title: Bacteriophages antagonize cGAS-like immunity in bacteria

abstract:
The recently discovered cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is related to eukaryotic cGAS-STING anti-viral immunity and is present in diverse prokaryotes. However, our understanding of how CBASS detects, inhibits, and co-evolves with phages is limited because CBASS function has only been studied in reconstituted heterologous systems. Here, we identify a phage-encoded CBASS antagonist (acbIIA1, anti-cbass type II-A gene 1) necessary for phage replication in the presence of endogenous CBASS immunity in Pseudomonas aeruginosa. acbIIA1 homologs are encoded by numerous lytic and temperate phages infecting Gram-negative bacteria. Deletion of acbIIA1 renders multiple phages susceptible to CBASS, but phages can then escape immune function via mutations in the major capsid gene. These mutants suggest that CBASS is activated by, or targets, the late-expressed phage capsid. Together, we establish a native model system to study CBASS and identify a common phage-encoded CBASS antagonist, demonstrating that CBASS is a bona fide anti-phage immune system in nature.

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title: NtrC expression at lag-phase improves competitive fitness under low and fluctuating nutrient condition

abstract:
The enteric pathogens cycle between nutrient rich host and nutrient poor external environment. These pathogens compete for nutrients in the host as well as in external environment and also often experience starvation. In this context, we have studied the role of a global nitrogen regulator (NtrC) of Salmonella Typhimurium. The ntrC mutation caused extended lag phase and slow growth in minimal media. In lag phase the wild-type cells showed [~]60 fold more expression of ntrC as compared to log phase cells. The role of ntrC gene is often studied with respect to nitrogen scavenging in a low nitrogen growing condition. However, our observation indicates that, even in the adequate supply of nitrogen, the ntrC null mutants failed to adapt to new nutrient conditions and were slow to exit from the lag phase. Gene expression studies at lag phase showed down-regulation of the genes involved in carbon/nitrogen transportation and metabolism in {Delta}ntrC mutant. In the co-culture competition studies, we observed that ntrC knockout was unable to survive with the wild-type Salmonella and E. coli. We also observed that the {Delta}ntrC mutant did not survive long term nitrogen starvation (150 days). Critical analysis of starvation survival revealed that, ntrC gene is essential for recycling of nutrients from the dead cells. The nutrient recycling efficiency of {Delta}ntrC mutant was [~] 12 times less efficient than wild-type. Hence, the current work establishes that ntrC expression at the lag phase is essential for competitive fitness of Salmonella to survive in an environment having low and fluctuating nutrient conditions.

ImportanceSalmonella Typhimurium, both inside or outside of the host organism face enormous competition from other microorganisms. The competition may take place either in steady or in fluctuating climatic conditions. Thus, how Salmonella Typhimurium survive under such overlapping stress conditions, remained unclear. Therefore, here we report that, a global regulator NtrC, which is also a part of two-component system, activates the set of genes and operons involved in rapid adaptation and efficient nutrient recycling/scavenging. These properties, make cell able to compete with other microbes, under characteristic feast- or-famine life style of Salmonella Typhimurium. Therefore, this piece of work helps us to understand the starvation physiology of enteric bacterial pathogen Salmonella Typhimurium.

category: microbiology


title: Multiethnic Polygenic Risk Prediction in Diverse Populations through Transfer Learning

abstract: Polygenic risk scores (PRS) leverage the genetic contribution of an individuals genotype by estimating disease risk. Traditional PRS prediction methods are predominantly for European population. The accuracy of PRS prediction in non-European populations is diminished due to much smaller sample size of genome-wide association studies (GWAS). In this article, we introduced a novel method to construct PRS for non-European populations, abbreviated as TL-Multi, by conducting transfer learning framework to learn useful knowledge from European population to correct the bias for non-European populations. We considered non-European GWAS data as target data and European GWAS data as informative auxiliary data. TL-Multi borrowed useful information from auxiliary data to improve the learning accuracy of the target data while preserving the efficiency and accuracy. To demonstrate the practical applicability of the proposed method, we applied TL-Multi to predict systemic lupus erythematosus (SLE) risk in Hong Kong population by borrowing information from European population. TL-Multi achieved better prediction accuracy than alternative methods including Lassosum, meta-analysis and linkage disequilibrium (LD)-informed pruning and P-values thresholding for multiethnic PRS (PT-Multi), and substantially improved the prediction performance with moderate cross-population genetic correlation in both simulations and SLE application.

category: genetics


title: GTAGMe-seq: joint profiling of genetic variants, DNA methylation, GpC methyltransferase footprints, and 3D genome in the same DNA molecules

abstract: Cis-regulatory elements (CREs) coordinate to regulate the expression of their targeted genes, up to megabases away. However, the joint measurement of CREs activities and their interactions over large genomic distances is limited by the current sequencing approaches. We report a method, GTAGMe-seq, which simultaneously captures the single nucleotide polymorphisms, DNA methylation, GpC methyltransferase footprints, and chromosome conformation changes from the same DNA molecules, together with transcriptome in the same assay. GTAGMe-seq shows high concordance with the state-of-art mono-omics assays across different molecular measurements. It reveals the coordinated GpC methyltransferase footprints at distal genomic segments in spatial proximity and different types of long-range allele-specific methyltransferase footprints.

category: genomics


title: On the Geometry of Interacting Axon Tracts in Relation to Their Functional Properties

abstract: It is well known that demyelination associated with multiple sclerosis alters coupling between axons in a diseased nerve tract due to exposure of ion channels. In this work we ask the question whether the presence of electric fields as a coupling mechanism between nerve axons, has any impact on the expected conduction properties of the nerve under focal demyelination. Using an approximate geometry for a bundle of three axons, we allow electric fields generated at a node of Ranvier to influence other nodes of Ranvier on different axons. In order to introduce focal demyelination, we allow a new type of structure, intermediate between a node and an internodal segment. We find that the nerves conduction profiles are different with and without field-based coupling. This is important because it opens up the possibility for an experimental investigation to study whether field-based coupling predominates over current-based coupling. Fields and currents may also be measured using different methods and so there are implications for bio-instrumentation. From a systems perspective, the study is based on a novel equation which allows electric fields to act as sources of current at axons nodes of Ranvier, thereby generating new behaviors from the set of coupled partial differential equations that model the electrodynamics of the nerve. In addition to our simulation study of focal demyelination, we present a justification for a certain geometric W-matrix used in our phenomenological 2019 model. We also provide the first simulations of a tract of tortuous axons, showing the impact of tract tortuosity on conduction velocity. The tortuous model is analyzed geometrically to illustrate the mechanism of synchronization. Finally, this same model is shown to be castable as a learning machine - a formulation that has neuroscience implications.

category: neuroscience


title: ACE2 engagement exposes the fusion peptide to pan-coronavirus neutralizing antibodies

abstract: Coronaviruses use diverse Spike (S) glycoproteins to attach to host receptors and fuse with target cells. Using a broad screening approach, we isolated from SARS-CoV-2 immune donors seven monoclonal antibodies (mAbs) that bind to all human alpha and beta coronavirus S proteins. These mAbs recognize the fusion peptide and acquire high affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha and beta coronaviruses, including Omicron BA.1 variant and bat WIV-1, and reduce viral titers and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope which is concealed by prefusion-stabilizing  2P mutations and becomes exposed upon binding of ACE2 or ACE2-mimicking mAbs. This study identifies a new class of pan-coronavirus neutralizing mAbs and reveals a receptor-induced conformational change in the S protein that exposes the fusion peptide region.

category: immunology


title: The Structural and Biochemical Basis for FER Receptor Kinase Early Signaling Initiation in Arabidopsis

abstract: Accumulating evidence has indicated that receptor-like kinase (RLK) autophosphorylation and substrate phosphorylation triggered by RLK are early and essential events for RLK function. However, the structural and biochemical basis for these early events is largely unclear. Herein, we used RLK FERONIA (FER) as a model and crystallized its core kinase domain (FER-KD) in the dephosphorylated state. We found that FER-KD adopts an active conformation in its crystal structure. Moreover, FER-KD mutants with reduced or no catalytic activity also adopt an active conformation before phosphorylation. Collectively, these observations suggest that FER employs a phosphorylation-independent active state before ligand-induced phosphorylation and full activation. We further demonstrated that FER is a dual-specificity kinase and that autophosphorylation on Tyr residues lags somewhat behind Ser/Thr phosphorylation. More importantly, Tyr phosphorylation is essential for FER-KD to initiate substrate GRP7 phosphorylation. Our work provides a paradigm to study the mechanisms of the early steps of RLK signaling initiation and highlights its "active" form and Tyr phosphorylation-gated roles in response to signaling stimuli.

category: biochemistry


title: Sabatier principle for rationalizing enzymatic hydrolysis of a synthetic polyester

abstract:
Interfacial enzyme reactions are common in nature and in industrial settings, including the enzymatic deconstruction of poly(ethylene terephthalate) (PET) waste. Kinetic descriptions of PET hydrolases are necessary for both comparative analyses, discussions of structure-function relations and rational optimization of technical processes. We investigated whether the Sabatier principle could be used for this purpose. Specifically, we compared the kinetics of two well-known PET hydrolases, leaf-branch compost cutinase (LCC) and a cutinase from the bacterium T. fusca (TfC) when adding different concentrations of the surfactant cetyltrimethylammonium bromide (CTAB). We found that CTAB consistently lowered the strength of enzyme-PET interactions, while its effect on enzymatic turnover was strongly biphasic. Thus, at gradually increasing CTAB concentrations, turnover was initially promoted and subsequently suppressed. This correlation with maximal turnover at an intermediate binding strength is in accordance with the Sabatier principle. One consequence of these results is that both enzymes had too strong intrinsic interaction with PET for optimal turnover, especially TfC, which showed a 20-fold improvement of kcat at the maximum. LCC on the other hand had an intrinsic substrate affinity closer to the Sabatier optimum and the turnover rate was 5-fold improved at weakened substrate binding. Our results show that the Sabatier principle may indeed rationalize enzymatic PET degradation and support process optimization. Finally, we suggest that future discovery efforts should consider enzymes with weakened substrate binding, since strong adsorption seems to limit their catalytic performance.

