LCK Regulates Homologous Recombination DNA Repair Identifying a New Therapeutic Target for Sensitizing PARP Inhibitors in HR Proficient Ovarian Cancer Poly-ADP Ribose Polymerase (PARP) targeted therapy is clinically approved for the treatment of homologous recombination (HR) repair deficient tumors. The remarkable success in treatment of HR repair deficient cancers has not translated to HR-proficient cancer. Our studies identify the mechanism of non-receptor lymphocyte-specific protein tyrosine kinase (LCK) in HR repair in endometrioid epithelial ovarian cancer (eEOC). LCK expression is induced and activation in the nucleus in response to DNA damage insult. LCK inhibition attenuates expression of RAD51, BRCA1, and BRCA2 proteins necessary for HR-mediated DNA repair, sufficient to suppress RAD51 foci formation, and augments {gamma}H2AX foci formation. Mechanistically, DNA damage leads to direct interaction of LCK with RAD51 and BRCA1 in a kinase dependent manner. Attenuation of LCK sensitized HR-proficient eEOC cells to PARP inhibitor in cell culture and pre-clinical mouse studies. These findings identify a new mechanism for expanding utility of PARP inhibitors in HR proficient ovarian cancer.

Graphical Abstract

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An exploratory study on the role of the stiffness of breast cancer cells in their detachment from spheroids and migration in 3D collagen matrices BackgroundTumour-cell detachment is a critical early event in the metastatic cascade. However, the role of the cells mechanical properties in detachment and migration is not well understood. This exploratory study aimed to assess how intracellular stiffness changes these processes.

MethodsMDA-MB-231 cells were embedded as 10,000-cell spheroids in 2 and 4 mg/ml collagen matrices. Intracellular stiffness was assessed with mitochondria tracking microrheology of cells that migrated distances equivalent to four and six times the cell diameter (dC) from the spheroid and compared to cells at the spheroid surface (0dC), representing medium, high and no migration, respectively.

Results and discussionThe mitochondrial mean square displacement and intracellular stiffness decreased during detachment and migration for both collagen concentrations (i.e. rigidities). The mean square displacement of 4dC and 6dC cells was similar, whereas cell stiffness was lower for 4dC than for 6dC cells. With increasing matrix rigidity, the intracellular stiffness decreased for 0dC cells and did not change for 4dC and 6dC cells. It is proposed that decreased cell stiffness drives detachment and migration and increased matrix rigidity physically hinders migration, and cells need to become softer or remodel the environment to migrate. The independence of the stiffness of migrated cells from matrix rigidity suggests that cells remodel their environment through matrix proteins cleavage to migrate.

ConclusionsThe study revealed the collective effects of enhanced migratory conditions and increased matrix rigidity on the mechanical properties of the cells. The expression of matrix metalloproteinases and transforming growth factor {beta} and the role of cell volume on detachment and migration in matrices with varying pore sizes are proposed targets for further studies on metastatic cancer cells.
Identification of a stereotypic molecular arrangement of endogenous glycine receptors at spinal cord synapses Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in spinal cord tissue using quantitative super-resolution correlative light and electron microscopy (SR-CLEM). We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant packing densities of about 2000 GlyRs m-2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks. Characterization of methylation profiles in spontaneous preterm birth placental villous tissue Preterm birth is a global public health crisis which results in significant neonatal and maternal mortality. Yet little is known regarding the molecular mechanisms of idiopathic spontaneous preterm birth, and we have few diagnostic markers for adequate assessment of placental development and function. Previous studies of placental pathology and our transcriptomics studies suggest a role for placental maturity in idiopathic spontaneous preterm birth. It is known that placental DNA methylation changes over gestation. We hypothesized that if placental hypermaturity is present in our samples, we would observe a unique idiopathic spontaneous preterm birth DNA methylation profile potentially driving the gene expression differences we previously identified in our placental samples. Our results indicate the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern suggesting hypermaturity. Only seven significant differentially methylated regions fitting the idiopathic spontaneous preterm birth specific (relative to the controls) profile were identified, indicating unusually high similarity in DNA methylation between idiopathic spontaneous preterm birth and term birth samples. We identified an additional 1,718 significantly methylated regions in our gestational age matched controls were the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern, again indicating a striking level of similarity between the idiopathic spontaneous preterm birth and term birth samples. Pathway analysis of these regions revealed differences in genes within the WNT and Cadherin signaling pathways, both of which are essential in placental development and maturation. Taken together, these data demonstrate that the idiopathic spontaneous preterm birth samples are molecularly more mature than expected given their respective gestational age which likely impacts birth timing. Using total RNA quality metrics for time since deposition estimates in degrading bloodstains Determining the time since deposition (TSD) of bloodstains would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. The physicochemical changes occurring to biomolecules as a bloodstain degrades can be used to approximate the TSD of bloodstains. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has previously targeted mRNA molecules. Whole bovine blood was stored in plastic microcentrifuge tubes at 21{degrees}C or 4{degrees}C and tested over different timepoints spanning one week. Nine RNA metrics were visually assessed and quantified using linear and mixed models; the RNA Integrity Number equivalent (RINe) and the DV200 demonstrated strong negative trends over time and statistical independence. The RINe model fit was high (R2 = 0.60), and while including the biological replicate as a random effect increased the fit for all RNA metrics, no significant differences were found between biological replicates stored at the same temperature for the RINe and DV200 metrics. Importantly, this suggests that these standardized metrics can likely be directly compared between scenarios and individuals, with DV200 having an inflection point at [~]28 hrs. This study provides a novel approach for blood TSD estimates, producing metrics that are not affected by inter-individual variation and improving our understanding of the rapid degradation occurring in bloodstains.

