK1 and increased cyclin B1 presence in the nucleus, suggesting activation of the CDK1–cyclin B1 complex. Furthermore, treatment with the CDK1 inhibitor RO3306 rescues the USP7 inhibitor‐dependent increase of mitotic activity.These observations suggest that CDK1 has the potential to catalyze mitosis‐specific phosphorylation irrespective of cell cycle phase and that cells rely on USP7‐specific deubiquitination to suppress or reverse premature CDK1 activity. Surprisingly, despite the nuclear localization of cyclin B and decrease in inhibitory CDK1 Tyr‐15 phosphorylation, USP7 inhibitors failed to drive cells into mitosis. How might this be? Nuclear localization of cyclin B normally occurs just minutes before the onset of mitosis and nuclear envelope breakdown (Santos et al, 2012), yet the nucleus remains intact following USP7 inhibition. Moreover, the decrease in Tyr‐15 phosphorylation suggests the ATR‐ and WEE1‐dependent G2/M checkpoint is inactivated by USP7 inhibition. Do these data hint at the presence of an additional, unknown regulatory mechanism controlling mitotic entry independent of the G2/M checkpoint and nuclear localization of the CDK1–cyclin B complex?To determine whether CDK1 is the driver of USP7 inhibitor toxicity, Galarreta et al exposed cells to CDK1 inhibitors and observed a reduction in apoptosis. Furthermore, CDK1 inhibitors promote cell survival in cells treated with three structurally unrelated USP7 inhibitors. Finally, CDC25A‐deficient mouse embryonic stem cells, which constitutively express low levels of CDK1, resist USP7 inhibition. Together, these data suggest that the USP7 inhibitor‐dependent toxicity is the result of CDK1‐mediated cell death. The authors note that the phosphatase PP2A is an antagonist for CDK1 in addition to being a candidate USP7 substrate (Lecona et al, 2016; Wlodarchak & Xing, 2016), and thus, they turned their attention to elucidating the connection between USP7 and PP2A. Combining biochemical and immunofluorescence studies, Galarreta et al (2021) demonstrate that USP7 interacts with two subunits of PP2A, and this interaction increases in response to USP7 inhibition. Inhibiting USP7 furthermore triggers PP2A re‐localization from the cytoplasm to the nucleus and increases the phosphorylation levels of PP2A substrates, such as AKT and PRC1. DT‐061, a chemical activator of PP2A, reduces CDK1 phosphorylation events, suggesting that PP2A deregulation is a key mediator of USP7 inhibitor‐related toxicity. Using phosphoproteomics to analyze cells treated with a USP7 inhibitor or PP2A‐inhibiting okadaic acid, the authors reveal that both treatments share a significant number of altered phosphorylated targets—especially those related to mitosis, the cell cycle, and epitopes with a CDK‐dependent motif. Thus, the effects of USP7 inhibitors on CDK1 appear to be mediated through PP2A localization to the nucleus.These unexpected findings raise several questions that potentially impact the current view of cell cycle regulation. For example, how does USP7 regulate PP2A localization and is this important for reversing CDK1‐dependent phosphorylation of mitotic substrates prior to mitosis? Does PP2A accumulation in the nucleus explain the failure of USP7‐inhibited cells to enter mitosis despite cyclin B1 nuclear localization? A role for ubiquitin signaling as a regulator of CDK1 in interphase cells has not been reported, and accordingly, new investigations will be needed to unravel the mechanisms by which USP7 controls PP2A localization.Another important question that arises is whether or not CDK1 has sufficient basal activity to phosphorylate numerous mitotic proteins independent of cell cycle phase. The observation that USP7 and PP2A act to prevent the improper accumulation of CDK1‐dependent phosphorylation even in G1 phase cells suggests this to be the case. Alternatively, USP7 activity may be required to prevent abnormal pairing of CDK1 with a cyclin that is ubiquitously expressed across the cell cycle. If so, more research will be needed to uncover how ubiquitin signaling ensures CDK1 only pairs with cyclin A and cyclin B once they accumulate later in the cell cycle.Interestingly, USP7 inhibition also causes a rapid loss in DNA synthesis of S‐phase cells, prompting the authors to perform a time course experiment to decipher the order of events following treatment (i.e., does CDK1 activation precede or follow termination of DNA replication?). High‐throughput microscopy and flow cytometry analysis reveal an immediate reduction of DNA replication, an increase of CDK1 activity, and elevated DNA damage before a detectable increase in H3S10P. Long‐term exposure of USP7 inhibitors leads to DNA damage restricted only to cells with corresponding high levels of H3S10P and MPM2. Overall, these results illustrate how inhibition of USP7 activates CDK1, disrupting DNA replication and inducing DNA damage (Fig 1).Open in a separate windowFigure 1USP7 regulates CDK1In untreated cells, CDK1 is suppressed by USP7 and PP2A, and CDK1‐cyclin B is only active during the G2/M transition. In response to treatment, USP7 facilitates PP2A localization to the nucleus. This allows CDK1 to initiate premature mitotic activity throughout the cell cycle, resulting in increased DNA damage and cellular toxicity.The finding that USP7 inhibitors caused a rapid shutdown of DNA replication brings to mind the recent findings by several groups, that CDK1 activation occurs concomitantly with the S/G2 transition and that premature CDK1 activation in S‐phase terminates replication (Akopyan et al, 2014; Lemmens et al, 2018; Saldivar et al, 2018; Deng et al, 2019; Branigan et al, 2021). According to these studies, coupling of CDK1 activation to the S/G2 transition is regulated by ATR‐CHK1 signaling, a pathway activated by DNA replication to restrain CDK1 through Tyr‐15 phosphorylation. Galarreta et al''s observation that USP7 inhibition overrides ATR‐CHK1 (i.e., Tyr‐15 phosphorylation) highlights the fundamental importance of ubiquitin signaling, and potentially PP2A localization, for ensuring proper S‐to‐M progression and genome maintenance. Ultimately, the mechanistic details of Galarreta et al''s observations remain to be elucidated, and undoubtedly, their findings will inspire future investigations. Moreover, their discovery may lead to a new strategy targeting CDK1 to mitigate unwanted toxicities in the clinic.     

Discussions on the quality of antibodies are no reason to ban animal immunization

Debates about the source of antibodies and their use are confusing two different issues. A ban on life immunization would have no repercussions on the quality of antibodies. Subject Categories: S&S: Economics & Business, Methods & Resources, Chemical Biology

