3CAC: improving the classification of phages and plasmids in metagenomic assemblies using assembly graphs MotivationBacteriophages and plasmids usually coexist with their host bacteria in microbial communities and play important roles in microbial evolution. Accurately identifying sequence contigs as phages, plasmids, and bacterial chromosomes in mixed metagenomic assemblies is critical for further unravelling their functions. Many classification tools have been developed for identifying either phages or plasmids in metagenomic assemblies. However, only two classifiers, PPR-Meta and viralVerify, were proposed to simultaneously identify phages and plasmids in mixed metagenomic assemblies. Due to the very high fraction of chromosome contigs in the assemblies, both tools achieve high precision in the classification of chromosomes but perform poorly in classifying phages and plasmids. Short contigs in these assemblies are often wrongly classified or classified as uncertain.

ResultsHere we present 3CAC, a new three-class classifier that improves the precision of phage and plasmid classification. 3CAC starts with an initial three-class classification generated by existing classifiers and improves the classification of short contigs and contigs with low confidence classification by using proximity in the assembly graph. Evaluation on simulated metagenomes and on real human gut microbiome samples showed that 3CAC outperformed PPR-Meta and viralVerify in both precision and recall, and increased F1-score by 10-60 percentage points.

AvailabilityThe 3CAC software is available on https://github.com/Shamir-Lab/3CAC.

Contactrshamir@tau.ac.il

Supplementary informationSupplementary data are available at Bioinformatics online.
Deep learning tools and modeling to estimate the temporal expression of cell cycle proteins from 2D still images Automatic characterization of fluorescent labeling in intact mammalian tissues remains a challenge due to the lack of quantifying techniques capable of segregating densely packed nuclei and intricate tissue patterns. Here, we describe a powerful deep learning-based approach that couples remarkably precise nuclear segmentation with quantitation of fluorescent labeling intensity within segmented nuclei, and then apply it to the analysis of cell cycle dependent protein concentration in mouse tissues using 2D fluorescent still images. First, several existing deep learning-based methods were evaluated to accurately segment nuclei using different imaging modalities with a small training dataset. Next, we developed a deep learning-based approach to identify and measure fluorescent labels within segmented nuclei, and created an ImageJ plugin to allow for efficient manual correction of nuclear segmentation and label identification. Lastly, using fluorescence intensity as a readout for protein concentration, a three-step global estimation method was applied to the characterization of the cell cycle dependent expression of E2F proteins in the developing mouse intestine.
A novel camera trap design for studying wildlife in mountain glacier ecosystems yields new insight for glacier biodiversity in the Pacific Northwest, USA ContextThe global recession of glaciers and perennial snowfields is reshaping mountain ecosystems. Beyond physical changes to the landscape and altered downstream hydrology, the implications of glacier decline for biodiversity are poorly known. Before predictions can be made about how climate change will affect wildlife in glacier-associated ecosystems, a more thorough accounting of the role that glaciers play in species life histories is needed. However, typical approaches for documenting wildlife presence and behavior--remote camera traps--are difficult to use in glaciated terrain due to limited options for securing them (e.g., no trees) and dramatic seasonal changes in snowpack.

AimsIn this study, we sought to test a novel camera trap designed for glaciated mountain ecosystems. We also aimed to use this approach to gain insight into wildlife and human usage of a mountain glacier in western North America.

MethodsWe deployed an elevational transect of uniquely designed camera traps along the western margin of the Paradise Glacier, a rapidly receding mountain glacier on the south side of Mount Rainier, WA, USA. Our simple camera trap design consisted of a wildlife camera attached to a camouflaged cylindrical cooler filled with snow and rocks.

Key resultsOur camera design proved ideal for a mountain glacier ecosystem and from June to September 2021, we detected at least 16 vertebrate species (seven birds, nine mammals) over 770 trap nights using glacier-associated habitats. Humans, primarily skiers, were the most common species detected, but we also recorded 99 observations of wildlife (birds and mammals). These included three species of conservation concern in Washington: wolverine (Gulo gulo), Cascade red fox (Vulpes vulpes cascadensis), and white-tailed ptarmigan (Lagopus leucura).

