Growth‐dependent heterogeneity in the DNA damage response in Escherichia coli

In natural environments, bacteria are frequently exposed to sub‐lethal levels of DNA damage, which leads to the induction of a stress response (the SOS response in Escherichia coli). Natural environments also vary in nutrient availability, resulting in distinct physiological changes in bacteria, which may have direct implications on their capacity to repair their chromosomes. Here, we evaluated the impact of varying the nutrient availability on the expression of the SOS response induced by chronic sub‐lethal DNA damage in E. coli. We found heterogeneous expression of the SOS regulon at the single‐cell level in all growth conditions. Surprisingly, we observed a larger fraction of high SOS‐induced cells in slow growth as compared with fast growth, despite a higher rate of SOS induction in fast growth. The result can be explained by the dynamic balance between the rate of SOS induction and the division rates of cells exposed to DNA damage. Taken together, our data illustrate how cell division and physiology come together to produce growth‐dependent heterogeneity in the DNA damage response.     

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4‐amino‐4,6‐dideoxy‐d‐glucose, also known as d‐viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide‐linked sugar, which in the Mimivirus is thought to be UDP‐d‐glucose. The enzyme required for the installment of the amino group at the C‐4′ position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5′‐phosphate‐dependent enzyme, referred to as L136. For this analysis, three high‐resolution X‐ray structures were determined: the wildtype enzyme/pyridoxamine 5′‐phosphate/dTDP complex and the site‐directed mutant variant K185A in the presence of either UDP‐4‐amino‐4,6‐dideoxy‐d‐glucose or dTDP‐4‐amino‐4,6‐dideoxy‐d‐glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP‐d‐glucose or dTDP‐d‐glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three‐dimensional architecture was previously reported by this laboratory. As determined in this investigation,DesI shows a profound preference in its catalytic efficiency for the dTDP‐linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three‐dimensional model for a virally encoded PLP‐dependent enzyme and thus provides new information on sugar aminotransferases in general.     

Tracking exogenous intracellular casp‐3 using split GFP

Cytosolic protein delivery promises diverse applications from therapeutics, to genetic modification and precision research tools. To achieve effective cellular and subcellular delivery, approaches that allow protein visualization and accurate localization with greater sensitivity are essential. Fluorescently tagging proteins allows detection, tracking and visualization in cellulo. However, undesired consequences from fluorophores or fluorescent protein tags, such as nonspecific interactions and high background or perturbation to native protein''s size and structure, are frequently observed, or more troublingly, overlooked. Distinguishing cytosolically released molecules from those that are endosomally entrapped upon cellular uptake is particularly challenging and is often complicated by the inherent pH‐sensitive and hydrophobic properties of the fluorophore. Monitoring localization is more complex in delivery of proteins with inherent protein‐modifying activities like proteases, transacetylases, kinases, etc. Proteases are among the toughest cargos due to their inherent propensity for self‐proteolysis. To implement a reliable, but functionally silent, tagging technology in a protease, we have developed a caspase‐3 variant tagged with the 11th strand of GFP that retains both enzymatic activity and structural characteristics of wild‐type caspase‐3. Only in the presence of cytosolic GFP strands 1–10 will the tagged caspase‐3 generate fluorescence to signal a non‐endosomal location. This methodology facilitates easy screening of cytosolic vs. endosomally‐entrapped proteins due to low probabilities for false positive results, and further, allows tracking of the resultant cargo''s translocation. The development of this tagged casp‐3 cytosolic reporter lays the foundation to probe caspase therapeutic properties, charge–property relationships governing successful escape, and the precise number of caspases required for apoptotic cell death.     

Molecular basis of cross‐species ACE2 interactions with SARS‐CoV‐2‐like viruses of pangolin origin

Pangolins have been suggested as potential reservoir of zoonotic viruses, including SARS‐CoV‐2 causing the global COVID‐19 outbreak. Here, we study the binding of two SARS‐CoV‐2‐like viruses isolated from pangolins, GX/P2V/2017 and GD/1/2019, to human angiotensin‐converting enzyme 2 (hACE2), the receptor of SARS‐CoV‐2. We find that the spike protein receptor‐binding domain (RBD) of pangolin CoVs binds to hACE2 as efficiently as the SARS‐CoV‐2 RBD in vitro. Furthermore, incorporation of pangolin CoV RBDs allows entry of pseudotyped VSV particles into hACE2‐expressing cells. A screen for binding of pangolin CoV RBDs to ACE2 orthologs from various species suggests a broader host range than that of SARS‐CoV‐2. Additionally, cryo‐EM structures of GX/P2V/2017 and GD/1/2019 RBDs in complex with hACE2 show their molecular binding in modes similar to SARS‐CoV‐2 RBD. Introducing the Q498H substitution found in pangolin CoVs into the SARS‐CoV‐2 RBD expands its binding capacity to ACE2 homologs of mouse, rat, and European hedgehog. These findings suggest that these two pangolin CoVs may infect humans, highlighting the necessity of further surveillance of pangolin CoVs.     

Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses

Super‐resolution imaging has revealed that key synaptic proteins are dynamically organized within sub‐synaptic domains (SSDs). To examine how different inhibitory receptors are regulated, we carried out dual‐color direct stochastic optical reconstruction microscopy (dSTORM) of GlyRs and GABA_ARs at mixed inhibitory synapses in spinal cord neurons. We show that endogenous GlyRs and GABA_ARs as well as their common scaffold protein gephyrin form SSDs that align with pre‐synaptic RIM1/2, thus creating trans‐synaptic nanocolumns. Strikingly, GlyRs and GABA_ARs occupy different sub‐synaptic spaces, exhibiting only a partial overlap at mixed inhibitory synapses. When network activity is increased by 4‐aminopyridine treatment, the GABA_AR copy numbers and the number of GABA_AR SSDs are reduced, while GlyRs remain largely unchanged. This differential regulation is likely the result of changes in gephyrin phosphorylation that preferentially occurs outside of SSDs. The activity‐dependent regulation of GABA_ARs versus GlyRs suggests that different signaling pathways control the receptors'' sub‐synaptic clustering. Taken together, our data reinforce the notion that the precise sub‐synaptic organization of GlyRs, GABA_ARs, and gephyrin has functional consequences for the plasticity of mixed inhibitory synapses.     

Concordant geographic and genetic structure revealed by genotyping‐by‐sequencing in a New Zealand marine isopod

Population genetic structure in the marine environment can be influenced by life‐history traits such as developmental mode (biphasic, with distinct adult and larval morphology, and direct development, in which larvae resemble adults) or habitat specificity, as well as geography and selection. Developmental mode is thought to significantly influence dispersal, with direct developers expected to have much lower dispersal potential. However, this prediction can be complicated by the presence of geophysical barriers to dispersal. In this study, we use a panel of 8,020 SNPs to investigate population structure and biogeography over multiple spatial scales for a direct‐developing species, the New Zealand endemic marine isopod Isocladus armatus. Because our sampling range is intersected by two well‐known biogeographic barriers (the East Cape and the Cook Strait), our study provides an opportunity to understand how such barriers influence dispersal in direct developers. On a small spatial scale (20 km), gene flow between locations is extremely high, suggestive of an island model of migration. However, over larger spatial scales (600 km), populations exhibit a clear pattern of isolation‐by‐distance. Our results indicate that I. armatus exhibits significant migration across the hypothesized barriers and suggest that large‐scale ocean currents associated with these locations do not present a barrier to dispersal. Interestingly, we find evidence of a north‐south population genetic break occurring between Māhia and Wellington. While no known geophysical barrier is apparent in this area, it coincides with the location of a proposed border between bioregions. Analysis of loci under selection revealed that both isolation‐by‐distance and adaption may be contributing to the degree of population structure we have observed here. We conclude that developmental life history largely predicts dispersal in the intertidal isopod I. armatus. However, localized biogeographic processes can disrupt this expectation, and this may explain the potential meta‐population detected in the Auckland region.     

Nonproductive exposure of PBMCs to SARS‐CoV‐2 induces cell‐intrinsic innate immune responses

Cell‐intrinsic responses mounted in PBMCs during mild and severe COVID‐19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS‐CoV and SARS‐CoV‐2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT–PCR experiments and single‐cell RNA sequencing revealed JAK/STAT‐dependent induction of interferon‐stimulated genes (ISGs) but not proinflammatory cytokines. This SARS‐CoV‐2‐specific response was most pronounced in monocytes. SARS‐CoV‐2‐RNA‐positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS‐CoV‐2‐specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS‐CoV‐2‐ and, to a much lesser extent, SARS‐CoV particles stimulate JAK/STAT‐dependent, monocyte‐accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID‐19.     

