Population differences in the length and early-life dynamics of telomeres among European pied flycatchers Telomere length and shortening rate are increasingly used as biomarkers for long-term costs in ecological and evolutionary studies because of their relationships with survival and fitness. Both early-life conditions and growth, and later-life stressors can create variation in telomere shortening rate. Studies on between- population telomere length and dynamics are scarce, despite the expectation that populations exposed to varying environmental constraints would present divergent telomere length patterns. The pied flycatcher (Ficedula hypoleuca) is a passerine bird breeding across Eurasia (from Spain to western Siberia) and migrating through the Iberian Peninsula to spend the non-breeding period in sub-Saharan Africa. Thus, different populations show marked differences in migration distance. We studied the large-scale variation of telomere length and early-life dynamics in the pied flycatcher by comparing six European populations across a north- south gradient (Finland, Estonia, England, and Spain) predicting negative effect of migration distance on adult telomere length, and of nestling growth on nestling telomere dynamics. There were clear population differences in telomere length, with English birds from mid-latitudes having the longest telomeres. Telomere length did not thus show consistent latitudinal variation and was not linearly linked to differences in migration distance. Early-life telomere shortening rate tended to vary between populations. Fast growth was associated with shorter telomeres in the early life, but faster nestling growth affected telomeres more negatively in northern than southern populations. While the sources of between-population differences in telomere-related biology remain to be more intensively studied, our study illustrates the need to expand telomere studies at the between-population level.
A nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an olfactory chemoattractant for the grass puffer Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this unknown function TTX analog as an olfactory chemoattractant.
Common Dynamic Determinants Govern Quorum Quenching Activity in N-terminal Serine Hydrolases Growing concerns about microbial antibiotic resistance have motivated extensive research into ways of overcoming antibiotic resistance. Quorum quenching (QQ) processes disrupt bacterial communication via quorum sensing, which enables bacteria to sense the surrounding bacterial cell density and markedly affects their virulence. Due to its indirect mode of action, QQ is believed to exert limited pressure on essential bacterial functions and may thus avoid inducing resistance. Although many enzymes display QQ activity against various bacterial signaling molecules, their mechanisms of action are poorly understood, limiting their potential optimization as QQ agents. Here we evaluate the capacity of three N-terminal serine hydrolases to degrade N-acyl homoserine lactones that serve as signaling compounds for Gram-negative bacteria. Using molecular dynamics simulations of the free enzymes and their complexes with two signaling molecules of different lengths, followed by quantum mechanics/molecular mechanics molecular dynamics simulations of their initial catalytic steps, we clarify the molecular processes underpinning their QQ activity. We conclude that all three enzymes degrade bacterial signaling molecules via similar reaction mechanisms. Moreover, we experimentally confirmed the activity of two penicillin G acylases from Escherichia coli (ecPGA) and Achromobacter spp. (aPGA), adding these biotechnologically well-optimized enzymes to the QQ toolbox. We also observed enzyme- and substrate-dependent differences in the catalytic actions of these enzymes, arising primarily from the distinct structures of their acyl-binding cavities and the dynamics of their molecular gates. As a consequence, the first reaction step catalyzed by ecPGA with a longer substrate had an elevated energy barrier because its shallow acyl binding site could not accommodate a productive substrate-binding configuration. Conversely, aPGA in complex with both substrates exhibited unfavorable energetics in both reaction steps due to the dynamics of the residues gating the acyl binding cavity entrance. Finally, the energy barriers of the second reaction step catalyzed by Pseudomonas aeruginosa acyl-homoserine lactone acylase with both substrates were higher than in the other two enzymes due to the unique positioning of Arg297{beta} in this enzyme. The discovery of these dynamic determinants will guide future efforts to design robust QQ agents capable of selectively controlling virulence in resistant bacterial species. Reconstitution of minimal motility system based on Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium.

