Chromatin accessibility landscape and active transcription factors in primary human invasive lobular and ductal breast carcinomas Background: Invasive lobular breast carcinoma (ILC), the second most prevalent histological subtype of breast cancer, exhibits unique molecular features compared with the more common invasive ductal carcinoma (IDC). While genomic and transcriptomic features of ILC and IDC have been characterized, genome-wide chromatin accessibility pattern differences between ILC and IDC remain largely unexplored. Methods: Here, we characterized tumor-intrinsic chromatin accessibility differences between ILC and IDC using primary tumors from The Cancer Genome Atlas (TCGA) breast cancer assay for transposase-accessible chromatin with sequencing (ATAC-seq) dataset. Results: We identified distinct patterns of genome-wide chromatin accessibility in ILC and IDC. Inferred patient-specific transcription factor (TF) motif activities revealed regulatory differences between and within ILC and IDC tumors. EGR1, RUNX3, TP63, STAT6, SOX family, and TEAD family TFs were higher in ILC, while ATF4, PBX3, SPDEF, PITX family, and FOX family TFs were higher in IDC. Conclusions: This study reveals the distinct epigenomic features of ILC and IDC and the active TFs driving cancer progression that may provide valuable information on patient prognosis.
Nrf2 modulates the hybrid epithelial/mesenchymal phenotype and Notch signaling during collective cancer migration Hybrid epithelial/mesenchymal cells (E/M) are key players in aggressive cancer metastasis. It remains a challenge to understand how these cell states, which are mostly non-existent in healthy tissue, become stable phenotypes participating in collective cancer migration. The transcription factor Nrf2, which is associated with tumor progression and resistance to therapy, appears to be central to this process. Here, using a combination of single cell biosensors, immunocytochemistry, and computational modeling, we show that Nrf2 functions as a phenotypic stability factor for hybrid E/M cells by inhibiting a complete epithelial-mesenchymal transition (EMT) during collective cancer migration. We also demonstrate that Nrf2 and EMT signaling are spatially coordinated near the migrating front. In particular, computational analysis of an Nrf2-EMT-Notch network and experimental modulation of Nrf2 by pharmacological treatment or CRISPR/Cas9 gene editing reveal that Nrf2 stabilizes a hybrid E/M phenotype which is maximally observed in the interior region immediately behind the leading edge. We further demonstrate that the Nrf2-EMT-Notch network enhances Dll4 and Jagged1 expression near the leading edge, which correlates with the formation of leader cells and protruding tips. Altogether, our results provide direct evidence that Nrf2 acts as a phenotypic stability factor in restricting complete EMT and plays an important role in coordinating collective cancer migration.
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. Multivariate Genomewide Association Analysis by Iterative Hard Thresholding 1In genome-wide association studies (GWAS), analyzing multiple correlated traits is potentially superior to conducting multiple univariate analyses. Standard methods for multivariate GWAS operate marker-by-marker and are computationally intensive. We present a penalized regression algorithm for multivariate GWAS based on iterative hard thresholding (IHT) and implement it in a convenient Julia package MendelIHT.jl (https://github.com/OpenMendel/MendelIHT.jl). In simulation studies with up to 100 traits, IHT exhibits similar true positive rates, smaller false positive rates, and faster execution times than GEMMAs linear mixed models and mv-PLINKs canonical correlation analysis. On UK Biobank data, our IHT software completed a 3-trait joint analysis in 20 hours and an 18-trait joint analysis in 53 hours, requiring up to 80GB of computer memory. In short, our software enables geneticists to fit a single regression model that simultaneously considers the effect of all SNPs and dozens of traits. Bolstering fitness via opportunistic CO2 fixation: mixotroph dominance in modern groundwater The current understanding of organic carbon inputs into ecosystems lacking photosynthetic primary production is predicated on data and inferences derived almost entirely from metagenomic analyses. The elevated abundances of putative chemolithoautotrophs in groundwaters suggest that dark CO2 fixation is an integral component of subsurface trophic webs. To understand the impact of autotrophically-fixed carbon, the flux of CO2-derived carbon through various subpopulations of subsurface microbiota must first be resolved, both quantitatively and temporally. Here, we implement novel stable isotope cluster analysis to render a time-resolved and quantitative evaluation of 13CO-derived carbon flow through a groundwater microbiome stimulated with reduced sulfur compounds. We demonstrate that mixotrophs, not obligate chemolithoautotrophs, were the most abundant active organisms in groundwater microcosms. Species of Hydrogenophaga, Polaromonas, Dechloromonas, and other metabolically versatile mixotrophs drove the recycling of organic carbon and, when chance afforded, supplemented their carbon requirements via chemolithoautotrophy and uptake of available organic compounds. Mixotrophic activity facilitated the replacement of 43 and 80% of total microbial carbon stores with 13C in just 21 and 70 days, respectively. This opportunistic "utilize whatever pathways net the greatest advantage in fitness" strategy may explain the great abundances of mixotrophs in other oligotrophic habitats, like the upper ocean and boreal lakes. Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates survival of developing retinal neurons Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD+ homeostasis for health and function, the specific roles of subcellular NAD+ pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, large-scale transcriptomics reveals dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation. Negative DNA supercoiling makes protein-mediated looping deterministic and ergodic within the bacterial doubling time Protein-mediated DNA looping is fundamental to gene regulation and such loops occur stochastically in purified systems. Additional proteins increase the probability of looping, but these probabilities maintain a broad distribution. For example, the probability of lac repressor-mediated looping in individual molecules ranged 0-100%, and individual molecules exhibited representative behavior only in observations lasting an hour or more. Titrating with HU protein progressively compacted the DNA without narrowing the 0-100% distribution. Increased negative supercoiling produced an ensemble of molecules in which all individual molecules more closely resembled the average. Furthermore, in only twelve minutes of observation, well within the doubling time of the bacterium, most molecules exhibited the looping probability of the ensemble. DNA supercoiling, an inherent feature of all genomes, appears to impose time-constrained, emergent behavior on otherwise random molecular activity. The genome of the cereal pest Sitophilus oryzae: a transposable element haven BackgroundThe rice weevil Sitophilus oryzae is one of the most important agricultural pests, causing extensive damage to cereal in fields and to stored grains. S. oryzae has an intracellular symbiotic relationship (endosymbiosis) with the Gram-negative bacterium Sodalis pierantonius and is a valuable model to decipher host-symbiont molecular interactions.