HIGHLIGHTSO_LIAmplification-free analysis of total RNA in degrading bloodstains.
C_LIO_LIShort-term RNA degradation assessment using high-resolution size measurements.
C_LIO_LITotal RNA quality and quantity metrics were assessed across a one-week.
C_LIO_LITotal RNA quality metrics demonstrated the strongest timewise trends.
C_LIO_LIBiological replicates produced similar results for RNA quality metrics.
C_LI Major orchestration of shikimate, early phenylpropanoid and stilbenoid pathways by Subgroup 2 R2R3-MYBs in grapevine The stilbenoid pathway is responsible for the production of resveratrol and its derivatives in grapevine. A few transcription factors (TFs) have been previously identified as regulators of this pathway but the extent of this control is yet to be fully understood. Here we demonstrate how DNA affinity purification sequencing (DAP-Seq) allows for genome-wide TF binding site interrogation in a non-model species. We obtained 5,190 and 4,443 binding events assigned to 4,041 and 3,626 genes for MYB14 and MYB15, respectively (around 40% of peaks being located within -10kb of transcription start sites). DAP-Seq of MYB14 and MYB15 was combined with aggregate gene centred co-expression networks built from more than 1,400 transcriptomic datasets from leaves, fruits and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15 and MYB13, a third uncharacterised member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS ) targets, these three regulators bind to 30 out of 47 STS family genes. Moreover all three MYBs bind to several PAL, C4H and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and novel resveratrol-modifying gene candidates amongst which ROMT2 -3 were validated enzymatically. A high proportion of DAP-Seq bound genes was induced in the activated transcriptomes of transient MYB15 -overexpressing stilbenoid-producing grapevine leaves, validating our methodological approach for identifying gene regulatory networks of specialised metabolism. Overall, MYB genes from Subgroup 2 appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production. Active enhancers strengthen insulation by RNA-mediated CTCF binding at TAD boundaries Vertebrate genomes are partitioned into Topologically Associating Domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at TAD boundaries correlates with better insulation, however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer:promoter interactions and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters we show that active TAD enhancers but not promoters induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs. "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. Glucose inhibits haemostasis and accelerates diet-induced hyperlipidaemia in zebrafish larvae Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals. An estimate of the deepest branches of the tree of life from ancient vertically-evolving genes Core gene phylogenies provide a window into early evolution, but different gene sets and analytical methods have yielded substantially different views of the tree of life. Trees inferred from a small set of universal core genes have typically supported a long branch separating the archaeal and bacterial domains. By contrast, recent analyses of a broader set of non-ribosomal genes have suggested that Archaea may be less divergent from Bacteria, and that estimates of inter-domain distance are inflated due to accelerated evolution of ribosomal proteins along the inter-domain branch. Resolving this debate is key to determining the diversity of the archaeal and bacterial domains, the shape of the tree of life, and our understanding of the early course of cellular evolution. Here, we investigate the evolutionary history of the marker genes key to the debate. We show that estimates of a reduced Archaea-Bacteria (AB) branch length result from inter-domain gene transfers and hidden paralogy in the expanded marker gene set. By contrast, analysis of a broad range of manually curated marker gene datasets from an evenly sampled set of 700 Archaea and Bacteria reveal that current methods likely underestimate the AB branch length due to substitutional saturation and poor model fit; that the best-performing phylogenetic markers tend to support longer inter-domain branch lengths; and that the AB branch lengths of ribosomal and non-ribosomal marker genes are statistically indistinguishable. Furthermore, our phylogeny inferred from the 27 highest-ranked marker genes recovers a clade of DPANN at the base of the Archaea, and places CPR within Bacteria as the sister group to the Chloroflexota. Allosteric activation of SARS-CoV-2 RdRp by remdesivir triphosphate and other phosphorylated nucleotides The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), Nsp12, has a unique NiRAN domain that transfers nucleoside monophosphates to the Nsp9 protein. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation) Nsp12 exists in inactive state in which NiRAN/RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or NTPs partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN/RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically-linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and design of its inhibitors.