ConclusionsCollectively, our results provide proof-of-concept for a novel camera trap design that is ideal for treeless, perennially snow-covered landscapes and revealed a rich diversity of wildlife using mountain glacier habitat in the Pacific Northwest. We highlight the global need for similar studies to better understand the true scale of biodiversity that will be impacted by glacier recession in mountain ecosystems. CCL28 modulates neutrophil responses during infection with mucosal pathogens The chemokine CCL28 is highly expressed in mucosal tissues, but its role during infection is not well understood. Here we show that CCL28 promotes neutrophil accumulation to the gut of mice infected with Salmonella and to the lung of mice infected with Acinetobacter. Neutrophils isolated from the infected mucosa expressed the CCL28 receptors CCR3 and, to a lesser extent, CCR10, on their surface. The functional consequences of CCL28 deficiency were different between the two infections, as Ccl28-/- mice were highly susceptible to Salmonella gut infection, but highly resistant to otherwise lethal Acinetobacter lung infection. In vitro, unstimulated neutrophils harbored pre-formed intracellular CCR3 that was rapidly mobilized to the cell surface following phagocytosis or inflammatory stimuli. Moreover, CCL28 stimulation enhanced neutrophil antimicrobial activity, production of reactive oxygen species, and formation of extracellular traps, all processes that were largely dependent on CCR3. Consistent with the different outcomes in the two infection models, neutrophil stimulation with CCL28 boosted the killing of Salmonella but not of Acinetobacter. CCL28 thus plays a critical role in the immune response to mucosal pathogens by increasing neutrophil accumulation and activation, which can enhance pathogen clearance but also exacerbate disease depending on the mucosal site and the infectious agent. Resolving when (and where) the Thylacine went extinct Like the Dodo and Passenger Pigeon before it, the predatory marsupial Thylacine (Thylacinus cynocephalus), or  Tasmanian tiger, has become an iconic symbol of human-caused extinction. The last captive animal died in 1936, but even today reports of the Thylacines possible ongoing survival in remote regions of Tasmania are newsworthy and capture the publics imagination. Extirpated from mainland Australia in the mid-Holocene, the large island of Tasmania became the species final stronghold. Following European settlement in the 1800s, the Thylacine was heavily persecuted and pushed to the margins of its range, although many sightings were reported thereafter--even well beyond the 1930s. To gain a new depth of insight into the extinction of the Thylacine, we assembled an exhaustive database of 1,237 observational records from Tasmania (from 1910 onwards), quantified their uncertainty, and charted the patterns these revealed. We also developed a new method to visualize the species 20th-century spatio-temporal dynamics, to map potential post-bounty refugia and pinpoint the most-likely location of the final persisting subpopulation. A direct reading of the high-quality records (confirmed kills and captures, in combination with sightings by past Thylacine hunters and trappers, wildlife professionals and experienced bushmen) implies a most-likely extinction date within four decades following the last capture (i.e., 1940s to 1970s). However, uncertainty modelling of the entire sighting record, where each observation is assigned a probability and the whole dataset is then subject to a sensitivity analysis, suggests that extinction might have been as recent as the late 1980s to early 2000s, with a small chance of persistence in the remote south-western wilderness areas. Beyond the intrinsically fascinating problem of reconstructing the final fate of the Thylacine, the new spatio-temporal mapping of extirpation developed herein would also be useful for conservation prioritization and search efforts for other rare taxa of uncertain status. Unheeded SARS-CoV-2 proteins? A deep look into negative-sense RNA SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein coding-loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modelling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis.

Contactpetr.pecinka@osu.cz; tlb20@cam.ac.uk An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to the major UPF1 isoform, designated UPF1 SL. Using biochemical and transcriptome-wide approaches, we find that UPF1LL overcomes the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate long 3UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress. Robust retrieval of data stored in DNA by de Bruijn graph-based de novo strand assembly DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. The major technical challenges include various errors, such as the strand breaks, rearrangements, and indels that frequently arise during DNA synthesis, amplification, sequencing, and preservation. Through a de novo assembly strategy, we developed an algorithm based on the de Bruijn graph and greedy path search (DBGPS) to address these issues. DBGPS shows distinct advantages in handling DNA breaks, rearrangements, and indels. The robustness of DBGPS is demonstrated by accelerated aging, multiple independent data retrievals, deep error-prone PCR, and large data scale simulations. Remarkably, 6.8 MB of data can be retrieved accurately from a seriously corrupted sample that has been treated at 70 {degrees}C for 70 days. With DBGPS, we were able to achieve a logical density of 1.30 bits/cycle and a physical density of 295 PB/g.