ResultsWe sequenced the Sitophilus oryzae genome using a combination of short and long reads to produce the best assembly for a Curculionidae species to date. We show that S. oryzae has undergone successive bursts of transposable element (TE) amplification, representing 72% of the genome. In addition, we show that many TE families are transcriptionally active, and changes in their expression are associated with insect endosymbiotic state. S. oryzae has undergone a high gene expansion rate, when compared to other beetles. Reconstruction of host-symbiont metabolic networks revealed that, despite its recent association with cereal weevils (30 Kyear), S. pierantonius relies on the host for several amino acids and nucleotides to survive and to produce vitamins and essential amino-acids required for insect development and cuticle biosynthesis.

ConclusionsHere we present the genome of an agricultural pest beetle, which may act as a foundation for pest control. In addition, S. oryzae may be a useful model for endosymbiosis, and studying TE evolution and regulation, along with the impact of TEs on eukaryotic genomes. 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. Hnf1b-CreER causes efficient recombination of a Rosa26-RFP reporter in duct and islet δ cells The Hnf1b-CreERT2 BAC transgenic (Tg(Hnf1b-cre/ERT2)1Jfer) has been used extensively to trace the progeny of pancreatic ducts in developmental, regeneration, or cancer models. Hnf1b-CreERT2 transgenics have been used to show that cells from that form a duct-like plexus in the embryonic pancreas are bipotent duct-endocrine progenitors, whereas adult mouse duct cells are not a common source of {beta} cells in various regenerative settings. The interpretation of such genetic lineage tracing studies is critically dependent on a correct understanding of the cell type specificity of recombinase activity with each reporter system. We have re-examined the performance of Hnf1b-CreERT2 with a Rosa26-RFP reporter transgene. This showed inducible recombination of up to 96% adult duct cells, a much higher efficiency than previously used reporter transgenes. Despite this high duct-cell excision, recombination in  and {beta} cells remained very low, similar to previously used reporters. However, nearly half of somatostatin-expressing {delta} cells showed reporter activation, which was due to Cre expression in {delta} cells rather than to duct to {delta} cell conversions. The high recombination efficiency in duct cells indicates that the Hnf1b-CreERT2 model can be useful for both ductal fate mapping and genetic inactivation studies. The recombination in {delta} cells does not modify the interpretation of studies that failed to show duct conversions to other cell types, but needs to be considered if this model is used in studies that aim to modify the plasticity of pancreatic duct cells. Behavioral effects of ethanol in the Red Swamp Crayfish (Procambarus clarkii) Alcohol abuse and dependence remains one of the primary preventable sources of human mortality in the United States. Model laboratory species can be used to evaluate behavioral, neuropharmacological and other biological changes associated with alcohol exposure and to identify novel treatment modalities. This report describes methods for evaluating the behavioral effects of ethyl alcohol (ethanol; EtOH) in a crayfish model. Crayfish (Procambarus clarkii) were immersed in ethanol baths with concentrations ranging from 0.1 to 1.0 molar, and for durations of 10-30 minutes. Studies evaluated hemolymph alcohol concentration, locomotor behavior in an open field and anxiety-like behavior using a Light/Dark transfer approach. EtOH immersion produced dose dependent increases in hemolymph EtOH concentration (up to 249 mg/dL) and reductions in open field locomotor behavior that depended on EtOH concentration or exposure duration. Under baseline conditions, crayfish exhibit avoidance of the open parts of the locomotor arena and a preference for the covered portion, when available. Acute EtOH immersion decreased time spent in the covered portion of the Light/Dark arena, consistent with a decrease in anxiety-like behavior. EtOH immersion once per day for 5 days did not alter the acute locomotor behavioral response, however increased activity was observed 3 days after the repeated EtOH regimen. Overall, this study shows that this inexpensive, easily maintained species can be used for behavioral pharmacological experiments designed to assess the acute and repeated effects of EtOH. Characterising ontogeny of quantity discrimination in zebrafish A sense of non-symbolic numerical magnitudes is widespread in the animal kingdom and has been documented in adult zebrafish. Here we investigated the ontogeny of this ability using a group size preference task in juvenile zebrafish. Fish