There is an ongoing debate on how antibodies are being generated, produced and used (Gray, 2020; Marx, 2020). Or rather, there are two debates, which are not necessarily related to each other. The first one concerns the quality of antibodies used in scientific research and the repercussions for the validity of results (Bradbury & Pluckthun, 2015). The second debate is about the use of animals to generate and produce antibodies. Although these are two different issues, we observe that the debates have become entangled with arguments for one topic incorrectly being used to motivate the other and vice versa. This is not helpful, and we should disentangle the knot.Polyclonal antibodies are being criticized because they suffer from cross‐reactivity, high background and batch‐to‐batch variation (Bradbury & Pluckthun, 2015). Monoclonal antibodies produced from hybridomas are criticized because they often lack specificity owing to genetic heterogeneity introduced during hybridoma generation that impairs the quality of the monoclonals (Bradbury et al, 2018). These are valid criticisms and producing antibodies in a recombinant manner will, indeed, help to improve quality and specificity. But a mediocre antibody will remain a mediocre antibody, no matter how it is produced. Recombinant methods will just produce a mediocre antibody more consistently.Getting a good antibody is not easy and much depends on the nature and complexity of the antigen. And low‐quality antibodies are often the result of poor screening, poor quality control, incomplete characterization and the lack of international standards. Nevertheless, the technologies to ensure good selection and to guarantee consistent quality are much more advanced than a decade ago, and scientists and antibody producers should implement these to deliver high‐quality antibodies. Whether antibodies are generated by animal immunization or from naïve or synthetic antibody libraries is less relevant; they can all be produced recombinantly, and screening and characterization are needed in all cases to determine quality, and if the antibody is fit for purpose.But criticisms on the quality of many antibodies and pleas for switching to recombinant production of antibodies cannot be mixed up with a call to ban animal immunization. The EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) recently published a recommendation to stop using animals for generating and producing antibodies for scientific, diagnostic and even therapeutic applications (EURL ECVAM, 2020). This recommendation is mainly supported by scientists who seem to be biased towards synthetic antibody technology for various reasons. Their main argument is that antibodies derived from naïve or synthetic libraries are a valid (and exclusive) alternative. But are they?One can certainly select antibodies from non‐immune libraries, and, depending on the antigen and the type of application, these antibodies can be fit for purpose. In fact, a few of such antibodies have made it to the market as therapeutics, Adalimumab (Humira®) being a well‐known example. But up to now, the vast majority of antibodies continues to come from animal immunization (Lu et al, 2020). And there is a good reason for that. It is generally possible to generate a few positive hits in a naïve/synthetic library; and the more diverse the library, the more hits one is likely to get. But many decades of experience with immunization of animals—especially when they are outbred—shows that they generate larger amounts of antibodies with superior properties. And the more complex your antigen is, the more the balance swings towards animal immunization if you want to have a guarantee for success.There are different factors at work here. First, the immune system of mammals has evolved over millions of years to efficiently produce excellent antibodies against a very diverse range of antigens. Second, presenting the antigen multiple times in its desired (native) conformation to the animal immune system exploits the natural maturation process to fine‐tune the immune response against particular qualities. Another factor is that in vivo maturation seems to select against negative properties such as self‐recognition and aggregation. It also helps to select for important properties that go beyond mere molecular recognition (Jain et al, 2017). In industrial parlance, antibodies from animal immunization are more “developable” and have favourable biophysical properties (Lonberg, 2005). Indeed, the failure rate for antibodies selected from naïve or synthetic libraries is significantly higher.Of course, the properties of synthetic antibodies selected from non‐immune libraries can be further matured in vitro, for example by light chain shuffling or targeted mutagenesis of the complementarity determining region (CDR). While this method has become more sophisticated over the years, it remains a very complex and iterative process without guarantee that it produces a high‐quality antibody.Antibodies are an ever more important tool in scientific research and a growing area in human and veterinary therapeutics. Major therapeutic breakthroughs in immunology and oncology in the past decades are based on antibodies (Lu et al, 2020). The vast majority of these therapeutic antibodies were derived from animals. An identical picture appears when you look at the antibodies in fast‐track development to combat the current COVID‐19 crisis: again, the vast majority are either derived from patients or from animal immunizations. The same holds true for antibodies that are used in diagnostics and epidemiologic studies for COVID‐19.It is for that reason that we need the tools and methods that guarantee antibodies of the highest quality and provide the best chance for success. The COVID‐19 pandemic is only one illustration of this need. If we block access to these tools, both scientific research and society at large will be negatively impacted. We therefore should not limit ourselves to naïve and synthetic libraries. Animal immunization remains an inevitable method that needs to stay. But we all agree that these immunizations must be performed under best practice to further reduce the harm to animals.     

The challenge of removing waste from wastewater: let technology use nature!

Tertiary treatments capable of removing chemical and biological contaminants of emerging concern have been successfully developed and implemented at full scale, opening the possibility of using wastewater treatment plants as recycling units, capable of producing wastewater that can be reused in various activities, such as agriculture irrigation; However, tertiary treatments remove only part of the wastewater microbiota, leaving the opportunity for regrowth and/or reactivation of potentially hazardous microorganisms, facilitated by the poor competition among the surviving microorganisms; Under the motto ‘added by technology, lead by nature’, the treatment and storage of treated wastewater must find the balance to develop a protection shield against the impoverishment the microbial quality and the development of potentially hazardous bacteria.

No man ever steps in the same river twice, for it''s not the same river and he''s not the same man. Heraclitus