One-Sentence SummaryA de Bruijn graph-based de novo assembly algorithm for DNA data storage enables fast and robust data readouts even with DNA samples that have been severely corrupted. Topology and adenocarcinoma cell localization dataset on the labyrinthin biomarker. The discovery and characterization of tumor associated antigens is increasingly important to advance the field of immuno-oncology. Data on the, 1) topology, 2) amino acid (a.a.) homology analyses and 3) cell surface localization are presented as evidence in support of labyrinthin as a pan-adenocarcinoma marker. Multiple bioinformatics tools were used to collectively predict calcium binding domain(s), N-myristoylation sites, kinase II phosphorylation sites, and transmembrane domain(s) with predicted N- and C-termini orientations. Sequence homology comparisons were found and alignments made for labyrinthin (255 a.a.) vs. the intracellular aspartyl/asparaginyl beta-hydroxylase (ASPH; 758 a.a.) and the ASPH-gene related protein junctate (299 a.a.), which are both type II proteins. The epitopes for commonly used MCA 44-3A6 anti-labyrinthin and FB-50 anti-ASPH/junctate antibodies were present in all three proteins. The selective expression of labyrinthin was investigated by third party fluorescent activated cell sorter (FACS) analyses of MCA 44-3A6 binding to non-permeabilized adenocarcinoma and normal cell line models. Labyrinthin was detected on non-permeabilized A549 human lung adenocarcinoma cells, but not on normal WI-38 human lung fibroblasts nor primary cultures of normal human glandular-related cells. Microscopic images of immunofluorescent labelled MCA 44-3A6 binding to A549 cells at random cell cycle stages complement the FACS results by further showing that labyrinthin persisted on the cell surfaces along with some cell internalization within 20 minutes. The dataset presented herein is related to research article: "Labyrinthin: A distinct pan-adenocarcinoma diagnostic and immunotherapeutic tumor specific antigen" by Babich et al., Heliyon, 2022 [1].

Specifications Table

O_TBL View this table:
org.highwire.dtl.DTLVardef@19222bborg.highwire.dtl.DTLVardef@1264bcdorg.highwire.dtl.DTLVardef@13a8158org.highwire.dtl.DTLVardef@1dcf0org.highwire.dtl.DTLVardef@503a9f_HPS_FORMAT_FIGEXP  M_TBL C_TBL Value of the data O_LIBecause identification and characterization of neoantigens [3, 4, 5] are rare yet increasingly important for diagnostics and therapeutics, the identification of labyrinthin as a pan-adenocarcinoma target is significant for further study by clinicians and cancer researchers.
C_LIO_LIThe data provide a basis to determine to what extent labyrinthin is a distinguishing marker for adenocarcinomas vs. conventional methods.
C_LIO_LIThe results indicate that therapeutic strategies can be employed that take advantage of labyrinthins convenient presence on adenocarcinoma cell surfaces.
C_LIO_LIThe discovery of a calcium binding sequence in labyrinthin provides a basis to research if it is key to explaining the long-known phenomena of elevated calcium and irregular signaling in cancer cells [8, 9].
C_LIO_LIThe data reveal a need to explore distinctions between ASPH and labyrinthin, in particular because ASPH [10, 11] and labyrinthin-based treatments have already been FDA approved for clinical trials.
C_LI A Novel Proposal for an Index for Regional Cerebral Perfusion Pressure - A Theoretical Approach Using Fluid Dynamics Cerebral blood flow (CBF) / cerebral blood volume (CBV) ratio derived by [15O] H2O/ CO2 and CO positron emission tomography (PET) examination has been used empirically as an index for cerebral perfusion pressure (CPP). However, this equation lacks a theoretical basis and furthermore, has not been confirmed to be proportionate to CPP, as direct measurement of local CPP is not practical.