HighlightsO_LICodon-optimization of Nsp12 triggers misfolding and activity loss
C_LIO_LISlow translation, accessory Nsp7 and Nsp8 subunits, and NTPs rescue Nsp12
C_LIO_LINon-substrate nucleotides activate RNA chain synthesis, likely via NiRAN domain
C_LIO_LICrosstalk between two Nsp12 active sites that bind the same ligands
C_LI Bioactive constituents of Verbena officinalis regulate killing efficiency of primary human natural killer cells by accelerating killing processes BackgroundNatural killer (NK) cells play a key role in eliminating tumorigenic and pathogen-infected cells. Verbena officinalis (V. officinalis) has been used as a medical plant in traditional and modern medicine, exhibiting anti-tumor and anti-inflammation activity, but its role in NK cells remains unclear.

PurposeThe impact of bioactive constituents of V. officinalis on immune responses remains largely elusive. In this work we investigated the potential targets of V. officinalis and focused on killing efficiency and related functions of NK cells regulated by bioactive constituents of V. officinalis.

Study design/methodsWe used primary human NK cells from peripheral blood mononuclear cells. Potential regulatory roles of selected compounds in NK killing function-related genes were first analyzed by network pharmacology approaches. Potential engagement between compounds and NK receptors was examined by molecular docking assay. Killing efficiency of NK cells was determined with real-time killing assay and live-cell imaging. Proliferation was examined by CFSE staining. Expression of cytotoxic proteins was analyzed using flow cytometry. Lytic granule release was quantified by CD107a degranulation assay. Contact time required for killing and determination of serial killers were analyzed using live cell imaging results.

ResultsResults from network pharmacology approaches and molecular docking analyses predicted that the five bioactive compounds (acteoside, apigenin, kaempferol, verbenalin and hastatoside) from V. officinalis can regulated various key genes essential for NK effector functions and can bind to NK inhibitory receptors as well as activating receptors, indicating their potential role in regulating NK killing capacity. Analyses of NK killing efficiency revealed that out of these five compounds verbenalin exhibited an encouraging potency to enhance NK killing efficiency. Further investigation showed that verbenalin did not affect proliferation, expression of cytotoxic proteins, or lytic granule degranulation, but rather reduced contact time required for killing, enhanced total killing events per NK cell, and elevated the fraction of killing competent NK cells.