Access to wholesome drinking water is not only a major ambition but also a basic human right that since antiquity has called scientists, engineers and politicians for action. The recognition that human excreta compromise the quality of the sources of drinking water triggered the development of sewage drainage systems as far as 3500–2500 BC, in cities such as Ur and Babylon (Lofrano and Brown, 2010). Among these ancient cities, Rome, where the largest known ancient sewer (Cloaca Maxima) and the first roman aqueduct (Aqua Appia) were built (600–312 BC), stands up (Lofrano and Brown, 2010). Despite the unexpected regression observed during the Middle Ages, the rising of urban and industrial agglomerations, matched by a growing production of wastewater, has been triggering the development of wastewater treatment technologies since the industrial revolution (Lofrano and Brown, 2010).Unlike other industrial activities, whose high added value products enable high‐profit margins, wastewater treatment may be not prioritized, at least in world regions with limited income and capacity to invest in both infrastructure and operation systems. Consequently, most of the urban wastewater treatment plants (UWWTP) operating worldwide rely upon biological‐based low‐cost technologies. The conventional activated sludge (CAS) technology is one of the most commonly applied worldwide (Orhon, 2014). With a long development history itself, this aerobic biologic process, in full‐scale operation since 1914, is regarded as the conventional norm for wastewater treatment (Alleman and Prakasam, 1983; Orhon, 2014).A century ago the major challenge of environmental engineers was to develop a treatment system able to reduce the load of readily degradable organic matter and pathogens from sewage. CAS‐based treatment systems fully achieve these goals (Tchobanoglous et al., 2003). But more than one century of industrial innovation and development changed dramatically our lifestyle, and consequently, the type of pollutants discharged in wastewater. Nowadays, UWWTPs are also expected to remove excess of inorganic nitrogen (N) and phosphorus (P) nutrients, responsible for the eutrophication of the receptor water bodies, and a myriad of (potentially) hazardous chemical micropollutants, which may pose risk to the aquatic ecosystems and human health given their acute and chronic toxicity. These chemical micropollutants of emerging concern, which are found at very low concentrations (up to μg l⁻¹), include both natural and xenobiotic compounds such as pharmaceuticals, personal care products, steroid hormones, drugs of abuse, and pesticides, among others (European Commission, 2013; Ribeiro et al., 2015). In addition to the chemical micropollutants, UWWTPs are now also challenged to impede the release of high loads of biological contaminants of emerging concern, such as some pathogenic virus, protozoa, or bacteria in particular antibiotic‐resistant (ARB) harbouring antibiotic resistance genes (ARG), into the receptor water bodies (Dulio et al., 2018; European Commission, 2020).Effective wastewater treatment systems are indeed the primary and major barrier between human activities and the environment, with a pivotal role on the prevention of contamination of surface‐ and groundwater. Inevitably, water bodies such as rivers, lakes and aquifers bridge sectors of activity and geographies, for instance when used as sources of agriculture irrigation water, drinking water production or habitat and fountain for wildlife or food‐producing animals. Pressures to implement technologies able to efficiently remove both chemical and biological contaminants within the urban water cycle are exacerbated under the climate change scenario. Massive withdrawal and consumption coupled with unpredictable weather conditions, such as drought and flood events, has been leading not only to freshwater scarcity but also to the deterioration of water quality (WWAP, 2019; European Commission, 2020).Freshwater scarcity brought the new concept of UWWTPs as recycling units, capable of producing final effluents that can be safely and sustainably reused for different purposes, namely in agriculture, the sector with the largest consumption of freshwater (WWAP, 2019). But to be reused, treated wastewater must be safe. This means that the concentration of eventual chemical and/or biological pollutants in treated wastewater must not put at risk the environmental and human health. Hence, the degree of contamination of the treated wastewater determines its end use or site of discharge (European Commission, 1991, 2020; Becerra‐Castro et al., 2015).Upgrading technologies capable of removal of N and P nutrients from wastewater have been successfully developed and implemented. Nowadays, full‐scale UWWTP with trains favouring the recirculation of the mixed liquor between aerobic and anoxic tanks, where ammonification of organic‐N, nitrification and denitrification occur according to the oxygen availability in each compartment are commonly found; and an increasing number of UWWTP where, in addition to the trains referred to above, recirculation includes anaerobic reactors favouring P granules accumulation are also operating worldwide (Tchobanoglous et al., 2003). More recently, the simultaneous C, N, and P removal is assured through the aerobic granular sludge technology, given the spatial distribution of the microorganisms of the different metabolic groups in the different micro‐environments of the granules (Nancharaiah and Reddy, 2018).In contrast with the C, N and P removal, the biological removal of chemical micropollutants seems to be less efficient. Despite the ability of a vast number of microorganisms to degrade a wide diversity of micropollutants, the low concentration of these compounds in wastewater may contribute for their low bioavailability in the biological reactors. Consequently, the secondary final effluents of CAS‐based UWWTPs still contain numerous micropollutants at environmental worrisome concentrations (McEachran et al., 2018).Advanced Oxidation Technologies (AOTs) have been recommended among the best solutions for the removal of chemical micropollutants from the secondary effluents of CAS‐based UWWTPs. A vast number of scientific studies has been conducted in this area, in order to develop and optimize tertiary processes capable of the efficient removal of these contaminants from the effluents before discharge into the receptor water bodies (Ribeiro et al., 2015). Among these technologies, ozonation has high visibility, being implemented in full‐scale UWWTPs, for instance in Switzerland, a country that recently implemented legislation recommending advanced treatment of wastewater aiming at protecting the environment (Rizzo et al., 2019).One of the advantages of AOTs is their capacity to disinfect water (Rizzo et al., 2020). Hence, besides degrading undesirable chemical micropollutants, numerous scientific bench studies demonstrated that the mechanisms for microbial inactivation used by AOTs, such as the oxidative stress as it is generated by ozonation, are also capable of reducing the microbial load of wastewater, including ARB&ARGs (e.g. Rizzo et al., 2020). Such promising results opened the possibility of upgrading CAS‐based UWWTPs with a final AOT polishing step and using the facilities as recycling units of urban wastewater. Additional treatment may be required in a reuse scenario, and in that cases, the final treated wastewater may need to undergo an adsorption post‐AOT treatment step to eventually remove toxic degradation products (Rizzo et al., 2019) and to be stored for periods that may vary between few hours to some days, depending on the needs. Hence, some bench and full‐scale studies have been conducted to assess the microbiological quality of the wastewater after the final AOT treatment.Consistently, studies focused on the effect of AOTs conclude that the microbiota, including ARB&ARGs, surviving AOT treatment is capable of re‐regrowth during the storage period, sometimes to values reaching or surpassing those measured in the untreated secondary effluent (Zimmermann et al., 2011; Becerra‐Castro et al., 2016; Czekalski et al., 2016; Sousa et al., 2017; Moreira et al., 2018; Biancullo et al., 2019; Iakovides et al., 2019). Moreover, re‐regrowth is accompanied by the disturbance of the microbial community, with possible implications on the decrease of diversity, and the overgrowth of Proteobacteria (Becerra‐Castro et al., 2016; Moreira et al., 2018). Among these, bacterial groups described as potential vectors of antibiotic resistance, such as Pseudomonas, have been detected at high relative abundance (Alexander et al., 2016; Jäger et al., 2018; Moreira et al., 2018).The same phenomena occur when other technologies are applied in the wastewater treatment. Comparatively milder processes such as UV_{254 nm} irradiation or even coagulation lead to similar disturbances (Becerra‐Castro et al., 2016; Grehs et al., 2019). When comparing different technologies, a positive correlation between disinfection efficacy and the predominance of ubiquitous, potentially hazardous, bacteria in the treated stored wastewater seems to occur (Becerra‐Castro et al., 2016). Interestingly, clean built environments, where asepsis and frequent disinfection are the rule, are characterized by the predominance of Proteobacteria (Mahnert et al., 2019). Moreover, cleaning with aggressive agents seems to favour microbiomes encoding functions related with virulence, multi‐drug efflux, oxidative stress, as well as membrane transport and secretion, which empower cells to acquire nutrients in highly competitive nutrient‐poor environments (Mahnert et al., 2019).Such results are not unexpected. Any process reducing the diversity and abundance of microorganisms in a given ecosystem, through physical removal of the cells or physical and/or chemical inactivation of macromolecules or cellular processes, is expected to generate a habitat where intercellular competition for space and nutrients is reduced, offering the opportunity for those that randomly survived the process and that are most versatile and fast to grow, to proliferate. Therefore, among the survivors, those with high capacity to grow under the conditions prevailing in the disinfected or cleaned system will thrive. Conversely, the microorganisms with specific requirements (e.g. nutritional) or with slower grow rates will be outcompeted. Proteobacteria are well known for their genomic plasticity. Some proteobacterial species, such as Pseudomonas aeruginosa, colonize a wide diversity of environmental compartments, including mineral water, chlorinated drinking water, surface water and soils, and even human bodies (Grobe et al., 2001; Naze et al., 2010; Palleroni, 2015). Part of the success of this ubiquitous opportunistic pathogenic species rely upon its capacity to exchange genetic information through horizontal gene transfer (Kung et al., 2010). Hence, Pseudomonas aeruginosa harbour genetic information which allows cell development in a wide diversity of environmental conditions, including in the presence of a vast array of antimicrobial compounds. Therefore, besides carrying intrinsic antimicrobial resistance, P. aeruginosa strains are excellent vectors of ARG dissemination (Manaia, 2017). The predominance of microorganisms with these type of features in treated wastewater is thus not desirable, mainly if its further use in agriculture irrigation is envisaged, given the possibility of contamination of the food chain.In this context, it may be argued that the upgrading UWWTPs with a final disinfection step is not enough to transform these facilities into wastewater recycling units, and more studies should be carried out to design and implement storage systems capable of attenuating the imbalance of the bacterial community before reuse of the stored treated wastewater.Measures to restore the microbial richness and diversity of the disinfected wastewater would prevent the overgrowth of hazardous bacteria fitted to couple with very clean oligotrophic environments, such as P. aeruginosa, through competition. Such measures might include the inoculation of the disinfected wastewater with balanced natural microbial communities, with a rich and diverse phylogenetic and functional assembly of microorganisms (van Bruggen et al., 2019). In these communities, organisms belonging to a wide variety of species interact through complex relationships (mutualism, commensalism, competition, predation, parasitism) assuring metabolic redundancy and the integrity of nutrient cycles and energy flows (van Bruggen et al., 2019). Such communities are stable and resilient, that is, show little disturbance and restore rapidly upon alteration of the environmental conditions or invasion (van Bruggen et al., 2019). Hence, procedures such as diluting disinfected wastewater with non‐polluted surface water, mixing with pristine sediments or soils or discharge in wetlands would introduce a healthy microbiome in the treated wastewater. Under this circumstance, the exogenous microbiome would act as a protection shield for the proliferation of the hazardous microorganism surviving the disinfection process, in a similar way of the natural human microbiota, our first line of defence against the invasion of pathogens.Definitely, microbes must have a say on removing waste from wastewater. The next research steps should be oriented towards a better understanding of the biotic relationships occurring in the treated wastewater and technological implementation of systems that are able to nurture these important artisan communities.     