We have developed a new index for CPP using the Poiseuille equation based on a simple model. Our model suggests that CBF/CBV2 is proportionate to CPP and that it is mathematically a more accurate index than CBF/CBV. Sensing Local Field Potentials with a Directional and Scalable Depth Array: the DISC array A variety of electrophysiology tools are available to the neurosurgeon for diagnosis, functional therapy, and neural prosthetics. However, no tool can currently address these three critical needs: (i) access to all cortical regions in a minimally invasive manner; (ii) recordings with microscale, mesoscale, and macroscale resolutions simultaneously; and (iii) access to spatially distant multiple brain regions that constitute distributed cognitive networks. We present a novel device for recording local field potentials (LFPs) with the form factor of a stereo-electroencephalographic electrode but combined with radially positioned microelectrodes and using the lead body to shield LFP sources, enabling directional sensitivity and scalability, referred to as the DISC array. As predicted by our electro-quasistatic models, DISC demonstrated significantly improved signal-to-noise ratio, directional sensitivity, and decoding accuracy from rat barrel cortex recordings during whisker stimulation. Critically, DISC demonstrated equivalent fidelity to conventional electrodes at the macroscale and uniquely, revealed stereoscopic information about current source density. Directional sensitivity of LFPs may significantly improve brain-computer interfaces and many diagnostic procedures, including epilepsy foci detection and deep brain targeting. Leaf Aqueous Extract of Manilkara hexandra influenced Glucose Metabolism in fish: An observation with Labeo rohita Fingerlings as model organism Manilkara hexandra (Roxb.) Dubard (common name Khirni, family: Sapotaceae) is an evergreen traditionally used medicinal plant. The present study aimed to see the effects of leaf aqueous extract of M. hexandra (LAEMH) on digestive and glucose metabolic enzyme action and serum glucose level in rohu, Labeo rohita, fingerlings. Experimental fish were fed a basal diet (Group I), and diets supplemented with LAEMH at 300 mg (Group II) and 600 mg (Group III) kg-1body weight. A significant reduction in serum glucose was noticed in the treated groups when compared with the control group. The reduced amylase activity was noticed in the treated fingerlings in a dose-dependent manner. However, lipase and protease activities didnt differ significantly among the experimental groups. Reduced serum glucose level might be correlated with the decline in the activity of digestive amylase in fish. Group II (3.35{+/-}0.19 U) and Group III (3.49{+/-}0.13 U) were recorded with reduced glucose-6-phosphatase activities than the control group (4.4{+/-}0.39 U). Moreover, the study revealed a decline in fructose-1,6-phosphatase activities in the treated groups in comparison to the control group. The decline in the activities of the metabolic enzymes might be associated with the non-availability of glucose owing to reduced activity of the digestive amylase in the treated groups. In conclusion, the present study established the hypoglycaemic effect of the leaf aqueous extract of M. hexandra in a fish model. Synthetic reconstruction of the hunchback promoter specifies the role of Bicoid, Zelda and Hunchback in the dynamics of its transcription For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as the model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation.

By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoids partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate. Patient-specific network connectivity combined with a next generation neural mass model to test clinical hypothesis of seizurepropagation Dynamics underlying epileptic seizures span multiple scales in space and time, therefore, understanding seizure mechanisms requires identifying the relations between seizure components within and across these scales, together with the analysis of their dynamical repertoire. In this view, mathematical models have been developed, ranging from single neuron to neural population.

In this study we consider a neural mass model able to exactly reproduce the dynamics of heterogeneous spiking neural networks. We combine the mathematical modelling with structural information from non-invasive brain imaging, thus building large-scale brain network models to explore emergent dynamics and test clinical hypothesis. We provide a comprehensive study on the effect of external drives on neuronal networks exhibiting multistability, in order to investigate the role played by the neuroanatomical connectivity matrices in shaping the emergent dynamics. In particular we systematically investigate the conditions under which the network displays a transition from a low activity regime to a high activity state, which we identify with a seizure-like event. This approach allows us to study the biophysical parameters and variables leading to multiple recruitment events at the network level. We further exploit topological network measures in order to explain the differences and the analogies among the subjects and their brain regions, in showing recruitment events at different parameter values.

