Microsecond simulations and CD spectroscopy reveals the intrinsically disordered nature of SARS-CoV-2 Spike-C-terminal cytoplasmic tail (residues 1242-1273) in isolation All available SARS-CoV-2 spike protein crystal and cryo-EM structures have shown missing electron densities for cytosolic C-terminal regions. Generally, the missing electron densities point towards the intrinsically disordered nature of the protein region. This curiosity has led us to investigate the C terminal cytosolic region of the spike glycoprotein of SARS-CoV-2 in isolation. The cytosolic regions is supposed to be from 1235-1273 residues or 1242-1273 residues depending on what prediction tool we use. Therefore, we have demonstrated the structural conformation of cytosolic region and its dynamics through computer simulations up to microsecond timescale using OPLS and CHARMM forcefields. The simulations have revealed the unstructured conformation of cytosolic region. Also, in temperature dependent replica-exchange molecular dynamics simulations it has shown its conformational dynamics. Further, we have validated our computational observations with circular dichroism (CD) spectroscopy-based experiments and found its signature spectra at 198 nm which is also adding the analysis as its intrinsically disordered nature. We believe that our findings will surely help us understand the structure-function relationship of the spike proteins cytosolic region.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY
Absence of cardiolipin from the outer-leaflet of a mitochondrial inner-membrane mimic restricts Opa1-mediated fusion Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix-facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric cardiolipin distribution. Using this system, we find the presence of cardiolipin on the inter-membrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of cardiolipin in this leaflet may influence protein and membrane properties, and future applications for this approach.
Steady-state measures of visual suppression In the early visual system, suppression occurs between neurons representing different stimulus properties. This includes features such as orientation (cross-orientation suppression), eye-of-origin (interocular suppression) and spatial location (surround suppression), which are thought to involve distinct anatomical pathways. We asked if these separate routes to suppression can be differentiated by their pattern of gain control on the contrast response function measured in human participants using steady-state electroencephalography. Changes in contrast gain shift the contrast response function laterally, whereas changes in response gain scale the function vertically. We used a Bayesian hierarchical model to summarise the evidence for each type of gain control. A computational meta-analysis of 16 previous studies found the most evidence for contrast gain effects with overlaid masks, but no clear evidence favouring either response gain or contrast gain for other mask types. We then conducted two new experiments, comparing suppression from four mask types (monocular and dichoptic overlay masks, and aligned and orthogonal surround masks) on responses to sine wave grating patches flickering at 5Hz. At the occipital pole, there was strong evidence for contrast gain effects in all four mask types at the first harmonic frequency (5Hz). Suppression generally became stronger at more lateral electrode sites, but there was little evidence of response gain effects. At the second harmonic frequency (10Hz) suppression was stronger overall, and involved both contrast and response gain effects. Although suppression from different mask types involves distinct anatomical pathways, gain control processes appear to serve a common purpose, which we suggest might be to suppress less reliable inputs. "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. Epitope-tagging of the endogenous murine BiP/GRP78/Hspa5 locus allows direct analysis of the BiP interactome and protein misfolding in vivo BiP/GRP78, encoded by the Hspa5 gene, is the major HSP70 family member in the endoplasmic reticulum (ER) lumen, and controls ER protein folding. The essential functions of BiP in facilitating proper protein folding are mainly mediated through its dynamic interaction with unfolded or misfolded client proteins, and by serving as a negative regulator of the Unfolded Protein Response. A mechanistic understanding of the dynamics of BiP interaction with its protein partners is essential to understand ER biology, and therefore, we have sought to develop a tractable model to study misfolded protein interaction with BiP. For this purpose, we have used homologous recombination to insert a 3xFLAG epitope tag into the endogenous murine Hspa5 gene, just upstream from the essential KDEL signal necessary for ER