Strength is in engagement: The rise of an online scientific community during the COVID‐19 pandemic

Many scientists, confined to home office by COVID‐19, have been gathering in online communities, which could become viable alternatives to physical meetings and conferences. Subject Categories: S&S: Careers & Training, Methods & Resources, S&S: Ethics

As COVID‐19 has brought work and travel to a grinding halt, scientists explored new ways to connect with each other. For the gene regulation community, this started with a Tweet that quickly expanded into the “Fragile Nucleosome” online forum, a popular seminar series, and many intimate discussions connecting scientists all over the world. More than 2,500 people from over 45 countries have attended our seminars so far and our forum currently has ~ 1,000 members who have kick‐started discussion groups and mentorship opportunities. Here we discuss our experience with setting up the Fragile Nucleosome seminars and online discussion forum, and present the tools to enable others to do the same.Too often, we forget the importance of social interactions in science. Indeed, many creative ideas originated from impromptu and fortuitous encounters with peers, in passing, over lunch, or during a conference coffee break. Now, the ongoing COVID‐19 crisis means prolonged isolation, odd working hours, and less social interactions for most scientists confined to home. This motivated us to create the “Fragile Nucleosome” virtual community for our colleagues in the chromatin and gene regulation field.

… the ongoing COVID‐19 crisis means prolonged isolation, odd working hours and less social interactions for most scientists confined to home.

While the need to address the void created by the COVID‐19 pandemic triggered our actions, a large part of the international community already has had limited access to research networks in our field. Our initiative offered new opportunities though, in particular for those who have not benefited from extensive networks, showing how virtual communities can address disparities in accessibility. This should not be a stop‐gap measure during the pandemic: Once we come out from our isolation, we still need to address the drawbacks of in‐person scientific conferences/seminars, such as economic disparities, travel inaccessibility, and overlapping family responsibilities (Sarabipour, 2020). Our virtual community offers some solutions to the standing challenges (Levine & Rathmell, 2020), and we hope our commentary can help start conversations about the advantages of virtual communities in a post‐pandemic world.

… once we come out from our isolation we still need to address the drawbacks of in‐person scientific conferences/seminars, such as economic disparities, travel inaccessibility and overlapping family responsibilities…

     

Context matters: On the road to responsible biosafety technologies in synthetic biology

Biosafety is a major challenge for developing for synthetic organisms. An early focus on application and their context could assist with the design of appropriate genetic safeguards. Subject Categories: Synthetic Biology & Biotechnology, S&S: Economics & Business

One of the goals of synthetic biology is the development of robust chassis cells for their application in medicine, agriculture, and the food, chemical and environmental industries. These cells can be streamlined by removing undesirable features and can be augmented with desirable functionalities to design an optimized organism. In a direct analogy with a car chassis, they provide the frame for different modules or “plug‐in” regulatory networks, metabolic pathways, or safety elements. In an effort to ensure a safe microbial chassis upfront, safety measures are implemented as genetic safeguards to limit risks such as unwanted cellular proliferation or horizontal gene transfer. Examples of this technology include complex genetic circuits, sophisticated metabolic dependencies (auxotrophies), and altered genomes (Schmidt & de Lorenzo, 2016; Asin‐Garcia et al, 2020). Much like seat belts or airbags in cars, these built‐in measures increase the safety of the chassis and of any organisms derived from it. Indeed, when it comes to safety, synthetic biology can still learn from a century‐old technology such as cars about the significance of context for the development of biosafety technologies.Every car today has seat belts installed by default. Yet, seat belts were not always a standard component; in fact, they were not even designed for cars to begin with. The original 2‐point belts were first used in aviation and only slowly introduced for motorized vehicles. Only after some redesign, the now‐common 3‐point car seat belts would become the life‐saving equipment that they are today. A proper understanding of the context of their application was therefore one of the crucial factors for their success and wide adoption. Context matters: It provides meaning for and defines what a technological application is best suited for. What was true for seat belts may be also true for biosafety technologies such as genetic safeguards.

… when it comes to safety, synthetic biology can still learn from a century‐old technology such as cars about the significance of context for the development of biosafety technologies.

Society has a much higher awareness of technology’s risks compared to the early days of cars. Society today requires that technological risks are anticipated and assessed before an innovation or its applications are widely deployed. In addition, society increasingly demands that research and innovation take into account societal needs and values. This has led to, among others, the Responsible Research and Innovation (RRI; von Schomberg, 2013) concept that has become prominent in European science policy. In a nutshell, RRI requires that innovative products and processes align with societal needs, expectations, and values in consultation with stakeholders. RRI and similar frameworks suggest that synthetic biology must anticipate and respond not only to risks, but also to societal views that frame its evaluation and risk assessment.     

Glycolysis regulates Hedgehog signalling via the plasma membrane potential

The authors regret omitting the citation of a bioRxiv preprint study by preprint: Emmons‐Bell et al (2020), who independently discovered the role of ion channel‐dependent membrane depolarization for Smo membrane accumulation in the fly wing disc. This study used a different methodological approach and did not describe the mechanism of how membrane potential affects hedgehog signaling. The reference is herewith added.     

Biodiversity 2030: a road paved with good intentions: The new EU Commission's biodiversity Strategy risks to remain an empty husk without proper implementation

The EU''s Biodiversity Strategy for 2030 makes great promises about halting the decline of biodiversity but it offers little in terms of implementation. Subject Categories: S&S: Economics & Business, Ecology, S&S: Ethics

Earth is teeming with a stunning variety of life forms. Despite hundreds of years of exploration and taxonomic research, and with 1.2 million species classified, we still have no clear picture of the real extent of global biodiversity, with estimates ranging from 3 to 100 million species. A highly quoted—although not universally accepted—study predicted some 8.7 million species, of which about 2.2 million are marine (Mora et al, 2011). Although nearly any niche on the surface of Earth has been colonized by life, species richness is all but evenly distributed. A large share of the known species is concentrated in relatively small areas, especially in the tropics (Fig 1). Ultimately, it is the network of the interactions among life forms and the physical environment that make up the global ecosystem we call biosphere and that supports life itself.Open in a separate windowFigure 1Biological hotspots of the worldA total of 36 currently recognized hotspots make up < 3% of the planet''s land area but harbor half of the world''s endemic plant species and 42% of all terrestrial vertebrates. Overall, hotspots have lost more than 80% of their original extension. Credit: Richard J. Weller, Claire Hoch, and Chieh Huang, 2017, Atlas for the End of the World, http://atlas‐for‐the‐end‐of‐the‐world.com/. Reproduced with permission.Driven by a range of complex and interwoven causes–such as changes in land and sea use, habitat destruction, overexploitation of organisms, climate change, pollution, and invasive species–biodiversity is declining at an alarming pace. A report by the Intergovernmental Science‐Policy Platform on Biodiversity and Ecosystem Services (IPBES) issued a clear warning: “An average of around 25 per cent of species in assessed animal and plant groups are threatened, suggesting that around 1 million species already face extinction, many within decades, unless action is taken to reduce the intensity of drivers of biodiversity loss. Without such action, there will be a further acceleration in the global rate of species extinction, which is already at least tens to hundreds of times higher than it has averaged over the past 10 million years” (IPBES, 2019) (Fig 2). Although focused on a smaller set of organisms, a more recent assessment by WWF has reached similar conclusions. Their Living Planet Index, that tracks the abundance of thousands of populations of mammals, birds, fish, reptiles, and amphibians around the world, shows a stark decline in monitored populations (WWF, 2020). As expected, the trend of biodiversity decline is not homogeneous with tropical areas paying a disproportionately high price, mostly because of unrestrained deforestation and exploitation of natural resources.Open in a separate windowFigure 2The global, rapid decline of biodiversity(A) Percentage of species threatened with extinction in taxonomic groups that have been assessed comprehensively, or through a “sampled” approach, or for which selected subsets have been assessed by the IUCN Red List of Threatened Species. Groups are ordered according to the best estimate, assuming that data‐deficient species are as threatened as non‐data deficient species. (B) Extinctions since 1500 for vertebrate groups. (C) Red List Index of species survival for taxonomic groups that have been assessed for the IUCN Red List at least twice. A value of 1 is equivalent to all species being categorized as Least Concern; a value of zero is equivalent to all species being classified as Extinct. Data for all panels from www.iucnredlist.org. Reproduced from (IPBES, 2019), with permission.