We demonstrate, along the example of diffusion-weighted magnetic resonance imaging (MRI) connectomes of 20 healthy subjects and 15 epileptic patients, that individual variations in structural connectivity, when linked with mathematical dynamic models, have the capacity to explain changes in spatiotemporal organization of brain dynamics, as observed in network-based brain disorders. In particular, for epileptic patients, by means of the integration of the clinical hypotheses on the epileptogenic zone (EZ), i.e. the local network where highly synchronous seizures originate, we have identified the sequence of recruitment events and discussed their links with the topological properties of the specific connectomes. The predictions made on the basis of the implemented set of exact mean-field equations turn out to be in line with the clinical pre-surgical evaluation on recruited secondary networks. Thirteen independent genetic loci associated with preserved processing speed in a study of cognitive resilience in 330,097 individuals in the UK Biobank Cognitive resilience is the ability to withstand the negative effects of stress on cognitive functioning and is important for maintaining quality of life while aging. UK Biobank (UKB) does not have direct measurements of the same cognitive phenotype at distal timepoints. Therefore, we used number of education years (EY) as a proxy phenotype for past cognitive performance. Current cognitive performance was determined based on processing speed. This approach captured an average time span of 40 years between past and current cognitive performance in 330,097 individuals. A confounding factor was that EY is highly polygenic and masked the genetics of resilience. To overcome this, we employed Genomics Structural Equation Modelling (GenomicSEM) to perform a GWAS-by-subtraction using two GWAS, one GWAS of EY and resilience and a second GWAS of EY but not resilience. Subtracting one from the other generated a GWAS of Resilience. Replication of this approach was shown using independent discovery and replication samples within UKB and the full UKB GWAS results show significant genetic correlation with a GWAS of cognitive change in the independent Health and Retirement Study (N=9,526; P=1.5x10-3). We found 13 independent genetic loci for Resilience. Functional analyses showed enrichment in several brain regions and involvement of specific cell types, including GABAergic neurons (P=6.59x10-8) and glutamatergic neurons (P=6.98x10-6) in the cortex. Gene-set analyses implicated the biological process "neuron differentiation" (P=9.7x10-7) and the cellular component "synaptic part" (P=2.14x10-6). Mendelian randomization analysis showed a causative effect of white matter volume on cognitive resilience. These results enhance neurobiological understanding of resilience. Groping in the fog: soaring migrants exhibit wider scatter in flight directions and respond differently to wind under low visibility conditions Atmospheric conditions are known to affect flight propensity, behaviour during flight, and migration route in birds. Yet, the effects of fog have only rarely been studied although they could disrupt orientation and hamper flight. Fog could limit the visibility of migrating birds such that they might not be able to detect landmarks that guide them during their journey. Soaring migrants modulate their flight speed and direction in relation to the wind vector to optimize the cost of transport. Consequently, landmark-based orientation, as well as adjustments of flight speed and direction in relation to wind conditions, could be jeopardized when flying in fog. Using a radar system operated in a migration bottleneck (Strait of Messina, Italy), we studied the behaviour of soaring birds under variable wind and fog