showed group size preference from 21 days post fertilization (dpf) and reliably chose the larger group when presented with discriminations of between 1 vs. 3, 2 vs. 5 and 2 vs. 3 conspecifics but not 2 vs. 4 conspecifics. When the ratio between the number of conspecifics in each group was maintained at 1:2, fish could discriminate between 1 vs. 2 individuals and 3 vs. 6, but again, not when given a choice between 2 vs. 4 individuals. These findings are in agreement with studies in other species suggesting the systems involved in quantity representation do not operate separately from other cognitive mechanisms. Rather they suggest quantity processing in fish may be the result of an interplay between attentional, cognitive and memory-related mechanisms as in humans and other animals. Our results emphasise the potential of the use of zebrafish to explore the genetic and neural processes underlying the ontogeny and function of number cognition. Phylogenomic analyses of Alismatales shed light into adaptations to aquatic environments Land plants first evolved from freshwater algae, and flowering plants returned to water as early as the Cretaceous and multiple times subsequently. Alismatales is the largest clade of aquatic angiosperms including all marine angiosperms, as well as terrestrial plants. We used Alismatales to explore plant adaptation to aquatic environments by analyzing a data set that included 95 samples (89 Alismatales species) covering four genomes and 91 transcriptomes (59 generated in this study). To provide a basis for investigating adaptation, we assessed phylogenetic conflict and whole-genome duplication (WGD) events in Alismatales. We recovered a relationship for the three main clades in Alismatales as (Tofieldiaceae, Araceae) + core Alismatids. We also found phylogenetic conflict among the backbone of the three main clades that could be explained by incomplete lineage sorting and introgression. Overall, we identified 18 putative WGD events across Alismatales. One of them occurred at the most recent common ancestor of core Alismatids, and three occurred at seagrass lineages. We also found that lineage and life-form were both important for different evolutionary patterns for the genes related to freshwater and marine adaptation. For example, several light or ethylene-related genes were lost in the seagrass Zosteraceae, but are present in other seagrasses and freshwater species. Stomata-related genes were lost in both submersed freshwater species and seagrasses. Nicotianamine synthase genes, which are important in iron intake, expanded in both submersed freshwater species and seagrasses. Our results advance the understanding of the adaptation to aquatic environments and whole-genome duplications using phylogenomics. 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. Sound level context modulates neural activity in the human brainstem Optimal perception requires adaptation to sounds in the environment. Adaptation involves representing the acoustic stimulation history in neural response patterns, for example, by altering response magnitude or latency as sound-level context change. Neurons in the auditory brainstem of rodents are sensitive to acoustic stimulation history and sound-level context (often referred to as sensitivity to stimulus statistics), but the degree to which the human brainstem exhibits such neural adaptation is unclear. In six electroencephalography experiments with over 125 participants, we demonstrate that the response latency of the human brainstem is sensitive to the history of acoustic stimulation over a few tens of milliseconds. We further show that human brainstem responses adapt to sound-level context in, at least, the last 44 ms, but that neural sensitivity to sound-level context decreases when the time window over which acoustic stimuli need to be integrated becomes wider. Our study thus provides evidence of adaptation to sound-level context in the human brainstem and of the timescale over which sound-level information affects neural responses to sound. The research delivers an important link to studies on neural adaptation in non-human animals. Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies Dementia with Lewy bodies is pathologically defined by the cytoplasmic accumulation of alpha-synuclein within neuronal cells in the brain. Alpha-synuclein is predominately pre-synaptic, but has been reported present in various subcellular compartments in cell and animal models. In particular, nuclear alpha-synuclein is evident in-vitro and in disease models and has been associated with altered DNA integrity, gene transcription, nuclear homeostasis. However, owing to various factors, the presence of alpha-synuclein in the nuclei of human brain cells remains controversial, as does its role in synucleinopathies.