Driven by a range of complex and interwoven causes […] biodiversity is declining at an alarming pace.

Against this dire background, the EU has drafted a Biodiversity Strategy 2030, an ambitious framework aimed to tackling the key reasons behind biodiversity loss. The plan hinges around a few main elements, such as the establishment of protected areas for at least 30% of Europe''s lands and seas (Fig 3); a significant increase of biodiversity‐rich landscape features on agricultural land by establishing buffer zones like hedges and fallow fields; halting and reversing the decline of pollinators; and planting 3 billion trees by 2030 (https://ec.europa.eu/info/strategy/priorities‐2019‐2024/european‐green‐deal/actions‐being‐taken‐eu/eu‐biodiversity‐strategy‐2030_en). The budget for implementing these measures was set at €20 billion per year.Open in a separate windowFigure 3Natura 2000, the EU''s network of protected areasIn 2019, 18% of land in the EU was protected as Natura 2000, with the lowest share of protected land in Denmark (8%) and the highest in Slovenia (38%). In 2019, the largest national network of terrestrial Natura 2000 sites was located in Spain, covering 138,111 km², followed by France (70,875 km²) and Poland (61,168 km²). Reproduced from Eurostat: https://ec.europa.eu/eurostat/statistics‐explained/index.php?title=Main_Page “Nature is vital for our physical and mental wellbeing, it filters our air and water, it regulates the climate and it pollinates our crops. But we are acting as if it didn''t matter, and losing it at an unprecedented rate”, said Virginijus Sinkevičius, Commissioner for the Environment, Oceans and Fisheries, at the press launch of the new EU action (https://ec.europa.eu/commission/presscorner/detail/en/ip_20_884). “This new Biodiversity Strategy builds on what has worked in the past, and adds new tools that will set us on a path to true sustainability, with benefits for all. The EU''s aim is to protect and restore nature, to contribute to economic recovery from the current crisis, and to lead the way for an ambitious global framework to protect biodiversity around the planet”.Environmental groups and other stakeholders have welcomed the EU''s pledge in principle. “This is a unique opportunity to shape a new society in harmony with nature”, applauded Wetlands International. “We must not forget that the biodiversity and climate crisis is a much bigger and persistent challenge for humanity than COVID‐19”, (https://europe.wetlands.org/news/welcoming‐the‐eu‐biodiversity‐strategy‐for‐2030/). EuroNatur, a foundation focused on conservation, stated that the goals set out by the new strategy provide a strong basis for improving the state of nature in the EU (www.euronatur.org).Alongside the voices of praise, however, many have expressed concerns that the strategy could turn into a little more than a wish list. “The big issue of the strategy is that while setting a goal for financial funds, the EU does not specify where the money is supposed to come from. It only says it should include ‘EU funds and national and private funding’”, commented the European Wilderness Society, an environmental advocacy non‐profit organization headquartered in Tamsweg, Austria. “Goals are important, but do not create change without an organized and sustainable implementation. It''s a good and ambitious document, but what is also obvious is the lack of strategy of how to implement it, and a lack of discussion of why previous documents of this type failed” (https://wilderness‐society.org/ambitious‐eu‐biodiversity‐strategy‐2030/).

Alongside the voices of praise, however, many have expressed concerns that the strategy could turn into a little more than a wish list.

The Institute for European Environmental Policy (IEEP) is on the same page. The sustainability think‐tank based in Brussels and London noted that the outgoing EU 2020 biodiversity strategy showed major implementation problems, especially because of lack of engagement at national level and of ad hoc legislation supporting the meeting of key targets. Therefore, “[it] can be argued that a legally binding approach to the biodiversity governance framework is urgently needed unless Member States and other key stakeholders can show greater intrinsic ownership to deliver on agreed objectives”, (https://ieep.eu/news/first‐impressions‐of‐the‐eu‐biodiversity‐strategy‐to‐2030). In addition, IEEP remarked that money is an issue, since the €20 billion figure appears more as an estimate than a certified obligation.“The intentions of the Commission are good and the strategy contains a number of measures and targets that can really make a difference. However, implementation depends critically on the member states and experiences with the Common Agricultural Policy the past decade or so have taught us that many of them are more interested in short‐term economic objectives than in safeguarding the natural wealth of their country for future generations”, commented David Kleijn, an ecologist and nature conservation expert at the Wageningen University, the Netherlands. “I think it is important that we now have an ambitious Biodiversity Strategy but at the same time I have little hope that we will be able to achieve its objectives”.

I think it is important that we now have an ambitious Biodiversity Strategy but at the same time I have little hope that we will be able to achieve its objectives.

There is further criticism against specific measures, such as the proposal of planting 3 billion trees. “To have lots of trees planted in an area does not necessarily translate into an increase of biodiversity. Biodiverse ecosystems are the result of million years of complex multi‐species interactions and evolutionary processes, which are not as easy to restore”, explained plant ecologist Susana Gómez‐González, from the University of Cádiz, Spain. Planting a large number of trees is a too simplistic approach for saving European forests from the combined effects of excessive anthropic pressure and climate change, and could even have detrimental effects (see Box 1). More emphasis should be placed instead in reducing tree harvesting in sensitive areas and in promoting natural forest renewal processes (Gómez‐González et al, 2020). “For a biodiversity strategy, increasing the number of trees, or even increasing the forest area, should not be an objective; priority should be given to the conservation and restoration of natural ecosystems, forests and non‐forests”, Gómez‐González said.In other cases, it could be difficult, if not impossible, to reach some of the goals because of lack of information. For example, one of the roadmap''s targets is to restore at least 25,000 km of Europe''s rivers back to free‐flowing state. However, the number of barriers dispersed along European rivers will probably prevent even getting close to the mark. An international research team has collected detailed information on existing instream barriers for 147 rivers in 36 European countries, coming up with the impressive figure of over 1.2 million obstacles that inevitably impact on river ecosystems, affecting the transport and dispersion of aquatic organisms, nutrients, and sediments (Belletti et al, 2020). Existing inventories mainly focused on dams and other large barriers, while, in fact, a large number of artificial structures are much smaller, such like weirs, locks, ramps, and fords. As a result, river fragmentation has been largely underestimated, and the models used to plan flow restoration might be seriously flawed. “To avoid ‘death by a thousand cuts’, a paradigm shift is necessary: to recognize that although large dams may draw most of the attention, it is the small barriers that collectively do most of the damage. Small is not beautiful”, concluded the authors (Belletti et al, 2020).