localization of BiP. Tagging BiP in this way did not alter Hspa5 expression under basal or ER-stress induced conditions in hepatocytes ex vivo or in fibroblasts. Furthermore, the tag did not alter the cellular localization of BiP or its functionality. All of these findings in primary tissue culture were also confirmed in vivo in livers of heterozygous mice harboring one WT and one FLAG-tagged Hspa5 allele. Hepatocyte-specific BiP-FLAG modification did not alter liver function or UPR signaling. Importantly, immunoprecipitation with anti-FLAG antibody completely pulled down FLAG-tagged BiP from lysates of BiP-FLAG expressing livers. Affinity purification-mass spectrometry (AP-MS) of BiP-FLAG protein complexes isolated from the BiP-FLAG-expressing livers of tunicamycin (TM)- and vehicle-treated mice revealed a marked increase in interaction of glycoproteins with BiP-FLAG in response to inhibition of N-glycosylation due to TM-treatment, validating utility of our BiP-Flag mice as a tool to identify ER misfolded proteins in vivo. Significantly, our AP-MS analysis also provided in vivo evidence demonstrating that BiP-FLAG binds to UPR transducers IRE1 and PERK under basal conditions but is released upon TM-treatment to activate UPR. We have also employed this mouse model to demonstrate that proinsulin in pancreatic {beta} cells misfolds and interacts with BiP-FLAG in healthy mice. In summary, we generated a novel model that can be used to investigate the BiP interactome in vivo under physiological and pathophysiological conditions in a cell type-specific manner. This tool provides, for the first time, an unbiased approach to identify unfolded and misfolded BiP-client proteins, and a new approach to study ER protein misfolding in a cell-type and temporal manner to uncover the role of BiP in many essential ER processes. 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. Bias correction for inverse variance weighting Mendelian randomization BackgroundInverse-variance weighted two-sample Mendelian randomization (IVW-MR) is the most widely used approach that utilizes genome-wide association studies (GWAS) summary statistics to infer the existence and the strength of the causal effect between an exposure and an outcome. Estimates from this approach can be subject to different biases due to the use of weak instruments and winners curse, which can change as a function of the overlap between the exposure and outcome samples.

MethodsWe developed a method (MRlap) that simultaneously considers weak instrument bias and winners curse, while accounting for potential sample overlap. Assuming spike-and-slab genomic architecture and leveraging LD-score regression and other techniques, we could analytically derive, reliably estimate, and hence correct for the bias of IVW-MR using association summary statistics only.

ResultsWe tested our approach using simulated data for a wide range of realistic settings. In all the explored scenarios, our correction reduced the bias, in some situations by as much as 30 folds. When applied to real data on obesity-related exposures, we observed significant differences between IVW-based and corrected effects, both for non-overlapping and fully overlapping samples. Additionally, our results are consistent with the fact that the strength of the biases will decrease as the sample size increases. Finally, we also showed that the overall bias is also dependent on the genetic architecture of the exposure, and traits with low heritability and/or high polygenicity are more strongly affected.

ConclusionsOur method not only reduces bias in causal effect estimation but also enables the use of much larger GWAS sample sizes, by allowing for potentially overlapping samples. Non-Newtonian Blood Rheology Impacts Left Atrial Stasis in Patient-Specific Simulations The lack of mechanically effective contraction of the left atrium (LA) during atrial fibrillation (AF) disturbs blood flow, increasing the risk of thrombosis and ischemic stroke. Thrombosis is most likely in the left atrial appendage (LAA), a small narrow sac where blood is prone to stagnate. Slow flow promotes the formation of erythrocyte aggregates in the LAA, also known as rouleaux, causing viscosity gradients that are usually disregarded in patient-specific simulations. To evaluate these non-Newtonian effects, we built atrial models derived from 4D computed tomography scans of patients and carried out computational fluid dynamics simulations using the Carreau-Yasuda constitutive relation. We examined six patients, three of whom had AF and LAA thrombosis or a history of transient ischemic attacks (TIAs). We modeled the effects of hematocrit and rouleaux formation kinetics by varying the parameterization of the Carreau-Yasuda relation and modulating non-Newtonian viscosity changes based on residence time. Comparing