ConclusionsIn this work, we have identified verbenalin as the most potent bioactive constituent from V. officinalis to enhance NK cell killing efficiency via accelerating killing processes and converting non-killers to killing competent NK cells without affecting the lytic granule pathway. A biochemically-realisable relational model of the self-manufacturing cell As shown by Hofmeyr, the processes in the living cell can be divided into three classes of efficient causes that produce each other, so making the cell closed to efficient causation, the hallmark of an organism. They are the enzyme catalysts of covalent metabolic chemistry, the intracellular milieu that drives the supramolecular processes of chaperone-assisted folding and self-assembly of polypeptides and nucleic acids into functional catalysts and transporters, and the membrane transporters that maintain the intracellular milieu, in particular its electrolyte composition. Each class of efficient cause can be modelled as a relational diagram in the form of a mapping in graph-theoretic form, and a minimal model of a self-manufacturing system that is closed to efficient causation can be constructed from these three mappings using the formalism of relational biology. This Fabrication-Assembly or (F,A)-system serves as an alternative to Robert Rosens replicative Metabolism-Repair or (M,R)-system, which has been notoriously problematic to realise in terms of real biochemical processes. A key feature of the model is the explicit incorporation of formal cause, which arrests the infinite regress that plagues all relational models of the cell. The (F,A)-system is extended into a detailed relational model of the self-manufacturing cell that has a clear biochemical realisation. This (F,A) cell model allows the interpretation and visualisation of concepts such as the metabolism and repair components of Rosens (M,R)-system, John von Neumanns universal constructor, Howard Pattees symbol-function split via the symbol-folding transformation, Marcello Barbieris genotype-ribotype-phenotype ontology, and Tibor Gantis chemoton. Thermal modulation of Zebrafish exploratory statistics reveals constraints on individual behavioral variability BackgroundVariability is a hallmark of animal behavior. It contributes to survival by endowing individuals and populations with the capacity to adapt to ever-changing environmental conditions. Intra-individual variability is thought to reflect both endogenous and exogenous modulations of the neural dynamics of the central nervous system. However, how variability is internally regulated and modulated by external cues remains elusive. Here we address this question by analyzing the statistics of spontaneous exploration of freely swimming zebrafish larvae, and by probing how these locomotor patterns are impacted when changing the water temperatures within an ethologically relevant range.

ResultsWe show that, for this simple animal model, five short-term kinematic parameters - interbout interval, turn amplitude, travelled distance, turn probability and orientational flipping rate - together control the long-term exploratory dynamics. We establish that the bath temperature consistently impacts the means of these parameters, but leave their pairwise covariance unchanged. These results indicate that the temperature merely controls the sampling statistics within a well-defined kinematic space delineated by this robust statistical structure. At a given temperature, individual animals explore the behavioral space over a timescale of tens of minutes, suggestive of a slow internal state modulation that could be externally biased through the bath temperature. By combining these various observations into a minimal stochastic model of navigation, we show that this thermal modulation of locomotor kinematics results in a thermophobic behavior, complementing direct gradient-sensing mechanisms.