Box 1: Why many trees don''t (always) make a forestForests are cathedrals of biodiversity. They host by far the largest number of species on land, which provide food and essential resources for hundreds of millions of people worldwide. However, forests are disappearing and degrading at an alarming pace. The loss of these crucial ecosystems has given new impulses to a variety of projects aimed at stopping this devastation and possibly reversing the trend.Once it is gone, can you rebuild a forest? Many believe the answer is yes, and the obvious solution is to plant trees. Several countries have thus launched massive tree‐planting programs, notably India and Ethiopia, where 350 million trees have been planted in single day (https://www.unenvironment.org/news‐and‐stories/story/ethiopia‐plants‐over‐350‐million‐trees‐day‐setting‐new‐world‐record). The World Economic Forum has set up its own One Trillion Tree initiative (https://www.1t.org/) “to conserve, restore, and grow one trillion trees by 2030”. Launched in January last year at Davos, 1t.org was conceived as a platform for governments, companies and NGOs/civil society groups to support the UN Decade on Ecosystem Restoration (2021–2030). The initiative has been christened by renowned naturalist Jane Goodall, who commented: “1t.org offers innovative technologies which will serve to connect tens of thousands of small and large groups around the world that are engaged in tree planting and forest restoration”, (https://www.weforum.org/agenda/2020/01/one‐trillion‐trees‐world‐economic‐forum‐launches‐plan‐to‐help‐nature‐and‐the‐climate/).However, things are way more complicated than they appear: large‐scale tree planting schemes are rarely a viable solution and can even be harmful. “[A] large body of literature shows that even the best planned restoration projects rarely fully recover the biodiversity of intact forests, owing to a lack of sources of forest‐dependent flora and fauna in deforested landscapes, as well as degraded abiotic conditions resulting from anthropogenic activities”, commented Karen Holl from the University of Caliornia, Santa Cruz, and Pedro Brancalion from the University of São Paulo (Holl & Brancalion, 2020). A common problem of tree plantations, for example, is the low survival rate of seedlings, mostly because the wrong tree species are selected and due to poor maintenance after planting. Moreover, grasslands and savannas, which are often targeted for establishing new forests, are themselves treasure troves of biodiversity. Ending indiscriminate deforestation, improving the protection of existing forests, and promoting their restoration would therefore be a more efficient strategy to preserve biodiversity in the shorter term. If tree planting is indeed necessary, it should be well planned by selecting the right areas for reforestation, using suitable tree species that can maximize biodiversity, and involving local populations to maintain the plantations, Holl and Brancalion argue (Holl & Brancalion, 2020).

…even the best planned restoration projects rarely fully recover the biodiversity of intact forests, owing to a lack of sources of forest‐dependent flora and fauna in deforested landscapes…

The health of soil, where a high proportion of biodiversity is hosted, is another problem the new strategy should address in a more focused manner. “In my opinion, the EU Biodiversity Strategy is already a leap forward in terms of policy interest in soils in general and in soil biodiversity in particular. Compared with other nations/regions of the world, Europe is by far in the forefront regarding this issue”, commented Carlos António Guerra at the German Centre for Integrative Biodiversity Research (iDiv) in Leipzig, Germany, and Co‐leader of the Global Soil Biodiversity Observation Network (https://geobon.org/bons/thematic‐bon/soil‐bon/). “Nevertheless, the connection between soil biodiversity and ecological functions needs further commitments. Soils allow for horizontal integration of several policy agendas, from climate to agriculture and, very importantly, nature conservation. This is not explicit in the EU Biodiversity Strategy in regard to soils”. It remains to be seen if EU restoration plan will emphasize soil biodiversity, or consider it as a mere side effect of other initiatives, Guerra added. “A soil nature conservation plan should be proposed”, he said. “Only such a plan, that implies that current and future protected areas have to consider, describe and protect their soil biodiversity would make a significant push to help protect such a valuable resource”.More generally, research shows that the current paradigm of protection must be shifted to prevent further losses to biodiversity. In fact, an analysis of LIFE projects—a cornerstone of EU nature protection—found that conservation efforts are extremely polarized and strongly taxonomically biased (Mammola et al, 2020). From 1992 to 2018, investment in vertebrates was sixfold higher than that for invertebrates, with birds and mammals alone accounting for 72% of the targeted species and 75% of the total budget. In relative terms, investment per species for vertebrates has been 468 times higher than for invertebrates (Fig 4). There is no sound scientific reasoning behind this uneven conservation attention, but just popularity. “[T]he species covered by a greater number of LIFE projects were also those which attracted the most interest online, suggesting that conservation in the EU is largely driven by species charisma, rather than objective features”, the researchers wrote (Mammola et al, 2020).Open in a separate windowFigure 4Taxonomic bias in EU fauna protection effortsBreakdown of the number of projects (A) and budget allocation (B) across main animal groups covered by the LIFE projects (n = 835). (C) The most covered 30 species of vertebrates (out of 410) and invertebrates (out of 78) in the LIFE projects analyzed (n = 835). The vertical bar represents monetary investment and the blue scatter line the number of LIFE projects devoted to each species. Reproduced from (Mammola et al, 2020), with permission.     

Portugal’s prominence in malaria research: How a small country became a key player in European research on one of the world’s deadliest diseases

Despite its limited resources, Portugal has gained a prominent position in research on malaria. Several historical and personal factors have contributed to this achievement. Subject Categories: S&S: Economics & Business, S&S: History & Philosophy of Science, Microbiology, Virology & Host Pathogen Interaction

Despite a significant increase that started during the 1990s, Portugal’s scientific production remains rather modest compared with the overall research output in the European Union (EU). However, the country’s achievements in malaria research are truly remarkable and, in relative terms, far above its EU neighbors in most relevant accounts. The factors to explain this accomplishment include the fact that malaria was autochthonous in Portugal until 1973; the country’s colonial history and its close ties with its former colonies; and several outstanding scientists who each inspired generations of malariologists.For most of the 20th century, research in Portugal was underfunded, and the country’s overall contribution to science was modest at best. This started to change when Portugal joined the European Union (then the European Economic Community) in 1985 and gained further momentum in the 1990s with the creation of a dedicated Ministry of Science. As a consequence, the Portuguese scientific production increased significantly in terms of the number of scientific articles published. Nevertheless, public funding for research has remained well below that of many other EU countries, and far from the target of 3% of the country’s GDP, which limits Portugal’s overall scientific output. Yet, there is one field of research where Portugal has been making significant contributions, even long before 1985: malaria.

… there is one field of research where Portugal has been making significant contributions, even long before 1985: malaria.