ToC graphics O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Endothelial Caspase-9 Promotes Glial Changes, Inflammation, and Contrast Sensitivity Decline in Retinal Vascular Injury

abstract: Retinal glial cells-- microglia, astrocytes, and Muller glia--provide homeostatic support, regulate vascular blood flow, and react to injury by releasing inflammatory cytokines. Glial reactivity has been shown to be relevant for retinal vascular pathology and neuronal death. Non-apoptotic expression of endothelial caspase-9 (EC Casp9) was recently identified as a key mediator of retinal edema, hypoxic-ischemic injury, and neurodegeneration in retinal vein occlusion (RVO). In the current study we aimed to determine the glial responses that are modulated by EC Casp9 as a means to identify relevant neuro-immune mechanisms for the development of retinal edema and neurodegeneration. To this end we used a mouse model of RVO and a tamoxifen inducible EC Casp9 KO mouse line. We show that EC Casp9 leads to an increase in reactive microglia and to macrogliosis in a time-dependent manner. RVO induced an EC Casp9 dependent astroglial caspase-6 and cleavage of GFAP. Cytokine array analysis revealed that RVO increases expression of inflammatory cytokines out of which CX3CL1, IGF-1, IL-4, LIX, IL-1, M-CSF, TNF-, IL-1{beta}, IL-10, and VEGF-A, were regulated by EC Casp9. Moreover, we found that EC Casp9 deletion resulted in protection from contrast sensitivity decline one day post-RVO. These results demonstrate that caspase-9 in hypoxic endothelial cells regulates retinal inflammatory signaling in microglia, astrocytes and Muller cells and changes in visual function.


title: Homozygous CADPS2 mutations cause neurodegenerative disease with Lewy bodies in parrots

abstract:
BackgroundSeveral genetic models that recapitulate neurodegenerative features of Parkinsons disease (PD) exist, which have been largely based on genes discovered in monogenic PD families. However, spontaneous genetic mutations have not been linked to the pathological hallmarks of PD in non-human vertebrates.

ObjectiveTo describe the genetic and pathological findings of three yellow crowned parrot (Amazona ochrocepahala) siblings with a severe and rapidly progressive neurological phenotype.

MethodsThe phenotype of the three parrots included severe ataxia, head tilt, and stargazing, while their parents were phenotypically normal. Tests to identify avian viral infections and brain imaging studies were all negative. Due to their inability to survive independently, they were all euthanized at age 3 months and their brains underwent neuropathological examination and proteasome activity assays. Whole genome sequencing (WGS) was performed on the three affected parrots and their parents.

ResultsThe brains of affected parrots exhibited neuronal loss, spongiosis, and Lewy bodies in the neocortex, amygdala, hypothalamus, periaqueductal gray matter, dorsal vagal nucleus, in some cerebellar Purkinje cells, and in the basal ganglia. Proteasome activity was significantly reduced in the affected parrots compared to a control (p<0.05). WGS identified a single homozygous missense mutation (p.V559L) in a highly conserved amino acid residue within the pleckstrin homology (PH) domain of the Calcium Dependent Secretion Activator 2 (CADPS2) gene. Previous studies suggest that CADPS2 is expressed at high levels in the substantia nigra where it regulates BDNF release. Thus, disruption of CADPS2 function could impact survival of dopaminergic neurons. Furthermore, CADPS2 expression is in part regulated by two well established PD genes, LRRK2 and SNCA.

ConclusionsOur data suggest that a homozygous mutation in the CADPS2 gene causes a severe neurodegenerative phenotype with Lewy bodies in parrots. Although CADPS2 variants have not been reported to cause PD in humans, further investigation of the gene in model organisms might provide important insights into the pathophysiology of Lewy body disorders.


title: Selective IL13Rα2-Targeted Functionality of IL13-Ligand CARs is Enhanced by Inclusion of 4-1BB Co-Stimulation

abstract: ABSTRACTChimeric antigen receptor (CAR) T cell immunotherapy is emerging as a powerful strategy for cancer therapy; however, an important safety consideration is the potential for off-tumor recognition of normal tissue. This is particularly important as ligand-based CARs are optimized for clinical translation. Our group has developed and clinically translated an IL13(E12Y) ligand- based CAR targeting the cancer antigen IL13R2 for treatment of glioblastoma (GBM). There remains limited understanding of how IL13-ligand CAR design impacts the activity and selectivity for the intended tumor-associated target IL13R2 versus the more ubiquitous unintended target IL13R1. In this study, we functionally compared IL13(E12Y)-CARs incorporating different intracellular signaling domains, including first-generation CD3{zeta}- containing CARs (IL13{zeta}), second-generation 4-1BB- (CD137) or CD28-containing CARs (IL13- BB{zeta} or IL13-28{zeta}), and third-generation CARs containing both 4-1BB and CD28 (IL13-28BB{zeta}). In vitro co-culture assays at high tumor burden establish that 2nd generation IL13-BB{zeta} or IL13- 28{zeta} outperform first-generation IL13{zeta} and 3rd generation IL13-28BB{zeta} CAR designs, with IL13- BB{zeta} providing superior CAR proliferation and in vivo anti-tumor potency in human xenograft mouse models. IL13-28{zeta} displayed a lower threshold for antigen recognition, resulting in higher off-target IL13R1 reactivity both in vitro and in vivo. Syngeneic mouse models of GBM also demonstrate safety and anti-tumor potency of murine IL13-BB{zeta} CAR T cells delivered systemically after lymphodepletion. These findings support the use of IL13-BB{zeta} CARs for greater selective recognition of IL13R2 over IL13R1, higher proliferative potential, and superior anti-tumor responsiveness. This study exemplifies the potential of modulating factors outside the antigen targeting domain of a CAR to improve selective tumor recognition..


title: SARS-CoV-2 accessory protein ORF8 decreases antibody-dependent cellular cytotoxicity

abstract: SARS-CoV-2 Spike glycoprotein is the major target of host neutralizing antibodies and the most changing viral protein in the continuously emerging SARS-CoV-2 variants as a result of frequent viral evasion from host antibody responses. In addition, SARS-CoV-2 encodes multiple accessory proteins that modulate host antiviral immunity by different mechanisms. Among all SARS-CoV-2 accessory proteins, ORF8 is rapidly evolving and a deletion in this protein has been linked to milder disease. Here, we studied the effect of ORF8 on peripheral blood mononuclear cells (PBMC). Specifically, we found that ORF8 can bind monocytes as well as NK cells. Strikingly, ORF8 binds CD16a (Fc{gamma}RIIIA) with nanomolar affinity and decreases the overall level of CD16 at the surface of monocytes and, to a lesser extent, NK cells. Strikingly, this decrease significantly reduces the capacity of PBMCs and particularly monocytes to mediate antibody-dependent cellular cytotoxicity (ADCC). Overall, our data identifies a new immune-evasion activity used by SARS-CoV-2 to escape humoral responses.


title: Improved fluorescent Listeria spp. biosensors for analysis of antimicrobials by flow cytometry

abstract: The global increase in antibiotic resistances of pathogenic microorganisms require identification and characterization of novel antimicrobials. Bacterial biosensors expressing fluorescent proteins such as pHluorin variants are suitable for high-throughput screenings. Here, we present Listeria spp. pH-sensitive biosensors with improved fluorescence for single cell analysis of antimicrobials by flow cytometry.


title: CRISPRi screen for enhancing heterologous α-amylase yield in Bacillus subtilis

abstract: Enhancing yield during bacterial enzyme production could have positive economic and environmental impacts. For cell factories, such improvements in yields could potentially be obtained by fine-tuning the metabolic processes and their regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two -amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Complex operons that are regulated by a wide variety of transcription factors, non-coding and structured RNAs add to the challenge of finding yield-affecting candidates. Surprisingly, we found that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation (75% of novel RNA predictions had absolute logFC > 2). Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expressed annotations, including both novel candidate and prior annotated ncRNAs, we selected 53 non-coding candidates. The targeting with CRISPRi knockdowns transcription in a genomic region on both the sense and the antisense strand. Thus, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3 UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.


title: Orphan nuclear receptors promote alternative lengthening of telomeres (ALT) through ALT-associated PML bodies

abstract: Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism utilized by about 15% of cancers. Orphan nuclear receptors (NRs), such as COUP-TF1, COUP-TF2, EAR2, TR2, and TR4, associate with telomeres of ALT cells by binding to variant telomeric repeats. However, how these orphan NRs function in the ALT pathway remains to be characterized. Here, we have established an ALT-inducing cell model by tethering orphan NRs to telomeres in non-ALT BJ fibroblast cells. We demonstrate that recruitment of orphan NRs to telomeres is sufficient to initiate formation of ALT-associated promyelocytic leukemia nuclear bodies (APBs) and telomeric DNA synthesis at APBs. We found that the ability of orphan NRs to initiate APB formation and recombination is dependent on the orphan NR AF2 domain, the zinc-finger protein ZNF827, and PML protein. Depletion of orphan NRs in ALT cell lines reduced APB formation and telomeric DNA synthesis, confirming the role of orphan NRs in ALT cells. Furthermore, we found that ATRX/DAXX depletion, together with the telomeric localization of orphan NRs, induces APB formation, telomere clustering, and telomeric DNA synthesis more dramatically in non-ALT cells. Accordingly, we propose that these events in ALT, orphan NR recruitment to telomeres and ATRX/DAXX loss, operate in concert to activate the ALT pathway.

category: molecular biology


title: Structural insights into crista junction formation by the Mic60-Mic19 complex

abstract: Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil domain of the mitochondrial contact and cristae organizing system (MICOS) subunit Mic60 forms an elongated, bow tie-shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with curved CJ necks. Our study suggests that the Mic60-Mic19 subcomplex transverses CJs as a molecular strut, thereby controlling CJ architecture and function.

category: cell biology


title: In vitro reconstitution of calcium-dependent recruitment of the human ESCRT machinery in lysosomal membrane repair

abstract:
The endosomal sorting complex required for transport (ESCRT) machinery has been shown to be centrally involved in repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open a new avenue for mechanistic understanding of endolysosomal membrane repair.