conditions over two consecutive springs (2016 and 2017), discovering that migrating birds exhibited a wider scatter of flight directions and responded differently to wind under fog conditions. Birds flying through fog deviated more from the mean migration direction and increased their speed with increasing crosswinds. In addition, airspeed and groundspeed increased in the direction of the crosswind, causing the individuals to drift laterally. Our findings represent the first quantitative empirical evidence of flight behaviour changes when birds migrate through fog and explain why low visibility conditions could risk their migration journey. Integrative Modelling of Signalling Network Dynamics Identifies Cell Type-selective Therapeutic Strategies for FGFR4-driven Cancers Oncogenic FGFR4 signalling represents a potential therapeutic target in many cancer types, including triple negative breast cancer (TNBC) and hepatocellular carcinoma (HCC). However, resistance to single-agent therapy directed at FGFR4 remains a major challenge, prompting the need to identify more effective combinatorial therapeutic strategies. Here, we integrated computational network modelling and experimental validation to characterise dynamic reprogramming of the FGFR4 signalling network in TNBC following FGFR4 kinase inhibition. We found that AKT, which signals downstream of FGFR4, displayed a rapid and potent reactivation following FGFR4 targeting. Through model-based simulation and systematic prediction of the effect of co-targeting specific network nodes, we predicted, and validated experimentally, strong synergism of co-targeting FGFR4 and particular ErbB kinases or AKT, but not the upstream kinase PI3K. Further, incorporation of protein expression data from hundreds of cancer cell types enabled us to adapt our model to other diverse cellular contexts, leading to the prediction that while AKT rebound occurs frequently, it is not a general phenomenon. Instead, ERK is reactivated in a subset of cell types, including the FGFR4-driven HCC cell line Hep3B. This was subsequently corroborated, and moreover, co-targeting FGFR4 and MEK in Hep3B cells markedly enhanced inhibition of cell proliferation. Overall, these findings provide novel insights into the dynamics of drug-induced network remodelling in cancer cells, highlight the impact of protein expression heterogeneity on network response to targeted therapy and identify candidate cell type-selective combination treatments for FGFR4-driven cancer. Reconstruction of nitrogenase predecessors suggests origin from maturase-like proteins The evolution of biological nitrogen fixation, uniquely catalyzed by nitrogenase enzymes, has been one of the most consequential biogeochemical innovations over lifes history. Though understanding the early evolution of nitrogen fixation has been a longstanding goal from molecular, biogeochemical, and planetary perspectives, its origins remain enigmatic. In this study, we reconstructed the evolutionary histories of nitrogenases, as well as homologous maturase proteins that participate in the assembly of the nitrogenase active-site cofactor but are not able to fix nitrogen. We combined phylogenetic and ancestral sequence inference with an analysis of predicted functionally divergent sites between nitrogenases and maturases to infer the nitrogen-fixing capabilities of their shared ancestors. Our results provide phylogenetic constraints to the emergence of nitrogen fixation and are consistent with a model wherein nitrogenases emerged from maturase-like predecessors. Though the precise functional role of such a predecessor protein remains speculative, our results highlight evolutionary contingency as a significant factor shaping the evolution of a biogeochemically essential enzyme.