Among many other achievements, Portuguese laboratories have delivered important contributions to malaria research in areas as diverse as drug development, discovery and repurposing, genetic diversity of Plasmodium parasites, mechanisms of drug resistance, co‐infection between Plasmodium and other parasites, host–Plasmodium interactions, nutrient sensing and acquisition by malaria parasites, modulation of Plasmodium liver infection, immune and inflammatory responses to Plasmodium infection, diagnosis, vaccines, the role of microbiota on malaria transmission, pathogenesis of placental and cerebral malaria and acute lung injury, mechanisms of tolerance to malaria, malaria epidemiology, and vector genetics (see Further Reading for examples). Portugal’s percentage of scientific papers published in the field of malaria during the past decade relative to the total number of published articles is the highest in the EU (Fig 1A). Naturally, Portugal cannot compete with larger or more affluent countries in terms of the absolute numbers of articles published on malaria. Yet, the country ranks 5^th in this regard, closely following the Netherlands, Belgium, Sweden, and Denmark, four countries that have been investing much more and much longer in scientific research (Fig 1B). In fact, if one takes into account the funding for R&D in the EU nations, Portugal ranks ahead of every other country in terms of the number of malaria papers published relative to the investment made in science at the national level (Fig 1C).Open in a separate windowFigure 1Malaria research in Portugal and in the EU(A) Percentage of papers on the subject of malaria relative to the total number of papers from each of the indicated countries from 2009 to present. (B) Number of malaria research articles per 1,000 researchers in each of the indicated countries. (C) Number of malaria research articles per 100,000 Euros of gross domestic expenditure on R&D in each of the indicated countries. Total R&D personnel and intramural R&D expenditure data are from 2017. Papers were quantified through searches of PubMed for articles with affiliation to each of the indicated countries, published from 2009 to present, by use of the terms “malaria” or “Plasmodium”. Data on R&D investments from Eurostat.This raises the question of why Portugal, a rather small country with only a few decades of research history and an overall moderate scientific performance, fares relatively so well when it comes to research on malaria. I argue that there are three independent, albeit interrelated factors to explain this feat.A lasting reality demanding an appropriate responseThe first factor was the presence of autochthonous malaria in Portugal until the second half of the 20th century and the establishment of research institutions largely dedicated to studying and fighting the disease. Until the end of the World War II, malaria was endemic throughout much of Southern Europe; Italy, Greece, and Portugal were particularly affected. From 1955 to 1969, the WHO conducted its Global Malaria Eradication Programme, which successfully eliminated malaria in several regions of the world, including Southern Europe. The specific history of malaria eradication in Portugal is described in great detail by Bruce‐Chwatt (Bruce‐Chwatt, 1977) and highlights the intense efforts by multiple state‐sponsored institutions dedicated to studying and combating the disease.Even before the war, in 1931, the Malaria Research Station (Estação Experimental de Combate ao Sezonismo, EECS) was created in Benavente, the goals of which included the collection and analysis of blood samples from infected individuals, treatment of malaria patients, identification of mosquito populations, and malaria prophylaxis. In 1938, the Malaria Institute (Instituto de Malariologia, iMal) was founded in Águas de Moura to investigate the epidemiology of the disease, promoting adequate treatment and implementing vector control measures (Saavedra, 2010). Nonetheless, it was not until 1973 that malaria was eventually eliminated in Portugal, three years after Italy, and only one year before Greece.

… it was not until 1973 that malaria was eventually eliminated in Portugal, three years after Italy, and only one year before Greece.

Yet, the threat of malaria reemergence meant ongoing vigilance, and iMal paved the way for the creation of the Centre for the Study of Malaria and Parasitology (Centro de Estudos de Malária e Parasitologia), in 1973, later to become the Centre for the Study of Zoonoses (Centro de Estudos de Zoonoses) in 1987, and the Centre for Vector and Infectious Disease Studies (Centro de Estudos de Vetores e Doenças Infeciosas) in 1993. In addition, the Portuguese School of Tropical Medicine (later called National School of Public Health and Tropical Medicine, ENSPMT, now the Institute of Tropical Medicine and Hygiene, IHMT), founded in 1902, was one of only four institution of its kind in the world (Amaral, 2008). Since its inception, its mission has been the teaching and research in tropical medicine, biomedical sciences, and international health and, to this day, a significant part of its research continues to focus on malaria.A close bond with AfricaAnother major factor for Portugal’s prominent position in malaria research is its colonial past and the country’s close ties with its former colonies. During its period of maritime expansion in the 15^th and 16^th centuries, Portugal colonized many territories from Asia to the Americas and Africa. Most, if not all, of these territories were, and for a large part still are, endemic for malaria. Former colonies, such as Brazil or the Portuguese territories in India, gained their independence during the 19^th century, but maintained close ties with Portugal.However, several African countries, specifically Angola, Cape Verde, Guinea‐Bissau, Mozambique, and S. Tomé & Príncipe, remained under Portuguese rule until well into the second half of the 20^th century (Miller, 1975). In fact, while most African nations gained their independence from European countries during the 1950s and 1960s, Portugal’s dictatorship held on to and suppressed its African overseas territories, which led to armed uprisings in Angola and Guinea‐Bissau in 1961, and in Mozambique in 1964 (Miller, 1975). During the ensuing colonial wars, thousands of Portuguese soldiers were sent to these countries, where they were exposed not only to the horrors of war, but also to malaria (Campos, 2017). The Portuguese military actions in Africa finally came to an end in 1974 after the peaceful Carnation Revolution, which established democracy in Portugal and ended the colonization of all Portuguese‐held African territories.Over the next few years, hundreds of thousands of military personnel and former residents of the ex‐colonies, known as “retornados”, moved back to Portugal, leading to an increase in the number of imported malaria cases (Bruce‐Chwatt, 1977). Since then, these numbers have subsided, but the close ties that Portugal maintains with its former colonies mean that travel to and from malaria‐endemic regions remains high, contributing to the prevalence of imported malaria cases (Piperaki, 2018). It also means that malaria is not such a distant threat for most Portuguese; even today, many younger people have direct contact with family members or friends who have experienced malaria, bringing the reality of this scourge closer to home than in many other EU countries.

… even today, many younger people have direct contact with family members or friends who have experienced malaria, bringing the reality of this scourge closer to home than in many other EU countries.

Remarkable and inspiring figuresThe third and final factor is the enormous and lasting influence of various uniquely inspiring figures from several generations of malaria researchers. Indeed, the history of Portuguese malaria research is rich in prominent scientists who shaped the national research landscape. Attempting to highlight specific names among the many doctors, epidemiologists, and scientists from the past and present is a naturally risky exercise that runs the risk of overlooking important figures. Nevertheless, the crucial contribution of a few representatives of four generations of Portuguese scientists is beyond dispute.Ricardo Jorge (1858–1939) was a renowned epidemiologist responsible for the 1899–1901 National Sanitary Plan, which marked the introduction of modern sanitary concepts in Portugal and changed national public health. In 1903, Jorge was the first to collect reliable and extensive data on the incidence of malaria and its seasonal distribution (JORGE, 1903). He was Portugal’s Health Inspector‐General from 1899 to 1926, succeeded by José Alberto de Faria (1888–1958), another key figure who, with the support of the Rockefeller Foundation (Saavedra, 2014), created the EECS in Benavente, the first step for advancing knowledge about malaria in Portugal (Bruce‐Chwatt, 1977).Well within the 20^th century, Francisco Cambournac (1903–1994) and Fausto Landeiro (1896–1949) were arguably the most important contributors to Portuguese malariology during that period. Following extensive training in some of the most reputed parasitology schools in Europe, Cambournac became Director of Benavente’s EECS in 1933, and Landeiro occupied that position from 1938 to 1949. Cambournac founded the iMal in Águas de Moura, serving as its Director from 1939 to 1954, and became Director of the WHO’s African region from 1954 to 1964 (Lobo, 2012).Cambournac and Landeiro published extensively on the epidemiology, entomology, and control of malaria during the 1930s and 1940s, and gave a comprehensive account of the status of the disease in Portugal during that period. Cambournac’s 237‐page long review (Cambournac, 1942) provided all the epidemiological and other data needed for future planning of control and eradication of malaria in the country, the success of which is widely acknowledged to his immense work (Bruce‐Chwatt, 1977).During the 1960s and early 1970s, the National School of Public Health and Tropical Medicine, ENSPMT, now the Institute of Tropical Medicine and Hygiene, IHMT, played an important role not only in Portuguese research on malaria and other tropical diseases, but also in the cooperation with Portugal’s overseas territories at the time. The 1974 revolution and the decolonization in Africa led to a reshaping of this cooperation, which became increasingly centered on reinforcing the newly independent countries’ health systems, on their capacity to carry out research on endemic diseases, and on training programs in tropical and preventive medicine (Havik, 2015). Virgílio do Rosário, professor at the IHMT and, later, head of the Institute’s Centre for Malaria and Other Tropical Diseases (CMDT), played a pivotal role in this process. Do Rosário was the founder of several national and international networks for studying malaria and neglected diseases in various regions around the world. He inspired a whole generation of future malaria researchers, making him an inescapable figure among Portuguese malariologists in the second half of the 20^th century.At the dawn of the 21^st century, many Portuguese scientists, who had benefitted from the country’s investment in science in the 1980s and 1990s to acquire international training, came back home to set up their own research groups. Among them was Maria Mota, who returned from New York University to Portugal in 2002 to become a group leader, initially at the Instituto Gulbenkian de Ciência (IGC), and subsequently at the Instituto de Medicina Molecular (iMM). Mota’s research on the liver stage of infection by Plasmodium parasites has had an enormous impact and yielded a plethora of outstanding publications. She became Director of iMM in 2014, and commonly features among the most influential women in Portugal. Mota is also a gifted and engaging communicator, who has helped to garner public attention to malaria research and to the fight against the disease. As a great scientist and public advocate for malaria research, Mota has inspired numerous scientists, several of whom have become independent malaria researchers themselves, both in Portugal and internationally.