Newly designed Protein Transduction Domain (PTD)‐mediated BMP‐7 is a potential therapeutic for peritoneal fibrosis

While the bone morphogenetic protein‐7 (BMP‐7) is a well‐known therapeutic growth factor reverting many fibrotic diseases, including peritoneal fibrosis by peritoneal dialysis (PD), soluble growth factors are largely limited in clinical applications owing to their short half‐life in clinical settings. Recently, we developed a novel drug delivery model using protein transduction domains (PTD) overcoming limitation of soluble recombinant proteins, including bone morphogenetic protein‐7 (BMP‐7). This study aims at evaluating the therapeutic effects of PTD‐BMP‐7 consisted of PTD and full‐length BMP‐7 on epithelial‐mesenchymal transition (EMT)‐related fibrosis. Human peritoneal mesothelial cells (HPMCs) were then treated with TGF‐β1 or TGF‐β1 + PTD‐BMP‐7. Peritoneal dialysis (PD) catheters were inserted into Sprague‐Dawley rats, and these rats were infused intra‐peritoneally with saline, peritoneal dialysis fluid (PDF) or PDF + PTD‐BMP‐7. In vitro, TGF‐β1 treatment significantly increased fibronectin, type I collagen, α‐SMA and Snail expression, while reducing E‐cadherin expression in HPMCs (P < .001). PTD‐BMP‐7 treatment ameliorated TGF‐β1‐induced fibronectin, type I collagen, α‐SMA and Snail expression, and restored E‐cadherin expression in HPMCs (P < .001). In vivo, the expressions of EMT‐related molecules and the thickness of the sub‐mesothelial layer were significantly increased in the peritoneum of rats treated with PDF, and these changes were significantly abrogated by the intra‐peritoneal administration of PTD‐BMP‐7. PTD‐BMP‐7 treatment significantly inhibited the progression of established PD fibrosis. These findings suggest that PTD‐BMP‐7, as a prodrug of BMP‐7, can be an effective therapeutic agent for peritoneal fibrosis in PD patients.     

Rivers,not refugia,drove diversification in arboreal,sub‐Saharan African snakes

The relative roles of rivers versus refugia in shaping the high levels of species diversity in tropical rainforests have been widely debated for decades. Only recently has it become possible to take an integrative approach to test predictions derived from these hypotheses using genomic sequencing and paleo‐species distribution modeling. Herein, we tested the predictions of the classic river, refuge, and river‐refuge hypotheses on diversification in the arboreal sub‐Saharan African snake genus Toxicodryas. We used dated phylogeographic inferences, population clustering analyses, demographic model selection, and paleo‐distribution modeling to conduct a phylogenomic and historical demographic analysis of this genus. Our results revealed significant population genetic structure within both Toxicodryas species, corresponding geographically to river barriers and divergence times from the mid‐Miocene to Pliocene. Our demographic analyses supported the interpretation that rivers are indications of strong barriers to gene flow among populations since their divergence. Additionally, we found no support for a major contraction of suitable habitat during the last glacial maximum, allowing us to reject both the refuge and river‐refuge hypotheses in favor of the river‐barrier hypothesis. Based on conservative interpretations of our species delimitation analyses with the Sanger and ddRAD data sets, two new cryptic species are identified from east‐central Africa. This study highlights the complexity of diversification dynamics in the African tropics and the advantages of integrative approaches to studying speciation in tropical regions.     

Predictable fold switching by the SARS‐CoV‐2 protein ORF9b

Extant fold‐switching proteins remodel their secondary structures and change their functions in response to environmental stimuli. These shapeshifting proteins regulate biological processes and are associated with a number of diseases, including tuberculosis, cancer, Alzheimer''s, and autoimmune disorders. Thus, predictive methods are needed to identify more fold‐switching proteins, especially since all naturally occurring instances have been discovered by chance. In response to this need, two high‐throughput predictive methods have recently been developed. Here we test them on ORF9b, a newly discovered fold switcher and potential therapeutic target from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). Promisingly, both methods correctly indicate that ORF9b switches folds. We then tested the same two methods on ORF9b1, the ORF9b homolog from SARS‐CoV‐1. Again, both methods predict that ORF9b1 switches folds, a finding consistent with experimental binding studies. Together, these results (a) demonstrate that protein fold switching can be predicted using high‐throughput computational approaches and (b) suggest that fold switching might be a general characteristic of ORF9b homologs.     