Here, we close this gap and provide a unique demonstration confirming the presence of nuclear alpha-synuclein in post-mortem brain tissue obtained from cases of dementia with Lewy bodies as well as from controls via immunohistochemistry, immunoblot, and label-free mass-spectrometry.

Discrete intra-nuclear alpha-synuclein puncta reactive against phosphorylated serine 129-alpha-synuclein and pan-alpha-synuclein antibodies were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations from Dementia with Lewy bodies cases and matched controls. Subsequent biochemical analysis of subcellular fractionated tissue confirmed alpha-synuclein as present in a nuclear fraction at levels ~ 10-fold lower than in the cytoplasm. Critically, however, an increase in monomeric nuclear alpha-synuclein phosphorylated as serine 129 was observed in cases of dementia with Lewy bodies alongside higher molecular weight pan- and phosphorylation reactive alpha-synuclein species, consistent with the formation of intranuclear phosphorylated alpha-synuclein oligomers. Furthermore, the presence of nuclear alpha-synuclein was confirmed via label free mass spectrometry, as 6 unique alpha-synuclein derived peptide sequences were identified in nuclear fractions (71.4% sequence coverage).

Collectively, our data confirm the presence of nuclear alpha-synuclein in human brain tissue and describe nuclear pathology associated with dementia with Lewy bodies. These findings address a major controversy in the synucleinopathy field by confirming the presence of nuclear alpha-synuclein in autoptic human brain tissue and, for the first time, identify that alpha-synuclein is aggregated into novel and potentially pathological assemblies in the nucleus as part of the disease process associated with dementia with Lewy bodies and thus may contribute to the disease phenotype. Probing Patterning in Microbial Consortia with picCASO: a Cellular Automaton for Spatial Organisation Microbial consortia exhibit spatial patterning across diverse environments. Since probing the self-organization of natural microbial communities is limited by their inherent complexity, synthetic models have emerged as attractive alternatives. In this study, we develop novel frameworks of bacterial communication and explore the emergent spatiotemporal organization of microbes. Specifically, we build quorum sensing-mediated models of microbial growth that are utilized to characterize the dynamics of communities from arbitrary initial configurations and establish the effectiveness of our communication strategies in coupling the growth rates of microbes. Our simulations indicate that the behavior of quorum sensing-coupled consortia can be most effectively modulated by the rates of secretion of AHLs. Such a mechanism of control enables the construction of desired relative populations of constituent species in spatially organized populations. Our models accurately recapitulate previous experiments that have investigated pattern formation in synthetic multi-cellular systems. Additionally, our software tool enables the easy implementation and analysis of our frameworks for a variety of initial configurations and simplifies the development of sophisticated gene circuits facilitating distributed computing. Overall, we demonstrate the potential of spatial organization as a tunable parameter in synthetic biology by introducing a communication paradigm based on the location and strength of coupling of microbial strains.