non-Newtonian and Newtonian simulations indicates that slow flow in the LAA increases blood viscosity, altering secondary swirling flows and intensifying blood stasis. While some of these effects can be subtle when examined using instantaneous metrics like shear rate or kinetic energy, they are manifested in the blood residence time, which accumulates over multiple heartbeats. Our data also reveal that LAA blood stasis worsens when hematocrit increases, offering a potential new mechanism for the clinically reported correlation between hematocrit and stroke incidence. In summary, we submit that hematocrit-dependent non-Newtonian blood rheology should be considered in calculating patient-specific blood stasis indices by computational fluid dynamics. Experimental evolution reveals post-transcriptional regulation as a novel driver of Leishmania fitness gain The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network driving parasite fitness, with genome instability causing highly reproducible, gene dosage-dependent changes in protein abundance linked to post-transcriptional regulation. These in turn were associated with a gene dosage-independent reduction in abundance of flagellar transcripts and a coordinated increase in abundance of coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of fitness-adapted ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings. Cellular Fitness Phenotypes of Cancer Target Genes from Oncobiology to Cancer Therapeutics To define the growing significance of cellular targets of cancer drugs, we examined the fitness dependency of cellular targets or effectors of cancer drug targets across human cancer cells from 19 cancer types. We observed that the deletion of 35 out of 47 cellular mediators or targets of oncology drugs did not result in the expected loss of cell fitness in appropriate cancer types for which drugs targeting or utilizing these molecules were approved. Additionally, our analysis recognized 43 cellular targets as fitness genes in several cancer types in which these drugs were not approved, and thus, providing clues repurposing approved oncology drugs in cancer types. For example, we found the widespread upregulation and fitness dependency of the components of the mevalonate and purine biosynthesis pathways (currently targeted by bisphosphonates, statins, and pemetrexed in certain cancers) and an association between the overexpression of these targets and reduction in the overall survival duration of patients with breast and other hard-to-treat cancers, for which such drugs are not approved. In brief, the present analysis raised cautions about off-target and undesirable effects of certain oncology drugs in a subset of cancers where the in-tended cellular effectors of drug might not be fitness genes and offers a potential rationale for repurposing certain approved oncology drugs for targeted therapeutics in additional cancer types. Cytokinin oxidase/dehydrogenase family genes exhibit functional divergence and overlap in rice growth and development Cytokinins play key roles in plant growth and development; hence, cytokinin biosynthesis and degradation have been extensively studied. Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes that regulate oxidative cleavage to maintain cytokinin homeostasis. In rice, 11 OsCKX genes have been identified to date; however, most of their functions remain unknown. Here, we comprehensively analyzed the expression patterns and functions of OsCKX genes. Using CRISPR/Cas9 technology, we constructed mutants of all OsCKX genes to determine the functions of OsCKXs in rice development. The results revealed that the single osckx and higher-order osckx4 osckx9 mutant lines showed functional overlap and subfunctionalization. Notably, the osckx1 osckx2 and osckx4 osckx9 double mutants displayed contrasting phenotypic changes in tiller number and panicle size compared to the wild type. Moreover, we identified several genes with significantly altered expression in osckx4 and osckx9 single and double mutant plants. Many differentially expressed genes were found to be associated with auxin and cytokinin pathways. Additionally, the cytokinins in osckx4 osckx9 mutants were increased compared to the wild type. Overall, our findings provide new insights into the functions of OsCKX genes in rice growth and may be used as a foundation for future studies aimed at improving rice yield. Key words: Cytokinin, expression pattern, OsCKX, panicle, phenotype, rice, tiller