SIGNIFICANCE STATEMENTThe origin of nitrogenase-catalyzed nitrogen fixation was a transformative event in lifes history, garnering long-term study from molecular, biogeochemical, and planetary perspectives. Reconstruction of ancestral nitrogenases suggests that the protein sequence space capable of yielding a nitrogen-fixing enzyme in the past was likely more constrained than previously thought. Specifically, here we show that nitrogenases likely evolved from ancestors that resemble maturases, homologs that today participate in nitrogenase cofactor assembly, contrary to the commonly accepted view that maturases evolved from a nitrogenase ancestor. We further submit that the molecular architecture that may have been required for nitrogenase origins was unlikely to have been shaped by the same environmental drivers often implicated in the evolution of nitrogen fixation. If this decoupling is found to be a recurring pattern in metabolic origins, then the presented results would undercut the common, systems-focused rationale of using ancient environmental conditions to explain the timing of critical and singular biogeochemical innovations in lifes past. Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible. In support of this, RNA polymerase transcribed a looped segment more slowly than an unlooped segment and paused more briefly at LacI obstacles on positively supercoiled templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase appears to facilitate elongation along topologically-complex, protein-coated templates. Flavobacterium johnsoniae Tyrosine Ammonia Lyase (FjTAL) in-silico Structure Prediction and Molecular Docking to L-Tyrosine, p-Coumaric Acid (pHCA) and Caffeic Acid Responsible for the conversion of L-tyrosine to p-coumaric acid in Flavobacterium johnsoniae, FjTAL has drawn the attention of many biochemical engineers who wish to carry out a sustainable biosynthetic scheme for the production of aromatic compounds. In this study, with the aid of various computational tools, the secondary and tertiary structures of FjTAL have been predicted. The results suggest that FjTAL forms a homo-tetramer when active as a cytosolic enzyme and it is mostly consisted of alpha helices. With the aid of molecular docking, one can hypothesize that FjTAL is likely to bind to L-tyrosine, p-coumaric acid, and caffeic acid with a similar molecular mechanism and thus, p-coumaric acid and caffeic acid may exhibit a negative feedback response toward the enzyme and inhibit its activity competitively. Two distinct binding pockets have been discovered, one of which contains highly conserved residues among several species. The residues which form the prosthetic group 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) also emerge in the evolutionary conserved binding pocket. The other discovered cavity, could either be a second binding site for the ligands or simply an artifact of the molecular docking task. wnt16 exerts pleiotropic effects on bone and lean mass in zebrafish Osteoporosis, a disease of bone fragility, and sarcopenia, a condition of reduced muscle mass and strength, commonly occur in the same individual--a condition termed osteosarcopenia. Genetic variants at the CPED1-WNT16 locus have pleiotropic effects on human bone mineral density (BMD) and lean tissue mass. However, our current understanding of Wnt16 and its influence on bone fails to explain effects on lean mass at this locus. In this study, we sought to better understand the biological mechanism underlying pleiotropy at the CPED1-WNT16 locus on BMD and lean mass. Using single cell analysis, microCT imaging, and genetic approaches in zebrafish, we show that wnt16 exerts dual influence on muscle and bone, and provide evidence that this occurs through a function in somite development, a period in which precursors for both of these tissues are specified. We show that mutations in wnt16 alter vertebral size and lean mass during axial skeletogenesis. We show that wnt16+ cells transcriptionally resemble cells within the amniote dermomyotome, and provide evidence they function as myogenic precursors during embryonic development. We also provide evidence that WNT16 is a gene of major effect on lean mass at the CPED-WNT16 locus. Our studies support the potential for wnt16 to exert pleiotropic effects on bone and lean mass through a function in myogenic precursors. Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. 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. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. A novel Fiji/ImageJ plugin for the rapid analysis of blebbing cells When confined, cells have recently been shown to undergo a phenotypic switch to what has been termed, fast amoeboid (leader bleb-based) migration. However, as this is a nascent area of research, few tools are available for the rapid analysis of cell behavior. Here, we demonstrate that a novel Fiji/ImageJ-based plugin, Analyze_Blebs, can be used to quickly obtain cell migration parameters and morphometrics from time lapse images. As validation, we show that Analyze_Blebs can detect significant differences in cell migration and morphometrics, such as the largest bleb size, upon introducing different live markers of F-actin, including F-tractin and LifeAct tagged with green and red fluorescent proteins. We also demonstrate, using flow cytometry, that live markers increase total levels of F-actin. Furthermore, that F-tractin increases cell stiffness, which was found to correlate with a decrease in migration, thus reaffirming the importance of cell mechanics as a determinant of Leader Bleb-Based Migration (LBBM).

Highlight summaryA new plugin, Analyze_Blebs, enables the rapid analysis of cell migration and morphometrics of fast amoeboid cells. Morphometrics combined with cell stiffness are found to be predictive of confined, fast amoeboid (leader bleb-based), migration. The SLIM1 transcription factor is required for arsenic resistance in Arabidopsis thaliana The transcriptional regulators of arsenic-induced gene expression remain largely unknown. Sulfur assimilation is tightly linked with arsenic detoxification. Here we report that mutant alleles in the SLIM1 transcription factor are substantially more sensitive to arsenic than cadmium. Arsenic treatment caused high levels of oxidative stress in the slim1 mutants, and slim1 alleles were impaired in both thiol and sulfate accumulation. We further found enhanced arsenic accumulation in roots of slim1 mutants. Transcriptome analyses indicate an important role for SLIM1 in arsenic-induced tolerance mechanisms. The present study identifies the SLIM1 transcription factor as an essential component in arsenic tolerance and arsenic-induced gene expression. Our results suggest that the severe arsenic sensitivity of the slim1 mutants is caused by altered redox status.