ConclusionsThis study establishes the existence of a well-defined locomotor space accessible to zebrafish larvae during spontaneous exploration, and quantifies self-generated modulation of locomotor patterns. Intra-individual variability reflects a slow diffusive-like probing of this space by the animal. The bath temperature in turn restricts the sampling statistics to sub-regions, endowing the animal with basic thermophobicity. This study suggests that in Zebrafish, as well as in other ectothermic animals, ambient temperature could be used to efficiently manipulate internal states in a simple and ethological way. A Hierarchy of Biomolecular Proportional-Integral-Derivative Feedback Controllers for Robust Perfect Adaptation and Dynamic Performance Proportional-Integral-Derivative (PID) feedback controllers have been the most widely used controllers in industry for almost a century due to their good performance, simplicity, and ease of tuning. Motivated by their success in various engineering disciplines, PID controllers recently found their way into synthetic biology, where the design of feedback molecular control systems has been identified as an important goal. In this paper, we consider the mathematical realization of PID controllers via biomolecular interactions. We propose an array of topologies that offer a compromise between simplicity and high performance. We first demonstrate that different Proportional-Integral (PI) controllers exhibit different capabilities for enhancing the dynamics and reducing variance (cell-to-cell variability). Next, we introduce several derivative controllers that are realized based on incoherent feedforward loops acting in a feedback configuration. Alternatively, we show that differentiators can be realized by placing molecular integrators in a negative feedback loop--an arrangement that can then be augmented by PI components to yield PID feedback controllers. We demonstrate that the derivative component can be exploited for enhancing system stability, dramatically increasing the molecular control systems dynamic performance, and for reducing the noise effect on the output of interest. The PID controller features are established through various deterministic and stochastic analyses as well as numerical simulations. Finally, we provide an experimental demonstration using a recently developed hybrid setup, the cyberloop, where the controller is implemented in silico to control a biological genetic circuit in vivo. The large array of novel biomolecular PID controllers introduced here forms a basis for the design and construction of advanced high-performance biomolecular control systems that robustly regulate the dynamics of living systems. Rescue of stalled clathrin-mediated endocytosis by asymmetric Arp2/3-mediated actin assembly Actin assembly facilitates vesicle formation in several trafficking pathways. Clathrin-mediated endocytosis (CME) shows elevated actin assembly dependence under high membrane tension. Why actin assembly at CME sites occurs heterogeneously even within the same cell, and how assembly forces are harnessed, are not fully understood. Here, endocytic dynamics, actin presence, and geometry of CME proteins from three different functional modules, were analyzed using three-dimensional (3D) super-resolution microscopy, live-cell imaging, and machine-learning-based computation. When hundreds of CME events were compared, sites with actin assembly showed a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. N-WASP is recruited to one side of CME sites where it is positioned to stimulate asymmetric actin assembly. We propose that asymmetric actin assembly rescues stalled CME sites by pulling vesicles into the cell much like a bottle opener pulls off a bottle cap. Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell Endothelial and skeletal muscle lineages arise from common embryonic progenitors. Despite their shared developmental origin, adult endothelial cells (ECs) and muscle stem cells (MuSCs) (satellite cells) have been thought to possess distinct gene signatures and signaling pathways. Here we shift this paradigm by uncovering how adult MuSC behavior is affected by the expression of a subset of EC transcripts. We used several computational analyses including single-cell RNAseq to show that MuSCs express low levels of canonical EC markers. We demonstrate that MuSC survival is regulated by one such prototypic endothelial signaling pathway (VEGFA-FLT1). Using pharmacological and genetic gain- and loss-of-function studies, we identify the FLT1-AKT1 axis as the key effector underlying VEGFA-mediated regulation of MuSC survival. All together, our data support that the VEGFA-FLT1-AKT1 pathway promotes MuSC survival during muscle regeneration, and highlights how the minor expression of select transcripts is sufficient for affecting cell behavior. Onlinemeta: A Web Server For Meta-Analysis Based On R-shiny Meta-analysis is a common statistical method used to summarize multiple studies that cover the same topic. It can provide less biased results and explain heterogeneity between studies. Although there exists a variety of meta-analysis software, they are rarely both convenient to use and capable of comprehensive analytical functions. As a result, we established a meta-analysis web tool called Meta-Analysis Online (Onlinemeta). Onlinemeta includes both risk bias analysis and meta-analysis. The risk bias analysis tool can produce heatmaps and histograms, whereas the meta-analysis tool can be used to analyze dichotomous variables, single-armed dichotomous variables, continuous variables, single-armed continuous variables, survival data, and diagnostic experiments. In addition, it can be used to generate forest plots, funnel plots, SROC curve, and crosshair plots. Onlinemeta can be found at the following address: https://smuonco.Shinyapps.io/Onlinemeta/. Phagocytic Uptake of Polymeric Particles by Immune cells Under Flow Conditions Particles injected intravenously are thought to be cleared by macrophages residing in the liver and spleen, but they also encounter circulating immune cells. It remains to be established if the circulating cells can take up particles while flowing, and if the uptake capacity is similar under static and flow conditions. Here, we use an in vitro peristaltic pump setup that mimics pulsatile blood flow to determine if immune cells take up particles under constant fluidic flow. We use polystyrene particles of varying sizes as the model of a polymeric particle for these studies. Our results show that the immune cells do phagocytose under flow conditions. We demonstrate that cell lines representing myeloid cells, primary human neutrophils and monocytes take up sub-micrometer-sized particles at similar or better rates under flow compared to static conditions. Experiments with whole human blood show that even under the crowding effects of red blood cells, neutrophils and monocytes take up particles while flowing. Together, these data suggest that circulating immune cells are likely to phagocytose intravenously injected particulates, which has implications for design of particles to evade or target these cells. Decreased Interfacial Dynamics Caused by the N501Y Mutation in the SARS-CoV-2 S1 Spike:ACE2 Complex Corona Virus Disease of 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has caused a massive health crisis across the globe, with some genetic variants gaining enhanced infectivity and competitive fitness, and thus significantly aggravating the global health concern. In this regard, the recent SARS-CoV-2 alpha variant, B.1.1.7 lineage, reported from the United Kingdom (UK), is of great significance in that it contains several mutations that increase its infection and transmission rates as evident from clinical reports. Specifically, the N501Y mutation in the SARS-CoV-2 spike S1 receptor binding domain (S1-RBD) has been shown to possess an increased affinity for ACE2, although the basis for this is not entirely clear yet. Here, we dissect the mechanism underlying the increased affinity using molecular dynamics (MD) simulations of the available ACE2-S1-RBD complex structure (6M0J) and show a prolonged and stable interfacial interaction of the N501Y mutant S1-RBD with ACE2 compared to the wild type S1-RBD. Additionally, we find that the N501Y mutant S1-RBD displays altered dynamics that likely aids in its enhanced interaction with ACE2. By elucidating a mechanistic basis for the increased affinity of the N501Y mutant S1-RBD for ACE2, we believe that the results presented here will aid in developing therapeutic strategies against SARS-CoV-2 including designing drugs targeting the ACE2-S1-RBD interaction.