HighlightThe osckx4 osckx9 double mutant had a significantly greater number of tillers, whereas the osckx1 osckx2 double mutant showed the opposite phenotypic change, compared to the wild type Aldosterone and Dexamethasone Activate African Lungfish Mineralocorticoid Receptor: Increased Activation After Removal of the Amino-Terminal Domain Aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, first appears in lungfish, which are lobe-finned fish that are forerunners of terrestrial vertebrates. Aldosterone activation of the MR regulates internal homeostasis of water, sodium and potassium, which was critical in the conquest of land by vertebrates. We studied transcriptional activation of the slender African lungfish MR by aldosterone, other corticosteroids and progesterone and find that aldosterone, 11-deoxycorticosterone, 11-deoxycortisol and progesterone have half-maximal responses (EC50s) below 1 nM and are potential physiological mineralocorticoids. In contrast, EC50s for corticosterone and cortisol were 23 nM and 66 nM, respectively. Unexpectedly, truncated lungfish MR, consisting of the DNA-binding, hinge and steroid-binding domains, had a stronger response to corticosteroids and progesterone than full-length lungfish MR, indicating that the N-terminal domain represses steroid activation of lungfish MR, unlike human MR in which the N-terminal domain contains an activation function. BLAST searches of GenBank did not retrieve a GR ortholog, leading us to test dexamethasone and triamcinolone for activation of lungfish MR. At 10 nM, both synthetic glucocorticoids are about 4-fold stronger than 10 nM aldosterone in activating full-length lungfish MR, leading us to propose that lungfish MR also functions as a GR. Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ-9-tetrahydrocannabinol In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development. The initial response of females towards congeneric males matches the propensity to hybridise in Ophthalmotilapia. AO_SCPLOWBSTRACTC_SCPLOWCichlid radiations often harbour closely related species with overlapping niches and distribution ranges. Such species sometimes hybridise in nature, which raises the question how can they coexist. This also holds for the Tanganyika mouthbrooders Ophthalmotilapia ventralis and O. nasuta. Earlier studies found indications of asymmetrical hybridisation with females of O. ventralis accepting males of O. nasuta, but not the other way around. We hypothesised that this was due to differences in the capacity for species recognition. Given the higher propensity of O. ventralis females towards hybridisation, we expect a reduced ability for species recognition in O. ventralis females, compared to O. nasuta females. We staged two experiments, one focusing on 22 female O. nasuta and one on 21 female O. ventralis. These fish were placed in one half of a tank and briefly exposed to a conspecific or a heterospecific male, a conspecific female, or nothing (control). Female response was evaluated by scoring six tracking parameters and by noting the occurrence of ten discrete behaviours before and during the encounter. Females always responded to the presence of another fish by approaching it. Remarkably, for both O. nasuta and O. ventralis, we did not find a different response between encounters with conspecific males and females. However, in agreement with our hypothesis, O. nasuta females behaved differently towards conspecific or heterospecific males, whereas O. ventralis females did not. When presented with a heterospecific male, O. nasuta females performed a lower number of  ram behaviours. Additionally, they never displayed the  flee behaviour, a component of the species mating repertoire that was seen in all but one of the presentations with a conspecific male. Our findings show that differences in species recognition at first encounter predict to a large degree the outcome of the mating process, even in the absence of mating behaviour. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. Faster short-read mapping with strobemer seeds constructed from syncmers Read alignment to genomes is a fundamental computational step used in many bioinformatic analyses, and often, it is the computational bottleneck. Therefore, it is desirable to perform the alignment step as fast as possible without compromising accuracy. Most alignment algorithms consider a seed-and-extend approach, where the time-consuming seeding step identifies and decides on candidate mapping locations. Recently, several advances have been made on seeding methods for fast sequence comparison.

We combine two such methods, syncmers and strobemers, in a novel seeding approach for constructing dynamic-sized fuzzy seeds and implement the method in a short-read aligner, strobealign. We benchmark strobealign to other traditional and recently proposed aligners on simulated and biological data and show that strobealign is an order of magnitude faster than traditional aligners such as BWA and Bowtie2 while being both faster and more accurate than more recently proposed aligners. Our aligner can free up substantial time and computing resources needed for read alignment in many pipelines.