Significance statementOne of the ways by which protein aggregates can propagate and lead to progression of a neurodegenerative disease is by damaging the membrane that is destined to degrade the misfolded, aggregated protein. ESCRT machinery has been implicated in sealing these damaged membranes, and the nature of the membrane recruitment trigger signal for this machinery is a major open question. Here, we show in vitro that ALG-2 can bring ESCRT machinery to membranes in a Ca2+-dependent manner.


title: A partnership of the lipid scramblase XK and of the lipid transfer protein VPS13A at the plasma membrane

abstract: Chorea-acanthocytosis and McLeod syndrome are diseases with shared clinical manifestations caused by mutations in VPS13A and XK, respectively. Key features of these conditions are the degeneration of caudate neurons and the presence of abnormally shaped erythrocytes. XK belongs to a family of plasma membrane (PM) lipid scramblases whose action results in exposure of PtdSer at the cell surface. VPS13A is an ER-anchored lipid transfer protein with a putative role in the transport of lipids at contacts of the ER with other membranes. Recently VPS13A and XK were reported to interact by still unknown mechanisms. So far, however, there is no evidence for a colocalization of the two proteins at contacts of the ER with the PM, where XK resides, as VPS13A was shown to be localized at contacts between the ER and either mitochondria or lipid droplets. Here we show that VPS13A can also localize at ER-PM contacts via the binding of its PH domain to a cytosolic loop of XK, that such interaction is regulated by an intramolecular interaction within XK and that both VPS13A and XK are highly expressed in the caudate neurons. Binding of the PH domain of VPS13A to XK is competitive with its binding to intracellular membranes that mediate other tethering functions of VPS13A. Our findings support a model according to which VPS13A-dependent lipid transfer between the ER and the PM is coupled to lipid scrambling within the PM. They raise the possibility that defective cell surface exposure of PtdSer may be responsible for neurodegeneration.


title: Evolution of CRISPR-associated Endonucleases as Inferred from Resurrected Proteins

abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 protein is an effector that plays a major role in a prokaryotic adaptive immune system, by which invading DNA can be targeted and cut for inactivation. The Cas9 endonuclease is directed to target sites by a guide RNA (gRNA) where Cas9 can recognize specific sequences (PAMs) in foreign DNA, which then serve as an anchoring point for cleavage of the adjacent RNA-matching DNA region. Although the CRISPR-Cas9 system has been widely studied and repurposed for diverse applications (notably, genome editing), its origin and evolution remain to be elucidated. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species as old as 2600 myr to the current day. Surprisingly, we demonstrate that these ancient forms were much more flexible in their PAM and gRNA scaffold requirements compared to modern day Cas9 enzymes. In addition, anCas portrays a gradual paleoenzymatic adaptation from nickase to double-strand break activity, suggesting a mechanism by which ancient CRISPR systems could propagate when harboring Cas enzymes with minimal PAMs. The oldest anCas also exhibit high levels of activity with ssDNA and ssRNA targets, resembling Cas nucleases in related system types. Finally, we illustrate editing activity of the anCas enzymes in human cells. The prediction and characterization of anCas proteins uncovers an unexpected evolutionary trajectory leading to ancient enzymes with extraordinary properties.

category: evolutionary biology


title: Characterizing Photosynthetic Biofuel Production: Isotopically non-stationary 13C metabolic flux analysis (INST-13CMFA) on limonene producingSynechococcus sp. PCC 7002

abstract: Synechococcus sp. PCC 7002 is a unicellular cyanobacterium capable of fast growth, even under high light intensity and high salinity. These attributes along with genetic tractability make Synechococcus sp. PCC 7002 an attractive candidate for industrial scale production of specialty and commodity chemicals. One such strain produces limonene, an energy dense diesel jet fuel drop-in additive, at a titer of 4 mg/L over a four-day incubation period. In this study, we use the state-of-the-art whole-cell characterization tool, isotopically non-stationary 13C metabolic flux analysis (INST-13CMFA) to determine intracellular fluxes through the pathways of central metabolism for the limonene producing strain and wild type strain of Synechococcus sp. PCC 7002. We find similar flux distribution in the Calvin-Benson-Bassham cycle, photorespiration, oxidative pentose phosphate pathway, and reductive tricarboxylic acid cycle. The key difference between strains is observed in the production of pyruvate. The limonene producing strain displays significantly higher flux through the amphibolic pathways of phosphoenolpyruvate carboxylase and the malic enzyme to synthesize pyruvate, while the wild type strain uses pyruvate kinase in a single step. Our findings suggest that this flux distribution is a mechanism to recover a physiologically optimal ratio of ATP to NADPH. The upregulation of this amphibolic pathway may act to restore the physiological ATP:NADPH ratio that has been disturbed by limonene biosynthesis.

category: bioengineering


title: Differential gene expression profiling reveals potential biomarkers and pharmacological compounds against SARS-CoV-2: insights from machine learning and bioinformatics approaches

abstract:
SARS-CoV-2 continues to spread and evolve worldwide, despite intense efforts to develop multiple vaccines and therapeutic options against COVID-19. Moreover, the precise role of SARS-CoV-2 in the pathophysiology of the nasopharyngeal tract (NT) is still unfathomable. Therefore, we used the machine learning methods to analyze 22 RNA-seq datasets from COVID-19 patients (n=8), recovered individuals (n=7), and healthy individuals (n=7) to find disease-related differentially expressed genes (DEGs). In comparison to healthy controls, we found 1960 and 153 DEG signatures in COVID-19 patients and recovered individuals, respectively. We compared dysregulated DEGs to detect critical pathways and gene ontology (GO) connected to COVID-19 comorbidities. In COVID-19 patients, the DEG- miRNA and DEG-transcription factors (TFs) interactions network analysis revealed that E2F1, MAX, EGR1, YY1, and SRF were the most highly expressed TFs, whereas hsa-miR-19b, hsa-miR-495, hsa-miR-340, hsa-miR-101, and hsa-miR-19a were the overexpressed miRNAs. Three chemical agents (Valproic Acid, Alfatoxin B1, and Cyclosporine) were abundant in COVID-19 patients and recovered individuals. Mental retardation, mental deficit, intellectual disability, muscle hypotonia, micrognathism, and cleft palate were the significant diseases associated with COVID-19 by sharing DEGs. Finally, we detected DEGs impacted by SARS-CoV-2 infection and mediated by TFs and miRNA expression, indicating that SARS-CoV-2 infection may contribute to various comorbidities. These pathogenetic findings can provide some crucial insights into the complex interplay between COVID-19 and the recovery stage and support its importance in the therapeutic development strategy to combat against COVID-19 pandemic.

IMPORTANCEDespite it has now been over two years since the beginning of the COVID-19 pandemic, many crucial questions about SARS-CoV-2 infection and the different COVID-19 symptoms it causes remain unresolved. An intriguing question about COVID-19 is how SARS-CoV-2 interplays with the host during infection and how SARS-CoV-2 infection can cause so many disease symptoms. Our analysis of three different datasets (COVID-19, recovered, and healthy) revealed significantly higher DEGs in COVID-19 patients than recovered humans and healthy controls. Some of these DEGs were found to be co-expressed in both COVID-19 patients. They recovered humans supporting the notion that DEGs level is directly correlated with the viral load, disease progression, and different comorbidities. The protein-protein interaction consisting of 24 nodes and 72 edges recognized eight hub-nodes as potential hub-proteins (i.e., RPL4, RPS4X, RPL19, RPS12, RPL19, EIF3E, MT-CYB, and MT-ATP6). Protein-chemical interaction analysis identified three chemical agents (e.g., Valproic Acid, Alfatoxin B1, and Cyclosporine) enriched in COVID-19 patients and recovered individuals. Mental retardation, mental deficiency, intellectual disability, muscle hypotonia, micrognathism, and cleft palate were the significant diseases associated with COVID-19 by sharing DEGs.

category: bioinformatics


title: Predicting variability in nascent RNA from transcription initiation kinetics

abstract: Nascent (actively transcribed) RNA accurately reflects transcriptional kinetics. Models predicting its fluctuation signature are difficult to solve because they are inherently non-Markovian--this is because elongation of nascent RNA takes approximately a fixed amount of time. Here, we derive an exact relationship between the distribution of the times between successive initiation events and the steady-state distribution of nascent RNA numbers. We verify the accuracy of the theory using live cell imaging data in prokaryotic cells and using simulations of eukaryotic transcription.

category: biophysics


title: Comparative Structural and Dynamics Study of Free and gRNA-bound FnCas9 and SpCas9 Proteins

abstract:
The introduction of CRISPR/Cas9 based gene editing has greatly accelerated therapeutic genome editing. However, the off-target DNA cleavage by CRISPR/Cas9 protein hampers its clinical translation thereby hindering its widespread use as a programmable genome editing tool. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. Here, we have compared the dynamics of a more specific naturally occurring Cas9 from Francisella novicida (FnCas9) to the most widely used, SpCas9 protein. Long-scale atomistic MD simulation of free and gRNA bound forms of both the Cas9 proteins was performed and their domain rearrangements and binding affinity with gRNA were compared to decipher the possible reason behind enhanced specificity of FnCas9 protein. The greater binding affinity with gRNA, high domain electrostatics and more volatility of FnCas9 than SpCas9 may explain its increased specificity and lower tolerance for mismatches.