As a great scientist and public advocate for malaria research, Mota has inspired numerous scientists, several of whom have become independent malaria researchers themselves…

These historical, epidemiological, and humane factors have made Portugal an important player in malaria research, from the basic science of the parasite to the pathology of the disease, and from epidemiology to clinical research and drug development. However, these great achievements, and the role played by individual inspiring scientists, should not be taken for granted, but rather serve as an argument for nurturing and supporting research on malaria by future generations of scientists and political decision‐makers. A small country with fairly limited financial and human resources cannot reasonably aspire to excel in every area of research, but it can efficiently direct and focus its investment on those that are more likely to generate success. The history of Portuguese malaria research clearly demonstrates this and warrants its continued support as a top priority for national science policies.Further ReadingImportant contributions to malaria research by Portuguese laboratories during the past decade Drug development, discovery and repurposing Oliveira R, Guedes RC, Meireles P, Albuquerque IS, Goncalves LM, Pires E, Bronze MR, Gut J, Rosenthal PJ, Prudencio M, Moreira R, O''Neill PM, Lopes F (2014) Tetraoxane‐pyrimidine nitrile hybrids as dual stage antimalarials. J Med Chem 57: 4916–4923da Cruz FP, Martin C, Buchholz K, Lafuente‐Monasterio MJ, Rodrigues T, Sonnichsen B, Moreira R, Gamo FJ, Marti M, Mota MM, Hannus M, Prudencio M (2012) Drug screen targeted at Plasmodium liver stages identifies a potent multistage antimalarial drug. J Infect Dis 205: 1278–1286Hanson KK, Ressurreicao AS, Buchholz K, Prudencio M, Herman‐Ornelas JD, Rebelo M, Beatty WL, Wirth DF, Hanscheid T, Moreira R, Marti M, Mota MM (2013) Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins. Proc Natl Acad Sci USA 110: E2838–E2847Machado M, Sanches‐Vaz M, Cruz JP, Mendes AM, Prudencio M (2017) Inhibition of Plasmodium Hepatic Infection by Antiretroviral Compounds. Front Cell Infect Microbiol 7: 329 Genetic diversity of Plasmodium parasites Guerra M, Neres R, Salgueiro P, Mendes C, Ndong‐Mabale N, Berzosa P, de Sousa B, Arez AP (2017) Plasmodium falciparum Genetic Diversity in Continental Equatorial Guinea before and after Introduction of Artemisinin‐Based Combination Therapy. Antimicrob Agents Chemother 61Mendes C, Salgueiro P, Gonzalez V, Berzosa P, Benito A, do Rosario VE, de Sousa B, Cano J, Arez AP (2013) Genetic diversity and signatures of selection of drug resistance in Plasmodium populations from both human and mosquito hosts in continental Equatorial Guinea. Malar J 12: 114 Mechanisms of drug resistance Escobar C, Pateira S, Lobo E, Lobo L, Teodosio R, Dias F, Fernandes N, Arez AP, Varandas L, Nogueira F (2015) Polymorphisms in Plasmodium falciparum K13‐propeller in Angola and Mozambique after the introduction of the ACTs. PLoS One 10: e0119215Ferreira A, Marguti I, Bechmann I, Jeney V, Chora A, Palha NR, Rebelo S, Henri A, Beuzard Y, Soares MP (2011) Sickle hemoglobin confers tolerance to Plasmodium infection. Cell 145: 398–409Veiga MI, Osorio NS, Ferreira PE, Franzen O, Dahlstrom S, Lum JK, Nosten F, Gil JP (2014) Complex polymorphisms in the Plasmodium falciparum multidrug resistance protein 2 gene and its contribution to antimalarial response. Antimicrob Agents Chemother 58: 7390–7397 Host‐Plasmodium interactions Portugal S, Carret C, Recker M, Armitage AE, Goncalves LA, Epiphanio S, Sullivan D, Roy C, Newbold CI, Drakesmith H, Mota MM (2011) Host‐mediated regulation of superinfection in malaria. Nat Med 17: 732–737Real E, Rodrigues L, Cabal GG, Enguita FJ, Mancio‐Silva L, Mello‐Vieira J, Beatty W, Vera IM, Zuzarte‐Luis V, Figueira TN, Mair GR, Mota MM (2018) Plasmodium UIS3 sequesters host LC3 to avoid elimination by autophagy in hepatocytes. Nat Microbiol 3: 17–25Sa ECC, Nyboer B, Heiss K, Sanches‐Vaz M, Fontinha D, Wiedtke E, Grimm D, Przyborski JM, Mota MM, Prudencio M, Mueller AK (2017) Plasmodium berghei EXP‐1 interacts with host Apolipoprotein H during Plasmodium liver‐stage development. Proc Natl Acad Sci USA 114: E1138–E1147 Nutrient sensing and acquisition Itoe MA, Sampaio JL, Cabal GG, Real E, Zuzarte‐Luis V, March S, Bhatia SN, Frischknecht F, Thiele C, Shevchenko A, Mota MM (2014) Host cell phosphatidylcholine is a key mediator of malaria parasite survival during liver stage infection. Cell Host Microbe 16: 778–786Mancio‐Silva L, Slavic K, Grilo Ruivo MT, Grosso AR, Modrzynska KK, Vera IM, Sales‐Dias J, Gomes AR, MacPherson CR, Crozet P, Adamo M, Baena‐Gonzalez E, Tewari R, Llinas M, Billker O, Mota MM (2017) Nutrient sensing modulates malaria parasite virulence. Nature 547: 213–216Meireles P, Mendes AM, Aroeira RI, Mounce BC, Vignuzzi M, Staines HM, Prudencio M (2017) Uptake and metabolism of arginine impact Plasmodium development in the liver. Sci Rep 7: 4072 Modulation of Plasmodium liver infection Ruivo MTG, Vera IM, Sales‐Dias J, Meireles P, Gural N, Bhatia SN, Mota MM, Mancio‐Silva L (2016) Host AMPK Is a Modulator of Plasmodium Liver Infection. 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