TNF‐α/IFN‐γ synergy amplifies senescence‐associated inflammation and SARS‐CoV‐2 receptor expression via hyper‐activated JAK/STAT1

Older age and underlying conditions such as diabetes/obesity or immunosuppression are leading host risk factors for developing severe complications from COVID‐19 infection. The pathogenesis of COVID‐19‐related cytokine storm, tissue damage, and fibrosis may be interconnected with fundamental aging processes, including dysregulated immune responses and cellular senescence. Here, we examined effects of key cytokines linked to cellular senescence on expression of SARS‐CoV‐2 viral entry receptors. We found exposure of human umbilical vein endothelial cells (HUVECs) to the inflammatory cytokines, TNF‐α + IFN‐γ or a cocktail of TNF‐α + IFN‐γ + IL‐6, increased expression of ACE2/DPP4, accentuated the pro‐inflammatory senescence‐associated secretory phenotype (SASP), and decreased cellular proliferative capacity, consistent with progression towards a cellular senescence‐like state. IL‐6 by itself failed to induce substantial effects on viral entry receptors or SASP‐related genes, while synergy between TNF‐α and IFN‐γ initiated a positive feedback loop via hyper‐activation of the JAK/STAT1 pathway, causing SASP amplification. Breaking the interactive loop between senescence and cytokine secretion with JAK inhibitor ruxolitinib or antiviral drug remdesivir prevented hyper‐inflammation, normalized SARS‐CoV‐2 entry receptor expression, and restored HUVECs proliferative capacity. This loop appears to underlie cytokine‐mediated viral entry receptor activation and links with senescence and hyper‐inflammation.     

Ancestral ecological regime shapes reaction to food limitation in the Least Killifish,Heterandria formosa

Populations with different densities often show genetically based differences in life histories. The divergent life histories could be driven by several agents of selection, one of which is variation in per‐capita food levels. Its relationship with population density is complex, as it depends on overall food availability, individual metabolic demand, and food‐independent factors potentially affecting density, such as predation intensity. Here, we present a case study of two populations of a small live‐bearing freshwater fish, one characterized by high density, low predation risk, low overall food availability, and presumably low per‐capita food levels, and the other by low density, high predation risk, high overall food availability, and presumably high per‐capita food levels. Using a laboratory experiment, we examined whether fish from these populations respond differently to food limitation, and whether size at birth, a key trait with respect to density variation in this species, is associated with any such differential responses. While at the lower food level growth was slower, body size smaller, maturation delayed, and survival reduced in both populations, these fitness costs were smaller in fish from the high‐density population. At low food, only 15% of high‐density fish died, compared to 75% of low‐density fish. This difference was much smaller at high food (0% vs. 15% mortality). The increased survival of high‐density fish may, at least partly, be due to their larger size at birth. Moreover, being larger at birth enabled fish to mature relatively early even at the lower food level. We demonstrate that sensitivities to food limitation differ between study populations, consistent with selection for a greater ability to tolerate low per‐capita food availability in the high‐density population. While we cannot preclude other agents of selection from operating in these populations simultaneously, our results suggest that variation in per‐capita food levels is one of those agents.     

Cryo‐EM structures of perforin‐2 in isolation and assembled on a membrane suggest a mechanism for pore formation

Perforin‐2 (PFN2, MPEG1) is a key pore‐forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane‐bound pre‐pore complex that converts to a pore‐forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo‐electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre‐pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre‐assembled complete pre‐pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre‐pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β‐hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre‐pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.     

Female extra‐pair behavior is not associated with reduced paternal care in Thorn‐tailed Rayadito

Extra‐pair behavior is present in 76% of socially monogamous bird species with biparental care. This behavior may produce costs to females related to a reduction in paternal care. We estimated the percentage of extra‐pair offspring and quantified paternal care in 44 nests of Thorn‐tailed Rayadito (Aphrastura spinicauda) to assess whether males reduce their parental care when females obtain extra‐pair fertilizations. We used data from a sub‐Antarctic population of Rayadito located on Navarino Island (55°4′S, 67°40′W), southern Chile. We found no statistical support for a relationship between variation in paternal care and the percentage of extra‐pair offspring. We discuss how the inability of breeding males to assess their genetic paternity and potential restrictions on behavioral flexibility may explain this result. Additionally, if paternal care is subjected to sexual selection, this could limit a facultative response to female extra‐pair behavior by males. Finally, it is possible that a reduction in paternal care might not have evolved in this particular locality given the low frequency of extra‐pair paternity in our study population.     