HighlightsO_LIThe gRNA binding led to opening of the structure of Cas9 endonuclease and exposure of hydrophobic residues to accommodate it.
C_LIO_LIConcerted domain movement was observed in both SpCas9 (HNH-REC2 domains, CTD-Topo domains, and REC3-RuvC) and FnCas9 (HNH-REC3-REC1 and PI-WED-RuvC).
C_LIO_LIBoth Cas9s have shown a decrease in percentage helicity and increase in the percentage of structurally dynamic residues (more secondary structural transitions) post gRNA binding.
C_LIO_LIIn gRNA-bound form of FnCas9, PAM-interacting domain was found to have larger structural transition and increased coil%.
C_LIO_LIElectrostatic interactions have a major contribution in the high binding affinity of FnCas9 with gRNA.
C_LIO_LIArginine-helix has shown a pivotal role in the binding of gRNA in FnCas9 protein.
C_LI


title: Intracellular Aβ42 aggregation leads to cellular thermogenesis

abstract: The aggregation of A{beta}42 is a hallmark of Alzheimers disease. It is still not known what the biochemical changes are inside a cell which will eventually lead to A{beta}42 aggregation. Thermogenesis has been associated with cellular stress, the latter of which may promote aggregation. We perform intracellular thermometry measurements using fluorescent polymeric thermometers (FPTs) to show that A{beta}42 aggregation in live cells leads to an increase in cell-averaged temperatures. This rise in temperature is mitigated upon treatment with an aggregation inhibitor of A{beta}42 and is independent of mitochondrial damage that can otherwise lead to thermogenesis. With this, we present a diagnostic assay which could be used to screen small-molecule inhibitors to amyloid proteins in physiologically relevant settings. To interpret our experimental observations and motivate the development of future models, we perform classical molecular dynamics of model A{beta} peptides to examine the factors that hinder thermal disspation. We observe that this is controlled by the presence of ions in its surrounding environment, the morphology of the amyloid peptides and the extent of its hydrogen-bonding interactions with water. We show that aggregation and heat retention by A{beta} peptides are favoured under intracellular-mimicking ionic conditions, which could potentially promote thermogenesis. The latter will, in turn, trigger further nucleation events that accelerate disease progression.


title: Patient-associated mutations in Drosophila Alk perturb neuronal differentiation and promote survival

abstract: Activating Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) mutations occur in pediatric neuroblastoma and are associated with poor prognosis. To study ALK-activating mutations in a genetically controllable system we employed CRIPSR/Cas9, incorporating orthologues of the human oncogenic mutations ALKF1174L and ALKY1278S in the Drosophila Alk locus. AlkF1251L and AlkY1355S mutant Drosophila exhibit enhanced Alk signaling phenotypes, but unexpectedly depend on the Jelly belly (Jeb) ligand for activation. Both AlkF1251L and AlkY1355S mutant larval brains display hyperplasia, represented by increased numbers of Alk-positive neurons. Despite this hyperplasic phenotype, no brain tumors were observed in mutant animals. We show that hyperplasia in Alk mutants was not caused by significantly increased rates of proliferation, but rather by decreased levels of apoptosis in the larval brain. Using single-cell RNA sequencing (scRNA-seq), we identify perturbations during temporal fate specification in AlkY1355S mutant mushroom body lineages. These findings shed light on the role of Alk in neurodevelopmental processes and highlight the potential of activating Alk mutations to perturb specification and promote survival in neuronal lineages.

category: cancer biology


title: WDR5 promotes breast cancer growth and metastasis via KMT2-independent translation regulation

abstract: Metastatic breast cancer remains a major cause of cancer related deaths in women and there are few effective therapies against this advanced disease. Emerging evidence suggests that key steps of tumor progression and metastasis are controlled by reversible epigenetic mechanisms. Using an in vivo genetic screen, we identified WDR5 as an actionable epigenetic regulator that is required for metastatic progression in models of triple-negative breast cancer. We found that knockdown of WDR5 in breast cancer cells independently impaired their tumorigenic as well as metastatic capabilities. Mechanistically, WDR5 promotes cell growth by increasing ribosomal gene expression and translation efficiency in a KMT2-independent manner. Consistently, pharmacological inhibition or degradation of WDR5 impedes cellular translation rate and the clonogenic ability of breast cancer cells. Furthermore, combination of WDR5-targeting with mTOR inhibitors leads to potent suppression of translation and proliferation of breast cancer cells. These results reveal novel therapeutic strategies to treat metastatic breast cancer.


title: PRMT5 is a therapeutic target in choroidal neovascularization

abstract: Ocular neovascular diseases including neovascular age-related macular degeneration (nvAMD) are widespread causes of blindness. Patients non-responsiveness to currently used biologics that target vascular endothelial growth factor (VEGF) poses an unmet need for novel therapies. Here, we identify protein arginine methyltransferase 5 (PRMT5) as a novel therapeutic target for nvAMD. PRMT5 is a well-known epigenetic enzyme. We previously showed that PRMT5 methylates and activates a proangiogenic and proinflammatory transcription factor, the nuclear factor kappa B (NF-{kappa}B), which has a master role in tumor progression, notably in pancreatic ductal adenocarcinoma and colorectal cancer. We identified a potent and specific small molecule inhibitor of PRMT5, PR5-LL-CM01, that dampens the methylation and activation of NF-{kappa}B. Here for the first time, we assessed the antiangiogenic activity of PR5-LL-CM01 in ocular cells. Immunostaining of human nvAMD sections revealed that PRMT5 is highly expressed in the retinal pigment epithelium (RPE)/choroid where neovascularization occurs, while mouse eyes with laser induced choroidal neovascularization (L-CNV) showed PRMT5 is overexpressed in the retinal ganglion cell layer and in the RPE/choroid. Importantly, inhibition of PRMT5 by PR5-LL-CM01 or shRNA knockdown of PRMT5 in human retinal endothelial cells (HRECs) and induced pluripotent stem cell (iPSC)-derived choroidal endothelial cells (iCEC2) reduced NF-{kappa}B activity and the expression of its target genes, such as tumor necrosis factor  (TNF-) and VEGF-A. In addition to inhibiting angiogenic properties of proliferation and tube formation, PR5-LL-CM01 blocked cell cycle progression at G1/S-phase in a dose-dependent manner in these cells. Thus, we provide the first evidence that inhibition of PRMT5 impedes angiogenesis in ocular endothelial cells, suggesting PRMT5 as a potential therapeutic target to ameliorate ocular neovascularization.

category: pharmacology and toxicology


title: Interfering with plant developmental timing promotes susceptibility to insect vectors of a bacterial parasite

abstract: Multi-trophic parasites often modulate host behaviors and development to attract other hosts. However, whether these modulations are controlled by specific parasite genes and are adaptive remains debated. Insect-transmitted phytoplasma parasites take control of their plant host by turning them into  Zombie plants, which feature spectacular vegetative organ proliferations and juvenilization. The phytoplasma effector SAP05 induces these symptoms by mediating the degradation of multiple plant SPL and GATA developmental transcription factors via recruiting the ubiquitin receptor RPN10. Here, we investigated if these SAP05-induced modulations impact the leafhopper vectors on which phytoplasmas depend for spread. SAP05 promotes leafhopper reproduction on Arabidopsis thaliana in a RPN10-dependent manner. Moreover, SAP05 homologs that degrade SPLs and both SPLs and GATAs promote leafhopper reproduction, whereas those that degrade only GATAs do not. Leafhoppers also produced more progeny on plants with misregulated MiR156, which suppresses SPL expression. SPLs control several developmental processes. Surprisingly, leaf color and shape changes and increased leaf numbers did not correlate to leafhopper progeny increases, whereas disturbing the plant phase transition processes did. Therefore, only specific phenotypes induced by a single parasite gene extend beyond one host to control another and promote host-host attractions of a multi-trophic parasite.

category: plant biology


title: HTL/KAI2 signalling substitutes for light to control plant germination

abstract: Deciphering signalling pathways is essential to understanding how organisms respond to environmental cues but elucidating how these signalling pathways evolve in new environments is less clear.1,2 Most plants, for example, monitor multiple environmental cues to optimize the time and place to germinate. Some root parasitic plants, however, germinate in response to small molecules like strigolactones (SLs) emanating from host roots3,4 whilst a number of ephemeral weeds germinate in response to chemicals called karrikins (KARs) released after a forest fire.5,6 Although these species represent distinct clades, they use the same HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2 (HTL/KAI2) signalling pathway to perceive strigolactones or karrikins, which suggests convergent evolution.3,5 Because specialist lifestyles are derived traits, it is not clear if HTL/KAI2 signalling in these species evolved from a specific germination-signalling pathway or whether this pathway had other functions that were co-opted for specialist germination circumstances. Here, we show HTL/KAI2 signalling in Arabidopsis bypasses the light requirement for germination. In part, this is because the HTL/KAI2 downstream component, SMAX1 impinges on PHYTOCHROME INTERACTING FACTOR 1/PHYTOCHROME INTERACTING FACTOR 3-LIKE 5 (PIF1/PIL5)-regulated hormone response pathways conducive to germination. We identified Arabidopsis accessions that can germinate in the dark, which had altered expression of HTL/KAI2 signalling components, suggesting that divergence in this signalling pathway occurs in nature. Moreover, Arabidopsis HTL/KAI2-regulated gene signatures were observed in germinating Striga seed. The ability of HTL/KAI2 signalling to substitute for light advances an explanation for how some specialist plants evolved their underground germination behaviour in response to specific environments.


title: A mini-TGA protein, lacking a functional DNA-binding domain, modulates gene expression through heterogeneous association with transcription factors

abstract: TGA transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigate the role of StTGA2.1, a potato TGA lacking the full bZIP, which we name a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirm that StTGA2.1 overexpression compensates for SA-deficiency. To understand the underlying mechanisms, we show that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigate the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirm that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases, which are known to play role in immune response. Finally, via structural modelling and molecular dynamics simulations, we hypothesise that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to novel functions and that such events should be studied carefully in other protein families.


title: Proteomic characterization of spinal cord synaptoneurosomes from Tg-SOD1/G93A mice supports a role for MNK1 and local translation in the early stages of amyotrophic lateral sclerosis

abstract: The isolation of synaptoneurosomes (SNs) represents a useful means to study synaptic events. However, the size and density of synapses varies in different regions of the central nervous system (CNS), and this also depends on the experimental species studied, making it difficult to define a generic protocol for SNs preparation. To characterize synaptic failure in the spinal cord (SC) in the Tg-SOD1/G93A mouse model of amyotrophic lateral sclerosis (ALS), we applied a method we originally designed to isolate cortical and hippocampal SNs to SC tissue. Interestingly, we found that the SC SNs were isolated in a different gradient fraction to the cortical/hippocampal SNs. We compared the relative levels of synaptoneurosomal proteins in control (Tg-SOD1) animals, with Tg-SOD1/G93A mice at onset and those that were symptomatic using iTRAQ proteomics. The results obtained suggest that an important regulator of local synaptic translation, MNK1 (MAP kinase interacting serine/threonine kinase 1), might well influence the early stages of ALS.