Availabilityhttps://github.com/ksahlin/strobealign. Hydrophobic mismatch is a key factor in protein transport on the Tat pathway The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoid, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits, TatA, TatB and TatC. TatA and TatB from all bacterial species possess short transmembrane alpha-helices (TMHs), both of which are only fifteen residues long in E. coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer. Here, by modifying the length of the TMHs of E. coli TatA and TatB, we assess the functional importance of the hydrophobic mismatch in the Tat transport mechanism. Surprisingly, both TatA and TatB with as few as 11 residues in their respective TMHs are still able to insert into the membrane bilayer, albeit with a decline in membrane integrity. Three different assays, both qualitative and quantitative, were conducted to evaluate the Tat activity of the TMH length mutants. Our experiments indicate that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off with any modification of this length. We believe our study supports a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation, and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes. Evaluation of US state pollinator plans using 3 evidence-based policymaking frameworks After the US federal government created a national pollinator protection plan in 2015, many states followed with their own. Since their goal is to promote pollinating insect conservation, we wanted to know whether the state plans are using best practices for evidence-based science policy. In early 2019 we found and downloaded every existing, publicly available US state pollinator protection plan. We then used content analysis to assess the goals, scope, and implementation of state-level pollinator protection plans across the US. This analysis was conducted using three distinct frameworks for evidence-based policymaking: US Department of Interior Adaptive Resources Management (ARM), US Environmental Protection Agency management pollinator protection plan (MP3) guidance, and Pew Trusts Pew-MacAthur Results First Project elements of evidence-based state policymaking (PEW) framework. Then we scored them using the framework criteria, to assess whether the plans were using known best practices for evidence based policymaking. Of the 31 states with a state pollinator plan, Connecticut was the state with the lowest total score across the three evaluation frameworks. The state with the highest overall scores, across the three frameworks, was Missouri. Most states did not score highly on the majority of the frameworks. Overall, many state plans were lacking policy elements that address monitoring, evaluation, and adjustment. These missing elements impact states ability to achieve their conservation goals. Our results indicate that states can improve their pollinator conservation policies to better match evidence-based science policy guidance, regardless of which framework is used. Multiple origins of nematode-Wolbachia symbiosis in supergroup F and convergent loss of bacterioferritin in filarial Wolbachia The intracellular endosymbiotic proteobacteria Wolbachia have evolved across the phyla nematoda and arthropoda. In Wolbachia phylogeny, supergroup F is the only clade with members from both arthropod and filarial nematode hosts and therefore can provide unique insights into their evolution and biology. In this study, 4 new supergroup F Wolbachia genomes have been assembled using a metagenomic assembly and binning approach, wMoz and wMpe from the human filarial parasites Mansonella ozzardi and Mansonella perstans, and wOcae and wMoviF from the blue mason bee Osmia caerulescens and the sheep ked Melophagus ovinus respectively. A comprehensive phylogenomic analysis revealed two independent origins of filarial Wolbachia in supergroup F, likely from ancestral arthropod hosts, as well as a convergent pseudogenization and loss of the bacterioferritin gene across all filarial Wolbachia. The new genomes provide a valuable resource for further studies on symbiosis, evolution, and the discovery of new antibiotics to treat mansonellosis.

Data depositionRaw Illumina reads for the M. perstans isolate Mpe1 and the M. ozzardi isolate Moz2 have been deposited in the NCBI SRA database under BioProject accession numbers PRJNA666672 and PRJNA666671 respectively. The assembled genome and PGAP annotations for the Wolbachia wMpe and wMoz are available from the NCBI GenBank database with accession numbers JACZHU000000000 and JADAKK000000000 respectively. The annotated genomes for wOcae and wMoviF are available as GenBank accessions JAHDJT000000000 and JAHRBA000000000 respectively, deposited under BioProject accessions PRJNA244005 and PRJNA739984 respectively. Multi-omics-based label-free metabolic flux inference reveals obesity-associated dysregulatory mechanisms in liver glucose metabolism Glucose homeostasis is maintained by modulation of metabolic flux. Enzymes and metabolites regulate the involved metabolic pathways. Dysregulation of glucose homeostasis is a pathological event in obesity. Analyzing metabolic pathways and the mechanisms contributing to obesity-associated dysregulation in vivo is challenging. Here, we introduce OMELET: Omics-Based Metabolic Flux Estimation without Labeling for Extended Trans-omic Analysis. OMELET uses metabolomic, proteomic, and transcriptomic data to identify changes in metabolic flux, and to quantify contributions of metabolites, enzymes, and transcripts to the changes in metabolic flux. By evaluating the livers of fasting ob/ob mice, we found that increased metabolic flux through gluconeogenesis resulted primarily from increased transcripts, whereas that through the pyruvate cycle resulted from both increased transcripts and changes in substrates of metabolic enzymes. With OMELET, we identified mechanisms underlying the obesity-associated dysregulation of metabolic flux in liver.