Evolutionary remodelling of N‐terminal domain loops fine‐tunes SARS‐CoV‐2 spike

The emergence of SARS‐CoV‐2 variants has exacerbated the COVID‐19 global health crisis. Thus far, all variants carry mutations in the spike glycoprotein, which is a critical determinant of viral transmission being responsible for attachment, receptor engagement and membrane fusion, and an important target of immunity. Variants frequently bear truncations of flexible loops in the N‐terminal domain (NTD) of spike; the functional importance of these modifications has remained poorly characterised. We demonstrate that NTD deletions are important for efficient entry by the Alpha and Omicron variants and that this correlates with spike stability. Phylogenetic analysis reveals extensive NTD loop length polymorphisms across the sarbecoviruses, setting an evolutionary precedent for loop remodelling. Guided by these analyses, we demonstrate that variations in NTD loop length, alone, are sufficient to modulate virus entry. We propose that variations in NTD loop length act to fine‐tune spike; this may provide a mechanism for SARS‐CoV‐2 to navigate a complex selection landscape encompassing optimisation of essential functionality, immune‐driven antigenic variation and ongoing adaptation to a new host.     

An efficient and precise method for generating knockout cell lines based on CRISPR‐Cas9 system

Although the efficiency and versatility of CRISPR‐Cas9 system has been greatly improved over conventional genome editing methods such as zinc finger or TALEN, it is still time‐consuming and labor‐intensive for screening knockout/knock‐in cell clones due to differences of the targeted location or efficacies of guide RNAs (gRNAs). Here, we adapted a targeted knock‐in strategy with CRISPR‐Cas9 system and characterized the efficiency for generating single or double knockout cell lines. Specifically, a homology‐arm based donor cassette consisting of genes encoding a fluorescence protein and antibiotic selection marker driven by a constitutive promoter was co‐transfected with a gRNA expressing unit. Based on FACS sorting and antibiotic drug selection, positive cell clones were confirmed by genotyping and at the protein expression level. The results indicated that more than 70% of analyzed clones identified by cell sorting and selection were successfully targeted in both single and double knockout experiments. The procedure takes less than three weeks to obtain knockout cell lines. We believe that this methodology could be applicable and versatile in generating knockout cell clones with high efficiency in most cell lines.     

Structural basis for the N‐degron specificity of ClpS1 from Arabidopsis thaliana

The N‐degron pathway determines the half‐life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N‐terminal residue (N‐degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N‐degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N‐degrons; however, ClpS1 from Arabidopsis thaliana (AtClpS1) shows a difference in that it binds strongly to Phe and Trp N‐degrons and only weakly to Leu. This difference in behavior cannot be explained without structural information due to the high sequence homology between bacterial and plant ClpS proteins. Here, we report the structure of AtClpS1 at 2.0 Å resolution in the presence of a bound N‐degron. The key determinants for α‐amino group recognition are conserved among all ClpS proteins, but the α3‐helix of eukaryotic AtClpS1 is significantly shortened, and consequently, a loop forming a pocket for the N‐degron is moved slightly outward to enlarge the pocket. In addition, amino acid replacement from Val to Ala causes a reduction in hydrophobic interactions with Leu N‐degron. A combination of the fine‐tuned hydrophobic residues in the pocket and the basic gatekeeper at the entrance of the pocket controls the N‐degron selectivity of the plant ClpS protein.     

Age is not just a number—Mathematical model suggests senescence affects how fish populations respond to different fishing regimes

Senescence is often described as an age‐dependent increase in natural mortality (known as actuarial senescence) and an age‐dependent decrease in fecundity (known as reproductive senescence), and its role in nature is still poorly understood. Based on empirical estimates of reproductive and actuarial senescence, we used mathematical simulations to explore how senescence affects the population dynamics of Coregonus albula, a small, schooling salmonid fish. Using an empirically based eco‐evolutionary model, we investigated how the presence or absence of senescence affects the eco‐evolutionary dynamics of a fish population during pristine, intensive harvest, and recovery phases. Our simulation results showed that the presence or absence of senescence affected how the population responded to the selection regime. At an individual level, gillnetting caused a larger decline in asymptotic length when senescence was present, compared to the nonsenescent population, and the opposite occurred when fishing was done by trawling. This change was accompanied by evolution toward younger age at maturity. At the population level, the change in biomass and number of fish in response to different fishery size‐selection patterns depended on the presence or absence of senescence. Since most life‐history and fisheries models ignore senescence, they may be over‐estimating reproductive capacity and under‐estimating natural mortality. Our results highlight the need to understand the combined effects of life‐history characters such as senescence and fisheries selection regime to ensure the successful management of our natural resources.