Author SummaryInteracting microorganisms that coexist in a given environment tend to have well-defined spatial arrangements. While the emergence of such organization is seen across different microbiomes in nature, it is hitherto not well understood. Decoding the inherent spatial patterning of microbes is constrained by the complexity of their natural habitats. Here, we take advantage of synthetic models of microbial communities to study the dynamics of emergent spatial organization. Our framework of bacterial communication utilizes modular synthetic devices to couple growth rates. In addition to uncovering potential principles of spatial organization, this work enables the construction of complex genetic circuits distributed across communicating strains. Additionally, we have developed a software tool, picCASO, that streamlines the investigation of microbial systems communicating through such frameworks. Large-scale analysis of SARS-CoV-2 synonymous mutations reveals the adaptation to the human codon usage during the virus evolution Many large national and transnational studies have been dedicated to the analysis of SARS-CoV-2 genome, most of which focused on missense and nonsense mutations. However, approximately 30% of the SARS-CoV-2 variants are synonymous, therefore changing the target codon without affecting the corresponding protein sequence.

By performing a large-scale analysis of sequencing data generated from almost 400,000 SARS-CoV-2 samples, we show that silent mutations increasing the similarity of viral codons to the human ones tend to fixate in the viral genome over-time. This indicates that SARS-CoV-2 codon usage is adapting to the human host, likely improving its effectiveness in using the human aminoacyl-tRNA set through the accumulation of deceitfully neutral silent mutations.