SignificanceThe emergence of the new SARS-CoV-2 lineage in the UK in December 2020 has further aggravated the COVID-19 pandemic due to an increased ability of the variant to infect human hosts, likely due to mutations in the viral S1 spike protein including the N501Y S1-RBD mutation that is located at the interface of S1-RBD and ACE2, the host cell receptor for SARS-CoV-2. Given its location at the interface, N501Y S1-RBD mutation can therefore potentially alter the interfacial interaction. Multiple, all-atom, explicit solvent MD simulations of the ACE2-S1-RBD complex carried here indicated a more stable interaction between the N501Y mutant S1-RBD and ACE2 through stabilizing interfacial interactions of residues at one end of the interface that are either sequentially or physically near the mutation site. These mechanistic details will aid in better understanding the mechanism by which the alpha variant has increased infectivity as well as in designing better therapeutics including ACE2-S1 spike protein inhibitors that will, in turn, help thwarting the current and future pandemic.

HighlightsO_LIN501 in the wild type SARS-CoV-2 S1-RBD forms unsustained hydrogen bonds with residues in the ACE2, namely Y41 and K353
C_LIO_LIY501 in the N501Y mutant SARS-CoV-2 S1-RBD is not capable of forming substantial hydrogen bonds with ACE2 within the time span of the current simulation
C_LIO_LIEvidence from analyzing the simulation results suggests that Y501 of S1-RBD could form other types of non-covalent interactions with ACE2, such as van der Waals interactions
C_LIO_LIN501Y S1-RBD mutation stabilizes the position of interfacial residues neighboring to the mutation site, as well as other non-interfacial residues that are distant from the mutation site
C_LIO_LIThese altered dynamics results in more stable interaction of S1-RBD with ACE2 which could be the main reason underlying the reported enhanced affinity of S1-RBD in the SARS-CoV-2 alpha variant (UK B.1.1.7 lineage) to ACE2
C_LI Characterization of divalent cation interactions with AASTY native nanodiscs Amphiphilic copolymers show promise in extracting membrane proteins directly from lipid bilayers into  native nanodiscs. However, many such copolymers are polyanionic and sensitive to divalent cations, limiting their applicability. We characterize the Ca2+ and Mg2+ sensitivity of poly(acrylic acid-co-styrene) (AASTY) copolymers with analytical UV and fluorescent size exclusion chromatography, enabling us to separate signals from nanodiscs, copolymers, and soluble aggregates. We find that divalent cations promote aggregation and precipitation of both free and lipid bound copolymers. We see that excess, free copolymer acts as a  cation sink that protects nanodiscs from Ca2+ induced aggregation. Removal of the free copolymer through dialysis induces aggregation that can be mitigated by KCl. Finally, we find that the nanodisc size is dynamic and dependent on lipid concentration. Our results offer insight to nanodisc behaviour, and can help guide experimental design, aimed at mitigating the shortcomings inherent in negatively charged nanodisc forming copolymers. Critical re-evaluation of the slope factor of the operational model of agonism Background and PurposeAlthough being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1985, Black et al. introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves.

Experimental ApproachFunctional analysis of OMA demonstrates that the slope factor implemented by Black et al. affects relationships among parameters of the OMA, leading to wrong estimates of operational efficacy and affinity of an agonist. In contrast, functional analysis of OMA with Hill coefficient as a slope factor has not this flaw.