title: TGFβ limits Myc-dependent TCR-induced metabolic reprogramming in CD8+ T cells

abstract: T cell activation is dependent upon the integration of antigenic, costimulatory and cytokine-derived signals and the availability and acquisition of nutrients from the environment. Furthermore, T cell activation is accompanied by reprogramming of cellular metabolism to provide the energy and building blocks for proliferation, differentiation and effector function. Transforming growth factor {beta} (TGF{beta}) has pleiotropic effects on T cell populations, having both an essential role in the maintenance of immune tolerance but also context-dependent pro-inflammatory functions. We set out to define the mechanisms underpinning the suppressive effects of TGF{beta} on mouse CD8+ T cell activation. RNA-sequencing analysis of TCR-stimulated T cells determined that Myc-regulated genes were highly enriched within gene sets downregulated by TGF{beta}. Functional analysis demonstrated that TGF{beta} impeded TCR-induced upregulation of amino acid transporter expression, amino acid uptake and protein synthesis. Furthermore, TCR-induced upregulation of Myc-dependent glycolytic metabolism was substantially inhibited by TGF{beta} treatment with minimal effects on mitochondrial respiration or mTOR activation. Thus, our data suggest that inhibition of Myc-dependent metabolic reprogramming represents a major mechanism underpinning the suppressive effects of TGF{beta} on CD8+ T cell activation.


title: Controlling taxa abundance improves metatranscriptomics differential analysis

abstract: A common task in analyzing metatranscriptomics data is to identify microbial metabolic pathways with differential RNA abundances across multiple sample groups. With information from paired metagenomics data, current differential methods control for either DNA or taxa abundances to address their strong correlation with RNA abundance. We discovered that when either DNA or taxa abundance is controlled for, RNA abundance still has a strong partial correlation with the other factor. Thus, both factors need to be controlled for in the differential model. In both simulation studies and a real data analysis, we demonstrated that controlling for both DNA and taxa abundances leads to superior performance compared to only controlling for one factor.


title: Obligate sexual reproduction of a homothallic fungus closely related to the Cryptococcus pathogenic species complex

abstract: Sexual reproduction is a ubiquitous and ancient trait of eukaryotic life. While sexual organisms are usually faced with the challenge of finding a compatible mating partner, species as diverse as animals, plants, and fungi have repeatedly evolved the ability to reproduce sexually without an obligate requirement for another individual. Here, we uncovered the underlying mechanism of self-compatibility (homothallism) in Cryptococcus depauperatus, a fungal species sister to the clinically relevant human fungal pathogens Cryptococcus neoformans and Cryptococcus gattii species complexes. In contrast to C. neoformans or C. gattii, which grow as a yeast in the asexual stage, and produce hyphae, basidia, and infectious spores during the sexual stage, C. depauperatus grows exclusively as hyphae decorated with basidia and abundant spores and appears to be continuously engaged in sexual reproduction. By combining the insights from comparative genomics and genetic analyses of mutants defective in key mating and meiosis genes, we demonstrate the sexual cycle of C. depauperatus involves meiosis, and reveal that self-compatibility is orchestrated by the expression, in the same cell, of an unlinked mating receptor (Ste3a) and pheromone ligand (MF) pair seemingly derived from opposite mating types of a heterothallic (self-sterile) ancestor. We identified a putative mating-type (MAT) determining region containing genes phylogenetically aligned with MATa alleles of other species, and a few MAT gene alleles scattered and unlinked throughout the genome, but no homologs of the mating-type homeodomain genes SXI1 (HD1) and SXI2 (HD2). Comparative genomic analyses suggested a dramatic remodeling of the MAT locus possibly owing to reduced selective constraints to maintain mating-type genes in tight linkage, associated with a transition to self-fertility. Our findings support C. depauperatus as an obligately sexual, homothallic fungal species and provide additional insight into the repeated transitions between modes of sexual reproduction that have occurred throughout the fungal kingdom.


title: Emerging Roles of Importin alpha/beta and EB1 in Differential Regulation of Kif18B Astral Microtubule Destabilization

abstract: Tight regulation of microtubule dynamics and microtubule organization is necessary for proper spindle assembly and chromosome segregation. The microtubule destabilizing Kinesin-8, Kif18B, controls astral microtubule dynamics and spindle positioning. Kif18B interacts with importin /{beta} as well as with the plus-tip tracking protein EB1, but how these associations modulate Kif18B activity or function is not known. We mapped the key binding sites on Kif18B, made residue-specific mutations, and assessed their impact on Kif18B function. Blocking EB1 interaction disrupted Kif18B MT plus-end accumulation and inhibited its ability to control astral microtubule length in cells. Blocking importin /{beta} interaction reduced Kif18B plus-end accumulation on astral MTs but did not inhibit its ability to control astral MT length. In vitro, importin /{beta} increased Kif18B binding to the microtubule lattice, which enhanced Kif18B accumulation at microtubule plus ends, and stimulated microtubule destabilization, suggesting that the importins spatially regulate Kif18B. In contrast, EB1 promoted microtubule destabilization without increasing lattice binding in vitro, which suggests that EB1 and importin /{beta} have distinct roles in the regulation of astral microtubule dynamics. We propose that Ran-regulation is important not only to control motor function near chromatin but also provides a spatial control mechanism to modulate microtubule binding of NLS-containing spindle assembly factors.


title: A conserved PLK1 docking site in TopBP1 maintains genome integrity during mitosis

abstract: TopBP1 is a large scaffold protein with multiple functions in genome integrity. We previously identified a novel role for TopBP1 during M phase by showing that TopBP1 reduces carry-over of DNA damage to daughter cells. This function emerges as a critical backup pathway in BRCA deficient cells, yet many aspects of TopBP1 regulation during mitosis are unclear. The mitotic kinase PLK1 has been reported to interact with TopBP1 but the functional relevance of this is unclear. Here, we identify and characterize a conserved PLK1 docking site in TopBP1. Endogenous deletion of the PLK1 docking site in TopBP1 results in increased number of mitotic TopBP1 foci, increased DNA damage in daughter cells, deficient mitotic DNA repair synthesis and increased frequency of binucleation. At the same time, cell cycle distribution and ATR activation are normal in cells with the PLK1 docking site deletion in TopBP1. Interestingly, mutation of this site in TopBP1 renders cells sensitive to PARP inhibitors but not to camptothecin hinting to different cellular effects of the two chemotherapeutics. Altogether, our data indicate that the PLK1-TopBP1 interaction is critical for the mitotic function of TopBP1.


title: Species Distribution Modeling of Aedes aegypti in Maricopa County, Arizona from 2014-2020

abstract:
Aedes aegypti mosquitoes, which are of great importance to public health, have recently and rapidly expanded their global range; however, little is understood about the distribution of these mosquitoes in desert environments. Trapping records for Ae. aegypti in Maricopa County, Arizona from 2014-2020 were used to construct species distribution models using Maxent. Satellite imagery and socioeconomic factors were used as predictors. Maps of predicted habitat suitability were converted to binary presence/absence maps, and consensus maps were created that represent "core" habitat for the mosquito over six years of time. Generally, population density was the most important predictor in the models while median income was the least important. This study is the first step toward understanding the distribution of the vector in a desert environment.

One-Sentence SummaryThis study uses satellite imagery and socioeconomic factors to identify core habitat over six years in a desert environment.

category: ecology


title: Genetic surveillance of SARS-CoV-2 Mpro reveals high sequence and structural conservation prior to the introduction of protease inhibitor Paxlovid

abstract:
SARS-CoV-2 continues to represent a global health emergency as a highly transmissible, airborne virus. An important coronaviral drug target for treatment of COVID-19 is the conserved main protease (Mpro). Nirmatrelvir is a potent Mpro inhibitor and the antiviral component of Paxlovid. The significant viral sequencing effort during the ongoing COVID-19 pandemic represented a unique opportunity to assess potential nirmatrelvir escape mutations from emerging variants of SARS-CoV-2. To establish the baseline mutational landscape of Mpro prior to the introduction of Mpro inhibitors, Mpro sequences and its cleavage junction regions were retrieved from [~]4,892,000 high-quality SARS-CoV-2 genomes in GISAID. Any mutations identified from comparison to the reference sequence (Wuhan-hu-1) were cataloged and analyzed. Mutations at sites key to nirmatrelvir binding and protease functionality (e.g., dimerization sites) were still rare. Structural comparison of Mpro also showed conservation of key nirmatrelvir contact residues across the extended Coronaviridae family (alpha-, beta-, and gamma-coronaviruses). Additionally, we showed that over time the SARS-CoV-2 Mpro enzyme remained under purifying selection and was highly conserved relative to the spike protein. Now, with the EUA approval of Paxlovid and its expected widespread use across the globe, it is essential to continue large-scale genomic surveillance of SARS-CoV-2 Mpro evolution. This study establishes a robust analysis framework for monitoring emergent mutations in millions of virus isolates, with the goal of identifying potential resistance to present and future SARS-CoV-2 antivirals.