HighlightsO_LIWe created OMELET to infer metabolic flux and its regulation from multi-omic data.
C_LIO_LIGluconeogenic and pyruvate cycle fluxes increased in fasting ob/ob mice.
C_LIO_LITranscripts increases mediated the increase in gluconeogenic fluxes in ob/ob mice.
C_LIO_LIIncreases in transcripts and substrates enhanced pyruvate cycle flux in ob/ob mice.
C_LI A novel two-generation approach to the population dynamics of gregarious parasitoids Parasitoids, as important natural enemies, help maintain balance in natural ecosystems. Their population dynamics is generally predicted from the number of individuals. Here, using gregarious parasitoids as models, we show that this traditional approach omits one important parameter: the mothers manipulation of offspring fertility due to the clutch size-body size- fertility correlation. As a result of this correlation, when females deliberately adjust the number of offspring laid in a host, they determine not only the number but also the body sizes and reproductive potentials of those offspring. For the first time in the model species Anaphes flavipes, we determined the parasitoids offspring fertility from clutch size. Using this, we experimentally clarified the advantage of specific clutch size combinations and we show that identical fertility in the F1 generation can lead to distinctly different fertility values in the F2 generation. Even with the same number of hosts, lower fertility in the F1 generation can cause higher fertility in the F2 generation. Based on these results, we propose a novel two-generation approach which includes the clutch size-body size-fertility correlation. Our novel approach provides a new perspective for determining the individual fitness levels of gregarious parasitoids with new options for the modelling of parasitoid population dynamics. The nucleus bypasses obstacles by deforming like a drop with surface tension mediated by lamin A/C Migrating cells must deform their stiff cell nucleus to move through pores and fibers in tissue. Lamin A/C is known to hinder cell migration by limiting nuclear deformation and passage through confining channels, but its role in nuclear deformation and passage through fibrous environments is less clear. We studied cell and nuclear migration through discrete, closely spaced, slender obstacles which mimic the mechanical properties of collagen fibers. Nuclei bypassed slender obstacles while preserving their overall morphology by deforming around them with deep local invaginations of little resisting force. The obstacles did not impede the nuclear trajectory or cause a rupture of the nuclear envelope. Nuclei likewise deformed around single collagen fibers in cells migrating in 3D collagen gels. In contrast to its limiting role in nuclear passage through confining channels, lamin A/C facilitated nuclear deformation and passage through fibrous environments; nuclei in lamin-null (Lmna-/-) cells lost their overall morphology and became entangled on the obstacles. Analogous to surface tension-mediated deformation of a liquid drop, lamin A/C imparts a surface tension on the nucleus that allows nuclear invaginations with little mechanical resistance, preventing nuclear entanglement and allowing nuclear passage through fibrous environments. Co-option of immune effectors by the hormonal signalling system triggering metamorphosis in Drosophila melanogaster Insect metamorphosis is regulated by the production, secretion and degradation of two peripheral hormones: 20-hydroxyecdysone (ecdysone) and juvenile hormone (JH). In addition to their roles in developmental regulation, increasing evidence suggests that these hormones are involved in innate immunity processes, such as phagocytosis and the induction of antimicrobial peptide (AMP) production. AMP regulation includes systemic responses as well as local responses at surface epithelia that contact with the external environment. At pupariation, Drosophila melanogaster increases dramatically the expression of three AMP genes, drosomycin (drs), drosomycin-like 2 (drsl2) and drosomycin-like 5 (drsl5). We show that the systemic action of drs at pupariation is dependent on ecdysone signalling in the fat body and operates via the ecdysone downstream target, Broad-Z4. In parallel, ecdysone also regulates local responses, specifically through the activation of drsl2 expression in the gut. Finally, we confirm the relevance of this ecdysone dependent AMP expression for the control of bacterial persistence by showing that flies lacking drs expression in the fat body have higher bacterial persistence over metamorphosis. Together, our data emphasize the importance of the association between ecdysone signalling and immunity using in vivo studies and establish a new role for ecdysone at pupariation, which impacts developmental success by regulating the immune system in a stage-dependent manner. We speculate that this co-option of immune effectors by the hormonal system may constitute a pre-emptive mechanism to control bacterial numbers in the pupa at the core of metamorphosis evolution. 