Key ResultsFitting of the OMA with Black et al. slope factor to concentration-response curves of several models (homotropic allosteric modulation, substrate inhibition and non-competitive auto-inhibition) gives wrong estimates of operational efficacy and affinity. In contrast, fitting of the OMA modified by the Hill coefficient to concentration-response curves of these several models gives correct estimates of operational efficacy and affinity.

Conclusion and ImplicationsThe OMA modified by the Hill coefficient can be used for correct ranking of agonism and subsequent analysis, like quantification of signalling bias. Comammox Nitrospira bacteria outnumber canonical nitrifiers irrespective of nitrogen source and availability Complete ammonia oxidizing bacteria coexist with canonical ammonia and nitrite oxidizing bacteria in a wide range of environments. Whether this is due to competitive or cooperative interactions, or a result of niche separation is not yet clear. Understanding the factors driving coexistence of nitrifiers is critical to manage nitrification processes occurring in engineered and natural ecosystems. In this study, microcosm-based experiments were used to investigate the impact of nitrogen source and loading on the population dynamics of nitrifiers in drinking water biofilter media. Shotgun sequencing of DNA followed by co-assembly and reconstruction of metagenome assembled genomes revealed clade A2 comammox bacteria were likely the primary nitrifiers within microcosms and increased in abundance over Nitrsomonas-like ammonia and Nitrospira-like nitrite oxidizing bacteria irrespective of nitrogen source type or loading. Changes in comammox bacterial abundance did not correlate with either ammonia or nitrite oxidizing bacterial abundance in urea amended systems where metabolic reconstruction indicated potential for cross feeding between ammonia and nitrite oxidizing bacteria. In contrast, comammox bacterial abundance demonstrated a negative correlation with nitrite oxidizers in ammonia amended systems. This suggests potentially weaker synergistic relationships between ammonia and nitrite oxidizers might enable comammox bacteria to displace nitrite oxidizers from complex nitrifying communities. Frequency modulated timer regulates mammalian hibernation Mammalian hibernators decrease basal metabolism and body temperature (Tb) to minimize energy expenditure in harsh seasons. During hibernation, Tb drops to low temperature (<10 {degrees}C) and remains constant for days, known as deep torpor in small mammalian hibernators. Spontaneous interbout arousals interrupt torpor bouts, when Tb recovers to euthermic state ~37 {degrees}C. Torpor-interbout arousal event repeats during several months of hibernation. However, little is known about mechanisms governing Tb fluctuation across torpor-interbout arousal cycles during hibernation. Recent improvement in data-logging techniques enables us to monitor Tb for more than hundred days with high precision, opening up new avenues for quantitative analysis to address the principle governing Tb fluctuation. Here, we analyzed Tb fluctuation across torpor-interbout arousal cycle of Syrian hamster, which can hibernate in response to chronic cold and short photoperiod under a laboratory condition, using generalized harmonic analysis and discovered a model with frequency modulation quantitatively reproducing Tb fluctuation. This analysis also identified an unexpectedly longer period of 120-430 days as the period that modulates another period of several days, generating Tb fluctuation for Syrian hamster. We propose that concerted action of two endogenous periods governs torpor-interbout arousal cycles during hibernation. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Identification and assessment of the antibacterial activity of Vigna mungo and Rhizobacteria. 1.ABSTRACTSZnO nanoparticles have received a lot of interest in recent years due to their unusual features. Antimicrobial properties of ZnO NPs However, the qualities of nanoparticles are determined by their size and form, making them suitable for a variety of applications. The current work looks at the synthesis, characterization, and antibacterial activity of ZnO NPs produced by Vigna Mungo and Rhizobacteria. Rhizobacteria isolated from V. mungo root nodule were morphologically, biochemically, and molecularly examined and identified as Rhizobium sp. strain and Bacillus flexus strain. The GC-MS analysis of methanol leaf extract of V. mungo was performed to detect and identify bioactive chemicals, and this indicated phytol as an antibacterial agent, while Squalene and Alpha - tocopherol had antioxidant and anti-tumour properties. Agar well diffusion experiment was used to determine the antibacterial properties of ZnO nanoparticles and Vigna Mungo leaf extract. This approach is widely documented, and standard zones of inhibition for sensitive and resistant values have been defined. The results demonstrated that both methanol extract and zinc oxide nanoparticles have strong antibacterial efficacy against the majority of the pathogens examined.