ImportanceThe recent authorization of oral SARS-CoV-2 antivirals, such as Paxlovid, has ushered in a new era of the COVID-19 pandemic. Emergence of new variants, as well as selective pressure imposed by antiviral drugs themselves, raise concern for potential escape mutations in key drug binding motifs. To determine the potential emergence of antiviral resistance in globally circulating isolates and its implications for the clinical response to the COVID-19 pandemic, sequencing of SARS-CoV-2 viral isolates before, during, and after the introduction of new antiviral treatments is critical. The infrastructure built herein for active genetic surveillance of Mpro evolution and emergent mutations will play an important role in assessing potential antiviral resistance as the pandemic progresses and Mpro inhibitors are introduced. We anticipate our framework to be the starting point in a larger effort for global monitoring of the SARS-CoV-2 Mpro mutational landscape.


title: Mechanism and consequence of daily modulation of cortical parvalbumin-positive inhibitory neurons

abstract: Parvalbumin-positive (PV) neurons, the main class of inhibitory neurons in the neocortex, play critical roles in maintaining normal brain function and are implicated in a variety of brain disorders. Here we found that their function is modulated in a time- and sleep-dependent manner naturally during the day. We first show that PV-evoked inhibition is stronger by the end of the light (ZT12) compared to the end of dark (ZT0) cycle. In addition, both PVs excitatory and inhibitory synaptic transmission slowly oscillate but in the opposite directions during the light/dark cycle. Whereas excitatory synapses are predominantly regulated by experience, inhibitory synapses are regulated by sleep. Mechanistically, we found that the daily regulation of PVs inhibitory synapses is mediated by acetylcholine activating M1 receptors. Consistent with our ex vivo findings, we show in vivo that PVs spontaneous activity display clear oscillation, which is opposite to that of the pyramidal neurons. Finally, we demonstrate that the daily changes in PV neural activity negatively correlate with the dLGN-evoked responses in V1, underscoring the physiological significance of PVs daily regulation.


title: Convergent impact of schizophrenia risk genes

abstract: Schizophrenia is a highly heritable psychiatric disorder with a complex genetic risk architecture that reflects the additive impact of hundreds of risk variants. While many schizophrenia-associated risk variants are thought to regulate the expression of target genes in a cell-type-specific manner, the mechanisms by which the effect of these myriad variants combine to contribute to risk remain unclear. Here we apply a CRISPR-based approach to evaluate in parallel twelve schizophrenia eGenes (that encompass common variation) in human glutamatergic neurons. Querying the shared neuronal impacts across risk genes uncovers a convergent effect concentrated on pathways of brain development and synaptic signaling. Our analyses reveal shared and divergent downstream effects of these twelve genes, independent of their previously annotated biological roles. General convergence of gene expression increases with increasing polygenicity, while the specificity of convergence increases between functionally similar genes. Convergent networks show brain-region and developmental period-specific enrichments, as well as disorder-specific enrichments for both rare and common variant target genes across schizophrenia, bipolar disorder, autism spectrum disorder, and intellectual disability. These gene targets are drug-able and potentially represent novel points of therapeutic intervention. Convergent signatures are also resolved in the post-mortem brain. Overall, convergence suggests a model to explain how non-additive interactions arise between risk genes and may explain cross-disorder pleiotropy of genetic risk for psychiatric disorders.


title: Longer metaphase and fewer chromosome segregation errors in modern human than Neandertal brain development

abstract: Since the ancestors of modern humans separated from those of Neandertals, around one hundred amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in the three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in the mouse, three substitutions in two of these proteins, KIF18a and KNL1, cause a prolongation of metaphase and a reduction in chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause a reduction in metaphase length and an increase in chromosome segregation errors in human brain organoids. Our data also show that, in these aspects, Neandertals were more similar to chimpanzees than to modern humans. Thus, the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neandertals.


title: Inhibiting presynaptic calcium channel mobility in the auditory cortex suppresses synchronized input processing

abstract:
The emergent coherent population activity from thousands of stochastic neurons in the brain is believed to constitute a key neuronal mechanism for salient processing of external stimuli and its link to internal states like attention and perception. In the sensory cortex, functional cell assemblies are formed by recurrent excitation and inhibitory influences. The stochastic dynamics of each cell involved is largely orchestrated by presynaptic CAV2.1 voltage-gated calcium channels (VGCCs). Cav2.1 VGCCs initiate the release of neurotransmitters from the presynaptic compartment and are therefore able to add variability into synaptic transmission which can be partly explained by their mobile organization around docked vesicles. To investigate the relevance of Cav2.1 channel surface mobility for the input processing in the primary auditory cortex (A1) in vivo, we make use of a new optogenetic system which allows us to acutely cross-link Cav2.1 VGCCs via a photo-cross-linkable cryptochrome mutant, CRY2olig. In order to map neuronal activity across all cortical layers of the A1, we performed laminar current-source density (CSD) recordings with varying auditory stimulus sets in transgenic mice with a citrine tag on the N-terminus of the VGCCs. Clustering VGCCs suppresses overall sensory-evoked population activity, particularly when stimuli lead to a highly synchronized distribution of synaptic inputs. Our findings reveal the importance of membrane dynamics of presynaptic calcium channels for sensory encoding by dynamically adjusting network activity across a wide range of synaptic input strength.

Statement of SignificanceVoltage Gated Calcium Channel (VGCC) mobility plays an important role in neuronal firing dynamics. Failure of these channels to function or be regulated has been linked to migraine and ataxia. We here link the microscopic process of VGCC mobility to the mesoscopic population dynamics as a mechanism to regulate and appropriately amplify synaptic inputs of different strengths to the mouse primary auditory cortex. We also demonstrate a novel and effective technique with which VGCC function can be further explored in meso- or macroscopic scales and with behaving subjects. We believe that this is of importance to the broader scientific community in aspects of non-linear scaling in the brain, potential translational applications, and basic research on cortical mechanisms of physiological function.


title: Glucocorticoids Mediate Transcriptome-wide Alternative Polyadenylation: Mechanisms and Functional Implications

abstract: Alternative polyadenylation (APA) is a common genetic regulatory mechanism that generates distinct 3' ends for RNA transcripts. Changes in APA have been associated with multiple biological processes and disease phenotypes. However, the role of hormones and their drug analogs in APA remains largely unknown. In this study, we investigated transcriptome-wide the impact of glucocorticoids on APA in 30 human B-lymphoblastoid cell lines. We found that glucocorticoids could regulate APA of a subset of genes, possibly by changing the expression of 142 RNA-binding proteins, some with known APA-regulating properties. Interestingly, genes with glucocorticoid-mediated APA were enriched in viral translation-related pathways, while genes with glucocorticoid-mediated expression were enriched in interferon and interleukin pathways, suggesting that glucocorticoid-mediated APA might result in functional consequences distinct from gene expression. Glucocorticoid-mediated APA was also cell-type-specific, suggesting an action of glucocorticoids that may be unique to immune regulation. We also observed evidence for genotype-dependent glucocorticoid-mediated APA, providing potential functional mechanisms for a series of common genetic variants that had previously been associated with immune disorders, but without a clear mechanism. In summary, this study reports a series of novel observations regarding the impact of glucocorticoids on APA, suggesting novel avenues for mechanistic studies of hormonal biology.


title: Jumping out of trouble: Evidence for a cognitive map in guppies (Poecilia reticulata)

abstract: Spatial cognitive abilities allow individuals to remember the location of food patches, predator hide-outs, or shelters. Animals typically incorporate learnt spatial information or use external environmental cues to navigate their surroundings. A spectacular example of how some fishes move is through aerial jumping. For instance, fish that are trapped within isolated pools, cut off from the main body of water during dry periods, may jump over obstacles and direct their jumps to return to safe locations. However, what information such re-orientation behaviour during jumping is based on remains enigmatic. Here we combine a lab and field experiment to test if guppies (Poecilia reticulata) incorporate learnt spatial information and external environmental cues (visual and auditory) to determine where to jump. In a spatial memory assay we found that guppies were more likely to jump towards deeper areas, hence incorporating past spatial information to jump to safety. In a matched vs. mismatched spatial cue experiment in the field, we found that animals only showed directed jumping when visual and auditory cues matched. We show that in unfamiliar entrapments guppies direct their jumps by combining visual and auditory cues, while in familiar entrapments they use a cognitive map. We hence conclude that jumping behaviour is a goal-directed behaviour, guided by different sources of information and involving important spatial cognitive skills.

category: animal behavior and cognition


title: 3D Organic Bioelectronics for Monitoring In Vitro Stem Cell Cultures

abstract: Three-dimensional in vitro stem cell models has enabled a fundamental understanding of cues that direct stem cell fate and be used to develop novel stem cell treatments. While sophisticated 3D tissues can be generated, technology that can accurately monitor these complex models in a high-throughput and non-invasive manner is not well adapted. Here we show the development of 3D bioelectronic devices based on the electroactive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) - PEDOT:PSS and their use for non-invasive, electrical monitoring of stem cell growth. We show that the electrical, mechanical and wetting properties as well as the pore size/architecture of 3D PEDOT:PSS scaffolds can be fine-tuned simply by changing the processing crosslinker additive. We present a comprehensive characterization of both 2D PEDOT:PSS thin films of controlled thicknesses, and 3D porous PEDOT:PSS structures made by the freeze-drying technique. By slicing the bulky scaffolds we show that homogeneous, porous 250 um thick PEDOT:PSS slices are produced, generating biocompatible 3D constructs able to support stem cell cultures. These multifunctional membranes are attached on Indium-Tin oxide substrates (ITO) with the help of an adhesion layer that is used to minimize the interface charge resistance. The optimum electrical contact result in 3D devices with a characteristic and reproducible, frequency dependent impedance response. This response changes drastically when human adipose derived stem cells grow within the porous PEDOT:PSS network as revealed by fluorescence microscopy. The increase of these stem cell population within the PEDOT:PSS porous network impedes the charge flow at the interface between PEDOT:PSS and ITO, enabling the interface resistance to be extracted by equivalent circuit modelling, used here as a figure of merit to monitor the proliferation of stem cells. The strategy of controlling important properties of 3D PEDOT:PSS structures simply by altering processing parameters can be applied for development of a number of stem cell in vitro models. We believe the results presented here will advance 3D bioelectronic technology for both fundamental understanding of in vitro stem cell cultures as well as the development of personalized therapies.


title: Transcriptome of the coralline alga Calliarthron tuberculosum (Corallinales, Rhodophyta) reveals convergent evolution of a partial lignin biosynthesis pathway

abstract: The discovery of lignins in the coralline red alga Calliarthron tuberculosum raised new questions about the deep evolution of lignin biosynthesis. Here we present the transcriptome of C. tuberculosum supported with newly generated genomic data to identify gene candidates from the monolignol biosynthetic pathway using a combination of sequence similarity-based methods. We identified candidates in the monolignol biosynthesis pathway for the genes 4CL, CCR, CAD, CCoAOMT, and CSE but did not identify candidates for PAL, CYP450 (F5H, C3H, C4H), HCT, and COMT. In gene tree analysis, we present evidence that these gene candidates evolved independently from their land plant counterparts, suggesting convergent evolution of a complex multistep lignin biosynthetic pathway in this red algal lineage. Additionally, we provide tools to extract metabolic pathways and genes from the newly generated transcriptomic and genomic datasets. Using these methods, we extracted genes related to sucrose metabolism and calcification. Ultimately, this transcriptome will provide a foundation for further genetic and experimental studies of calcifying red algae.