One-Sentence SummarySynonymous SARS-CoV-2 mutations related to the activity of different mutational processes may positively impact viral evolution by increasing its adaptation to human codon usage. Trade-offs in biosensor optimization for dynamic pathway engineering Recent progress in synthetic biology allows the construction of dynamic control circuits for metabolic engineering. This technology promises to overcome many challenges encountered in traditional pathway engineering, thanks to their ability to self-regulate gene expression in response to bioreactor perturbations. The central components in these control circuits are metabolite biosensors that read out pathway signals and actuate enzyme expression. However, the construction of metabolite biosensors is a major bottleneck for strain design, and a key challenge is to understand the relation between biosensor dose-response curves and pathway performance. Here we employ multiobjective optimization to quantify performance trade-offs that arise in the design and calibration of metabolite biosensors. Our approach reveals strategies for tuning dose-response curves along an optimal trade-off between production flux and the cost of an increased expression burden on the host. We explore properties of control architectures built in the literature, and identify their advantages and caveats in terms of performance and robustness to growth conditions and leaky promoters. We demonstrate the optimality of a control circuit for glucaric acid production in Escherichia coli, which has been shown to increase titer by 2.5-fold as compared to static designs. Our results lay the groundwork for the automated design of control circuits for pathway engineering, with applications in the food, energy and pharmaceutical sectors. Directed evolution of angiotensin-converting enzyme 2 (ACE2) peptidase activity profiles for therapeutic applications Angiotensin Converting-Enzyme 2 (ACE2) is currently being investigated for its ability to beneficially modulate the Angiotensin receptor (ATR) therapeutic axis to treat multiple human diseases, but its broad substrate scope and diverse physiological roles limit its potential as a therapeutic agent. In this work we engineer ACE2 variants with enhanced hydrolytic activity and specificity toward Angiotensin-II (Ang-II) that may lead to new therapeutic candidates with improved efficacy and reduced off-target side effects. We established a yeast display-based liquid chromatography screen that enabled use of directed evolution to discover ACE2 variants with improved Ang-II activity and specificity relative to the off-target peptide substrates Apelin-13 and Angiotensin-I (Ang-I). We screened ACE2 active site libraries to reveal three substitution-tolerant positions (M360, T371 and Y510) that can be mutated to enhance ACE2s activity profile and followed up on these hits with focused double mutant libraries to further improve the enzyme. Relative to wildtype ACE2, our top variant (T371L/Y510Ile) displayed a sevenfold increase in Ang-II turnover number (kcat), a sixfold diminished catalytic efficiency (kcat/Km) on Apelin-13, and an overall decreased activity on other ACE2 substrates that were not directly assayed in directed evolution screen. At physiologically relevant substrate concentrations, T371L/Y510Ile hydrolyzes more Ang-II than wildtype ACE2 with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to both established and unexplored ACE2 therapeutic applications and provide a foundation for further ACE2 engineering efforts. α-Phenylalanyl tRNA synthetase competes with Notch signaling through its N-terminal domain The alpha subunit of the cytoplasmic Phenylalanyl tRNA synthetase (-PheRS, FARSA in humans) displays cell growth and proliferation activities and its elevated levels can induce cell fate changes and tumor-like phenotypes that do neither dependent on the canonical function of charging tRNAPhe with phenylalanine nor on stimulating general translation. In intestinal stem cells of Drosophila midguts, -PheRS levels are naturally slightly elevated and human FARSA mRNA levels are elevated in multiple cancers. In the Drosophila midgut model, elevated -PheRS levels caused the accumulation of many additional proliferating cells resembling intestinal stem cells (ISCs) and enteroblasts (EBs). This phenotype resembles partially the tumor-like phenotype described as Notch RNAi phenotype for the same cells. Genetic interactions between -PheRS and Notch suggest that their activities neutralize each other and that elevated -PheRS levels attenuate Notch signaling whether Notch induces differentiation into enterocytes, neuroblast stem cell proliferation, or transcription of a Notch reporter. These non-canonical functions all map to the N-terminal part of -PheRS which accumulates naturally in the intestine. This truncated version of -PheRS (-S) also localizes to nuclei and displays weak sequence similarity to the Notch intracellular domain (NICD), suggesting that -S might compete with the NICD for binding to a common target. Supporting this hypothesis, the tryptophan (W) residue reported to be key for the interaction between the NICD and the Su(H) BTD domain is not only conserved in -PheRS and -S, but also essential for attenuating Notch signaling.

Author SummaryAminoacyl tRNA synthetases charge tRNAs with their cognate amino acid to ensure proper decoding of the genetic code during translation. Independent of its aminoacylation function, the alpha subunit of Drosophila cytoplasmic Phenylalanyl tRNA synthetase (-PheRS, FARSA in humans) has an additional activity that promotes growth and proliferation (Ho et al., 2021). Here we describe that elevated -PheRS levels also induce cell fate changes and tumorous phenotypes in Drosophila midguts. Excessive proliferating cells with stem and progenitor cell characteristics accumulate and the composition of the terminally differentiated cells changes, too. This phenotype together with observed genetic interactions between -PheRS and Notch levels show that -PheRS counteracts Notch signaling in many different tissues and developmental stages. This novel activity of -PheRS maps to its N-terminal part, which is naturally produced. The fragment contains a DNA binding domain, translocates into nuclei, and displays essential similarities to a Notch domain that binds to the downstream transcription factor. This suggests that it might be competing with Notch for binding to a common target. Not only because Notch plays important roles in many tumors, but also because FARSA mRNA levels are considerably upregulated in many tumors, this novel activity deserves more attention for cancer research. Blocking antibodies against integrin-a3, integrin-aM, and integrin-aMb2 de-differentiate myofibroblasts and reverse lung and kidney fibroses in a mouse model Fibrosis is involved in 45% of deaths in the United States, and no treatment exists to reverse the progression of the disease. Myofibroblasts are key to the progression and maintenance of fibrosis. We investigated features of cell adhesion necessary for monocytes to differentiate into myofibroblasts, seeking to identify pathways key to myofibroblast differentiation. Blocking antibodies against integrins 3, M, and M{beta}2 de-differentiate myofibroblasts in vitro, lower the pro-fibrotic secretome of myofibroblasts, and reverse lung and kidney fibrosis in vivo. Decorins collagen-binding peptide directs blocking antibodies (against integrins-3, -M, -M{beta}2) to both fibrotic lungs and fibrotic kidneys, reducing the dose of antibody necessary to reverse fibrosis. This targeted immunotherapy blocking key integrins may be an effective therapeutic for the treatment and reversal of fibrosis.