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. Re-convolving the compositional landscape of primary and recurrent glioblastoma reveals prognostic andtargetable tissue states Glioblastoma is an aggressive diffusely infiltrating neoplasm that spreads beyond surgical resection margins, where it intermingles with non-neoplastic brain cells. This complex microenvironment harbouring infiltrating glioma and non-neoplastic brain cells is the origin of tumor recurrence. Thus, understanding the cellular and molecular features of the glioma microenvironment is therapeutically and prognostically important. Here, we used single-nucleus RNA sequencing (snRNAseq) of primary and recurrent glioma to define three compositional  tissue-states rooted in cohabitation of cell-types and transcriptional states. These comprise a state featuring A) abundant normal brain microenvironment cells, B) reactive/inflamed brain microenvironment, and C) cellular/proliferative tumor. All these states exhibited variable degrees of infiltration by glioma cells. Spatial transcriptomics confirmed that the cell-types and transcriptomics states which compositionally cohabitate tissue states also colocalize in space. Tissue states are clinically significant because they correlate with radiographic, histopathologic, and prognostic features. We found that enrichment of tissue state B signature correlated with shorter survival. Importantly, we found that our compositionally defined tissue states are enriched in distinct metabolic pathways. One such pathway is fatty acid biosynthesis, which was enriched in tissue state B - a state enriched in recurrent glioblastoma and composed of astrocyte-like/mesenchymal glioma cells, reactive astrocytes resembling those seen in neurodegeneration, and monocyte-like myeloid cells. We showed that treating acute slices of GBM with a fatty acid synthesis inhibitor is sufficient to deplete the transcriptional signature of tissue state B. Our findings define a novel compositional approach to the glioma-infiltrated tissue which allows us to discover prognostic and targetable features, paving the way to new mechanistic and therapeutic discoveries. RIG-I immunotherapy overcomes radioresistance in p53-positive malignant melanoma Radiation therapy induces cytotoxic DNA damage, which results in cell-cycle arrest and activation of cell-intrinsic death pathways, but its application has been limited by the radioresistance of tumors, such as in malignant melanoma. RIG-I is a cytosolic immune receptor expressed in all somatic cells, including tumor cells, with a key role in sensing viral RNA. RIG-I specific oligonucleotide ligands elicit a robust cell-intrinsic antiviral response and immunogenic cell death in tumor cells and are being tested in clinical trials. Nonetheless, their potential to overcome radioresistance has not yet been explored. Here, we demonstrate that activation of RIG-I enhances the extent and immunogenicity of irradiation-induced tumor cell death in human and murine melanoma cell lines in vitro and improved survival in the murine B16 melanoma model. Pathway analysis of transcriptomic data revealed a central role for p53 downstream of the combination treatment, which was corroborated using p53-/- B16 cells. In vivo, the effect of irradiation on immune-cell activation and inhibition of tumor growth was absent in mice carrying p53-/- B16 tumors, while the response to RIG-I stimulation in those mice was maintained. Our results identify p53 as pivotal for the synergistic antitumoral effect of RIG-I and irradiation, resulting in potent induction of immunogenic tumor-cell death. Thus, the administration of RIG-I ligands in combination with radiotherapy is a promising therapeutic approach to treating radioresistant tumors with a functional p53 pathway, such as malignant melanoma.