title: Human Digital Twin: Automated Cell Type Distance Computation and 3D Atlas Construction in Multiplexed Skin Biopsies

abstract: Mapping the human body at single cell resolution in three-dimensions (3D) is an important step toward a "digital twin" model that captures important structure and dynamics of cell-cell interactions. Current 3D imaging methods suffer from low resolution and are limited in their ability to distinguish cell types and their spatial relationships. We present a novel 3D workflow: MATRICS-A (Multiplexed Image Three-D Reconstruction and Integrated Cell Spatial - Analysis) that generates a 3D map of cells from multiplexed images and calculates cell type distance from endothelial cells and other features of interest. We applied this workflow to multiplexed data from sequential skin sections from younger and older donors (n=10; 33-72 years) with biopsies from ten anatomical regions with different sun exposure effects (mild, moderate-marked). Up to 26 sequential sections from each sample underwent multiplexed imaging with 18 biomarkers covering 12 cell types (keratinocytes (granular, spinous, basal), epithelial and myoepithelial cells, fibroblasts, macrophages, T helpers, T killers, T regs, neurons and endothelial cells, markers of DNA damage and repair (p53, DDB2) and cell proliferation (Ki67). Following cell classification, the tissue and classified cells were reconstructed into 3D volumes. A significant inverse correlation between DDB2 positive cells and age was found (corr= -0.78, adj. p=0.047). This suggests reduced capacity for repair in non-cancer older sun-exposed individuals. While absolute immune cell count did not differ by age or sun exposure, the ratio of T Helper/T Killer cells was positively correlated with age (corr=0.82, adj. p=0.048) This is the first such 3D study in skin and paves the way for cataloging more cell types and spatial relationships in aging and disease in skin and other organs.


title: Nitrite lowers the oxygen cost of ATP supply in skeletal muscle cells by stimulating the rate of glycolytic ATP synthesis

abstract: Dietary nitrate lowers the oxygen cost of human exercise. This effect has been suggested to result from stimulation of coupling efficiency of skeletal muscle oxidative phosphorylation by reduced nitrate derivatives. In this paper, we report the acute effects of sodium nitrite on the bioenergetic behaviour of L6 myocytes. At odds with improved efficiency of mitochondrial ATP synthesis, extracellular flux analysis reveals that a [1/2]-hour exposure to NaNO2 (0.1 - 5 {micro}M) significantly decreases mitochondrial coupling efficiency in static myoblasts and tends to lower it in spontaneously contracting myotubes. Unexpectedly, NaNO2 stimulates the rate of glycolytic ATP production in both myoblasts and myotubes. Increased ATP supply through glycolysis does not emerge at the expense of oxidative phosphorylation, which means that NaNO2 acutely increases the rate of overall myocellular ATP synthesis, highly significantly so in myoblasts and tending towards significance in contractile myotubes. Notably, NaNO2 exposure shifts myocytes to a more glycolytic phenotype. Mitochondrial oxygen consumption does not decrease after NaNO2 exposure, and non-mitochondrial respiration tends to drop. When total ATP synthesis rates are normalised to total cellular oxygen consumption rates, it thus transpires that NaNO2 lowers the oxygen cost of ATP supply in L6 myocytes.


title: Cellular compartmentalisation and receptor promiscuity as a strategy for accurate and robust inference of position during morphogenesis

abstract: Precise spatial patterning of cell fate during morphogenesis requires accurate inference of cellular position. In making such inferences from morphogen profiles, cells must contend with inherent stochasticity in morphogen production, transport, sensing and signalling. Motivated by the multitude of signalling mechanisms in various developmental contexts, we show how cells may utilise multiple tiers of processing (compartmentalisation) and parallel branches (multiple receptor types), together with feedback control, to bring about fidelity in morphogenetic decoding of their positions within a developing tissue. By simultaneously deploying specific and nonspecific receptors, cells achieve a more accurate and robust inference. We explore these ideas in the patterning of Drosophila melanogaster wing imaginal disc by Wingless morphogen signalling, where multiple endocytic pathways participate in decoding the morphogen gradient. This distributed information processing at the scale of the cell highlights how local cell autonomous control facilitates global tissue scale design.


title: Liquid droplet aging and fibril formation of the stress granule protein TIA1 low complexity domain

abstract: Protein domains biased toward a few amino acid types are vital for the formation of biomolecular condensates in living cells. These membraneless compartments are formed by molecules exhibiting a range of molecular motions and structural order. Missense mutations increase condensate persistence lifetimes or structural order, properties that are thought to underlie pathological protein aggregation. We examined seeded fibrils of the T-cell restricted intracellular antigen-1 low complexity domain and determined residues 338-357 compose the rigid fibril core. Aging of wild-type and P362L mutant low complexity domain liquid droplets resulted in fibril assemblies that are structurally distinct from the seeded fibril preparation. The results show that most disease mutations lie outside the region that forms homogeneous fibril structure, the droplets age into conformationally heterogenous fibrils, and the P362L disease mutation does not favor a specific fibril conformation.


title: Behavioral and imaging analysis of Foxg1 heterozygous mice

abstract: FOXG1 Syndrome (FS) is a devastating neurodevelopmental disorder that is caused by a heterozygous loss-of-function (LOF) mutation of the FOXG1 gene, which encodes a transcriptional regulator important for telencephalic brain development. People with FS have marked developmental delays, impaired ambulation, movement disorders, seizures, and behavior abnormalities including autistic features. Current therapeutic approaches are entirely symptomatic, however the ability to rescue phenotypes in mouse models of other genetic neurodevelopmental disorders such as Rett syndrome, Angelman syndrome, and Phelan-McDermid syndrome by postnatal expression of gene products has led to hope that similar approaches could help modify the disease course in other neurodevelopmental disorders such as FS. While FoxG1 protein function plays a critical role in embryonic brain development, the ongoing adult expression of FoxG1 and behavioral phenotypes that present when FoxG1 function is removed postnatally provides support for opportunity for improvement with postnatal treatment. Here we generated a new mouse allele of Foxg1 that disrupts protein expression and characterized the behavioral and structural brain phenotypes in heterozygous mutant animals. These mutant animals display changes in locomotor behavior, gait, anxiety, social interaction, aggression, and learning and memory compared to littermate controls. Additionally, they have structural brain abnormalities reminiscent of people with FS. This information provides the framework for future studies to evaluate the potential for post-natal expression of FoxG1 to modify the disease course in this severe neurodevelopmental disorder.


title: S-adenosylmethionine synthases specify distinct H3K4me3 populations and gene expression patterns during heat stress

abstract: Methylation is a widely occurring modification that requires the methyl donor S- adenosylmethionine (SAM) and acts in the regulation of gene expression and other processes. SAM is synthesized from methionine, which is imported or generated through the 1-carbon cycle (1CC). Alterations in 1CC function have clear effects on lifespan and stress-responsive phenotypes, but specific mechanistic links have been difficult to identify because methylation is a widely distributed modification. Here we find that two SAM synthases in Caenorhabditis elegans, SAMS-1 and SAMS-4, contribute differently to modification of H3K4me3, gene expression and survival in the heat stress response. Both synthases are expressed in intestinal and hypodermal cells, which are major stress responsive tissues. We find that SAM is provisioned to distinct targets, even within the same methylation mark, depending on the enzymatic source. This suggests that determining how methyl donors are provided will broaden insight into 1CC functions in aging and stress.


title: Constitutive, calcium-independent endoplasmic reticulum-plasma membrane contact site oscillations and its implications in store-operated calcium entry

abstract: The endoplasmic reticulum and plasma membrane (ER-PM) contact site dynamics plays a central role for store-operated calcium entry (SOCE). ER localized calcium sensor STIM1 translocates to the contact sites, interacts with Orai and mediates calcium influx from the extracellular environment. Different species of phosphoinositides (PIPs) have been reported to be involved in contact site dynamics as well as STIM1 translocation. However, most of the studies were based on loss-of-function experiments or conditions that generate massive calcium store depletion. The kinetics of ER-PM contact site dynamics during physiological stimuli -induced calcium oscillations are not well understood. Using total internal reflection fluorescence microscopy (TIRFM), we investigated the relationship between dynamics of STIM1 as well as cortical ER (cER) proteins and calcium oscillations in rat basophilic leukemia (RBL) mast cells. Surprisingly, a significant percentage of cells displayed cyclic STIM1 and cER dynamics that were calcium-independent. Using specific lipid sensors, we showed that cyclic ER-PM contact site assembly was in phase with PI(4)P oscillation, but preceded phases of PI(4,5)P2 or PI(3,4,5)P3 oscillation. Optogenetic recruitment of the phosphoinositide 5-phosphatase from INPP5E, which decreased PI(4,5)P2 and increased PI(4)P levels on the plasma membrane, stimulated the translocation of STIM1 and inhibited calcium oscillations. Interestingly, prolonged stable translocation of STIM1 to the plasma membrane had an inhibitory effect on calcium oscillations. Collectively, our findings suggest that ER-PM contact sites formation is PI(4)P-dependent. In addition, reversibility of ER-PM contact sites dynamics and intermediate strength of ER-PM contact are needed for calcium oscillation.