SummaryBlocking antibodies against integrins-3, -M, and -M{beta}2 can be targeted to sites of fibrosis, reverse lung and kidney fibroses, and offer the potential to bring immunotherapy to fibrosis Life history adaptations to fluctuating environments: Combined effects of demographic buffering and lability of demographic parameters Demographic buffering and lability have been identified as adaptive strategies to optimise fitness in a fluctuating environment. These are not mutually exclusive, however we lack efficient methods to measure their relative importance for a given life history. Here, we decompose the stochastic growth rate (fitness) into components arising from nonlinear responses and variance-covariance of demographic parameters to an environmental driver, which allows studying joint effects of buffering and lability. We apply this decomposition for 154 animal matrix population models under different scenarios, to explore how these main fitness components vary across life histories. Faster-living species appear more responsive to environmental fluctuations, either positively or negatively. They have the highest potential for strong adaptive demographic lability, while demographic buffering is a main strategy in slow-living species. Our decomposition provides a comprehensive framework to study how organisms adapt to variability through buffering and lability, and to predict species responses to climate change. "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. Electrical Stimulated GLUT4 Signaling Attenuates Critical Illness-Associated Muscle Wasting BackgroundCritical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a byproduct of critical care, attributed to impaired recovery, long-term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood, however loss of mechanical stimuli contributes to critical illness associated muscle atrophy and weakness. Passive mechanical loading (ML) and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care.

MethodsRats were subjected to 8 days critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and Western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM.

ResultsFollowing 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, Myosin:Actin ratio and single muscle fiber maximum force normalized to cross-sectional area (specific force) were reduced by 40-50% (p< 0.0001). ES significantly reduced the loss of soleus muscle fiber cross-sectional area (CSA) and Myosin:Actin ratio by approximately 30% (p< 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signaling in the soleus muscle following critical care and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (p< 0.01). ES promoted phosphofructokinase and insulin signaling pathways to control levels (p< 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signaling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (p= 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately 2-fold (p= 0.06). Reduction of muscle protein degradation rather than protein synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (p= 0.006) and MuRF1 (p= 0.08) by approximately 50%, downstream of AMPK-FoxO3.

ConclusionsES maintained GLUT4 translocation through increased AMPK-TBC1D4 signaling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signaling and reduced muscle protein degradation in CIM. Multi-omic analysis of the Arabidopsis clock activator mutant rve 4 6 8 reveals connections to carbohydrate metabolism and proteasome regulation Plants are able to sense changes in their light environments, such as the onset of day and night, as well as anticipate these changes in order to adapt and survive. Central to this ability is the plant circadian clock, a molecular circuit that precisely orchestrates plant cell processes over the course of a day. REVEILLE proteins (RVEs) are recently discovered members of the plant circadian circuitry that activate the evening complex and PRR genes to maintain regular circadian oscillation. The RVE 8 protein and its two homologs, RVE 4 and 6, have been shown to limit the length of the circadian period, with rve 4 6 8 triple-knockout plants possessing an elongated period along with increased leaf surface area, biomass, cell size and delayed flowering relative to wild-type Col-0 plants. Here, using a multi-omics approach consisting of phenomics, transcriptomics, proteomics, and metabolomics we draw novel connections between RVE8-like proteins and a number of core plant cell processes. In particular, we reveal that loss of RVE8-like proteins results in altered carbohydrate, organic acid and lipid metabolism, including a starch excess phenotype at dawn. We further demonstrate that rve 4 6 8 plants have lower levels of 20S proteasome subunits and possess significantly reduced proteasome activity, potentially explaining the increase in cell-size observed in RVE8-like mutants. Overall, this robust, multi-omic dataset, provides substantial new insights into the far reaching impact RVE8-like proteins have on the diel plant cell environment.