title: Ancestral origins are associated with SARS-CoV-2 susceptibility and protection in a Florida patient population

abstract: COVID-19 is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The severity of COVID-19 is highly variable and related to known (e.g., age, obesity, immune deficiency) and unknown risk factors. The widespread clinical symptoms encompass a large group of asymptomatic COVID-19 patients, raising a crucial question regarding genetic susceptibility, e.g., whether individual differences in immunity play a role in patient symptomatology and how much human leukocyte antigen (HLA) contributes to this. To reveal genetic determinants of susceptibility to COVID-19 severity in the population and further explore potential immune-related factors, we performed a genome-wide association study on 284 confirmed COVID-19 patients (cases) and 95 healthy individuals (controls). We compared cases and controls of European (EUR) ancestry and African American (AFR) ancestry separately. We identified two loci on chromosomes 5q32 and 11p12, which reach the significance threshold of suggestive association (p<1x10-5 threshold adjusted for multiple trait testing) and are associated with the COVID-19 susceptibility in the European ancestry (index rs17448496: odds ratio [OR] = 0.173; 95% confidence interval [CI], 0.08-0.36 for G allele; p=5.15x 10-5 and index rs768632395: OR = 0.166; 95% CI, 0.07-0.35 for A allele; p= 4.25x10-6, respectively), which were associated with two genes, PPP2R2B at 5q32, and LRRC4C at 11p12, respectively. To explore the linkage between HLA and COVID-19 severity, we applied fine-mapping analysis to dissect the HLA association with mild and severe cases. Using In-silico binding predictions to map the binding of risk/protective HLA to the viral structural proteins, we found the differential presentation of viral peptides in both ancestries. Lastly, extrapolation of the identified HLA from the cohort to the worldwide population revealed notable correlations. The study uncovers possible differences in susceptibility to COVID-19 in different ancestral origins in the genetic background, which may provide new insights into the pathogenesis and clinical treatment of the disease.


title: High-throughput imaging and quantitative analysis uncovers the nature of plasmid positioning by ParABS

abstract: The faithful segregation and inheritance of bacterial chromosomes and low-copy number plasmids requires dedicated partitioning systems. The most common of these, ParABS, consists of ParA, a DNA-binding ATPase and ParB a protein that binds to centromeric-like parS sequences on the DNA cargo. The resulting nucleoprotein complexes are believed to move up a self-generated gradient of nucleoid-associated ParA. However, it remains unclear how this leads to the observed cargo positioning and dynamics. In particular, the evaluation of models of plasmid positioning has been hindered by the lack of quantitative measurements. Here, we use high-throughput imaging, analysis and modelling to determine the dynamical nature of these systems. We find that F plasmid is actively brought to specific subcellular home positions within the cell with dynamics akin to an over-damped spring. We identify ParA hopping on the nucleoid as the primary determinant of this geometry-sensing and develop a unified computational model that quantitatively explains the observed behaviour. Furthermore, we confirm our model prediction that cells with the lowest plasmid concentration transition to oscillatory dynamics and find similar behaviour in a distantly-related ParABS system. Our results indicate that ParABS regularly positions plasmids across the nucleoid but operates just below the threshold of an oscillatory instability, minimising ATP consumption. Overall, this work uncovers the dynamical nature of plasmid positioning by ParABS and provides insights relevant for chromosome-based systems.


title: Prediction of gene essentiality using machine learning and genome-scale metabolic models

abstract: The identification of essential genes, i.e. those that impair cell survival when deleted, requires large growth assays of knock-out strains. The complexity and cost of such experiments has triggered a growing interest in computational methods for gene essentiality prediction. In the case of metabolic genes, Flux Balance Analysis (FBA) is widely employed to predict essentiality under the assumption that cells maximize their growth rate. However, this approach implicitly assumes that knock-out strains optimize the same objectives as the wild-type, which excludes cases in which deletions cause large changes in cell physiology to meet other objectives for survival. Here we resolve this limitation with a novel machine learning approach that predicts essentiality directly from wild-type flux distributions. We first project the wild-type FBA solution onto a mass flow graph, a digraph with reactions as nodes and edge weights proportional to the mass transfer between reactions, and then train binary classifiers on the connectivity of graph nodes. We demonstrate the efficacy of this approach using the most complete metabolic model of Escherichia coli, achieving near state-of-the art prediction accuracy for essential genes. Our approach suggests that wild-type FBA solutions contain enough information to predict essentiality, without the need to assume optimality of deletion strains.

category: systems biology


title: Evaluation of the broth microdilution methodology for susceptibility testing of Mycobacterium tuberculosis in Peru

abstract: Peru is amongst the 30 countries with the highest burden of multidrug-resistant tuberculosis worldwide. In the fight against drug-resistant tuberculosis, the UKMYC6 microtiter plate was developed and validated by the CRyPTIC project. Our objective was to evaluate the use of the broth microdilution UKMYC6 plate for susceptibility testing of drug-resistant Mycobacterium tuberculosis (MTB) strains in Peru. 496 nationally-representative MTB strains determined drug-resistant by the routine agar proportion method (APM) were selected. MICs of 13 anti-tuberculosis drugs were determined for each strain using the microdilution UKMYC6 plates and compared with the APM result. MIC distributions for APM-susceptible and APM-resistant strains were demonstrated for rifampicin, isoniazid, kanamycin, and levofloxacin, with reasonable agreement (0.64[≤]k[≤]0.79) for rifampicin, ethambutol, ethionamide and kanamycin and the best agreement for isoniazid and levofloxacin (k>0.8). No strain presented MICs higher than the CRyPTIC Epidemiological cut-off values for the new (bedaquiline, delamanid) or repurposed (clofazimine, linezolid) drugs. The microbroth dilution method using the UKMYC6 microtiter plate allowed the complete susceptibility characterization, through the determination of MICs, of drug-resistant Mycobacterium tuberculosis strains in Peru. This methodology showed a good diagnostic performance for the drugs rifampicin, isoniazid, kanamycin and levofloxacin drugs.

category: microbiology


title: Influenza A virus RNA Polymerase targets the chromatin of innate immune response genes

abstract: The influenza A virus (IAV) profoundly affects host cell nuclear processes to accommodate for its transcription. In particular, the IAV RNA polymerase (FluPol) associates with host genes to snatch capped 5-ends from nascent messenger RNAs, that are then used to prime viral transcription. The extend of FluPol-host gene interaction and its actual consequence on host transcription are still poorly characterized. We show here, using genome-wide approaches, that the PA, PB1, and PB2 subunits of FluPol establish contacts with both chromatin and RNA transcripts at promoters, but also within the coding region of genes. These interactions also reach into downstream regions where recruitment of FluPol subunits correlates with previously reported transcription termination defects induced by IAV, indicative of an implication of the FluPol in this viral strategy to limit host cell defense by interfering with transcription re-initiation. The latter is further suggested by a bias in the FluPol genomic targets, enriched in genes associated with anti-viral defense. Together, our observations suggest that FluPol-chromatin binding contributes to targeted dampening of host immune response pathways.


title: Plastomes of Hyalomonas oviformis and Hyalogonium fusiforme evolved dissimilar architecture after loss of photosynthesis

abstract:
The loss of photosynthesis in land plants and algae is typically associated with parasitism but can also occur in free-living species, including chlamydomonadalean green algae. The plastid genomes (ptDNAs) of colorless chlamydomonadalean species are surprisingly diverse in architecture, including highly expanded forms (Polytoma uvella, Leontynka pallida) as well as outright genome loss (Polytomella species). Here, we explore the ptDNAs of Hyalomonas (Hm.) oviformis (SAG 62-27; formerly known as Polytoma oviforme) and Hyalogonium (Hg.) fusiforme (SAG 62-1c), each representing independent losses of photosynthesis within the Chlamydomonadales. The Hm. oviformis ptDNA is moderately sized (132 kb), smaller than that of its photosynthetic relative Hyalomonas chlamydogama SAG 11-48b (198.3 kb), with a reduced gene complement but still encoding the ATPase subunits. The Hg. fusiforme plastome, however, is the largest yet observed in colorless plants or algae (~463 kb) and has a coding repertoire that is almost identical to that of its photosynthetic relatives in the genus Chlorogonium. Furthermore, the ptDNA of Hg. fusiforme shows no clear evidence of pseudogenization, which is consistent with our analyses showing that Hg. fusiforme is the non-photosynthetic lineage of most recent origin among the known colorless Chlamydomonadales. Together, these new ptDNAs clearly show that, in contrast to parasitic algae, plastid genome compaction is not an obligatory route following the loss of photosynthesis in free-living algae, and that certain chlamydomonadalean algae have a remarkable propensity for genomic expansion, which can persist regardless of the trophic strategy.

One sentence summaryThe plastid genomes of two free-living chlamydomonadalean algae, Hyalomonas oviformis and Hyalogonium fusiforme, reveal different evolutionary stages following the loss of photosynthesis.

category: genomics


title: WWP2 osteoarthritis risk allele rs1052429-A confers risk by affecting cartilage matrix deposition via hypoxia associated genes

abstract:
ObjectiveTo explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele rs1052429-A on WWP2 expression in a human 3D in vitro model of cartilage.

MethodsCo-expression behavior of WWP2 with genes expressed in lesioned OA articular cartilage (N=35 samples) was explored. By applying lentiviral particle mediated WWP2 upregulation in 3D in vitro pellet cultures of human primary chondrocytes (N=8 donors) the effects of upregulation on cartilage matrix deposition was evaluated. Finally, we transfected primary chondrocytes with miR-140 mimics to evaluate whether miR-140 and WWP2 are involved in similar pathways.

ResultsUpon performing Spearman correlations in lesioned OA cartilage, 98 highly correlating genes (|{rho}|>0.7) were identified. Among these genes, we identified GJA1, GDF10, STC2, WDR1, and WNK4. Subsequent upregulation of WWP2 on 3D chondrocyte pellet cultures resulted in a decreased expression of COL2A1 and ACAN and an increase in EPAS1 expression. Additionally, we observed a decreased expression of GDF10, STC2, and GJA1. Proteomics analysis identified 42 proteins being differentially expressed with WWP2 upregulation, which were enriched for ubiquitin conjugating enzyme activity. Finally, upregulation of miR-140 in 2D chondrocytes resulted in significant upregulation of WWP2 and WDR1.

ConclusionsMimicking the effect of OA risk allele rs1052429-A on WWP2 expression initiates detrimental processes in the cartilage shown by a response in hypoxia associated genes EPAS1,GDF10, and GJA1 and a decrease in anabolic markers, COL2A1 and ACAN.