One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium. Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy As of October 2021, coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global emergency, and novel therapeutics are urgently needed. Here we describe human single chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. In two independent animal models, intranasal administration counteracted the infection. Due to high efficiency, remarkable stability, resilience to nebulization and low production cost, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status. A tool for assessing alignment of biomedical data repositories with open, FAIR, citation and trustworthy principles Increasing attention is being paid to the operation of biomedical data repositories in light of efforts to improve how scientific data is handled and made available for the long term. Multiple groups have produced recommendations for functions that biomedical repositories should support, with many using requirements of the FAIR data principles as guidelines. However, FAIR is but one set of principles that has arisen out of the open science community. They are joined by principles governing open science, data citation and trustworthiness, all of which are important aspects for biomedical data repositories to support. Together, these define a framework for data repositories that we call OFCT: Open, FAIR, Citable and Trustworthy. Here we developed an instrument using the open source PolicyModels toolkit that attempts to operationalize key aspects of OFCT principles and piloted the instrument by evaluating eight biomedical community repositories listed by the NIDDK Information Network (dkNET.org). Repositories included both specialist repositories that focused on a particular data type or domain, in this case diabetes and metabolomics, and generalist repositories that accept all data types and domains. The goal of this work was both to obtain a sense of how much the design of current biomedical data repositories align with these principles and to augment the dkNET listing with additional information that may be important to investigators trying to choose a repository, e.g., does the repository fully support data citation? The evaluation was performed from March to November 2020 through inspection of documentation and interaction with the sites by the authors. Overall, although there was little explicit acknowledgement of any of the OFCT principles in our sample, the majority of repositories provided at least some support for their tenets. Markov Chain Abstractions of Electrochemical Reaction-Diffusion in Synaptic Transmission for Neuromorphic Computing Progress in computational neuroscience towards understanding brain function is challenged both by the complexity of molecular-scale electrochemical interactions at the level of individual neurons and synapses and the dimensionality of network dynamics across the brain covering a vast range of spatial and temporal scales. Our work abstracts an existing highly detailed, biophysically realistic 3D reaction-diffusion model of a chemical synapse to a compact internal state space representation that maps onto parallel neuromorphic hardware for efficient emulation at a very large scale and offers near-equivalence in input-output dynamics while preserving biologically interpretable tunable parameters. A Facile Alternative Sustainable Process for the Extraction and Purification of Microbial Melanin Conventional techniques for the extraction of the melanin encounter several disadvantages such as poor extraction efficiency, excessive use of acid and alkali, and lengthy time duration. To extract melanin in a more sustainable manner, herein suitability of an aqueous solution of quaternary ammonium hydroxide known as tetrabutylammonium hydroxide (40% w/w TBAOH in water) to extract and purify the pigment from the endophyte Streptomyces hyderabadensis 7VPT5-5R was investigated. The solvent could extract 5.54{+/-}0.03 g/L of pure melanin against 3.31{+/-} 0.02 g/L of pure melanin by conventional method from bacterial culture resulting 66% increase in the yield of the pigment. The quaternary ammonium based solvent was further recycled for 5 consecutive cycles and production of melanin with yield ranging from 5.54{+/-}0.03 g/L to 5.47{+/-} 0.02 g/L was obtained. The yield of the pigment was 38-40% higher in the recycled solvent in comparison to the melanin yield extracted by conventional method. Considering the efficiency of the solvent for effective extraction and purification of melanin and its recyclability potential, it can be considered as a sustainable solvent for the extraction of the pigments from bacterial culture. Furthermore, the developed method for extraction of melanin from microorganisms as a sustainable source would help in reducing the cost of melanin leading to its efficient utilization in diverse fields. Diurnal activity budget and conservation implication of Hippopotamus amphibius at Bui National Park, Ghana Animals apportion time for their daily behavioral activities. We studied the activity budget of Hippopotamus amphibius at the Black Volta River in the Bui National Park, Ghana. We performed instantaneous scan sample using ground count survey between August 2020 to July 2021. We observed that H. amphibius spent most of their day time resting (54.75 %), followed by feeding (22.93 %), walking (19.2 %) and touching (3.12 %). We found significant difference in the time spent between the daily activities (H = 41.67; p < 0.0001). Except activities involving touching, we found no significant seasonal differences in feeding, resting and walking activities by H. amphibius. Understanding the behavioral activities of H. amphibius will enhance management and conservation of the animals. OncoLoop: A network-based precision cancer medicine framework At present, prioritizing cancer treatments at the individual patient level remains challenging, and performing co-clinical studies using patient-derived models in real-time is often not feasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework to predict and validate drug sensitivity in human tumors and their pre-existing high-fidelity (cognate) model(s) by leveraging perturbational profiles of clinically-relevant oncology drugs. As proof-of-concept, we applied OncoLoop to prostate cancer (PCa) using a series of genetically-engineered mouse models (GEMMs) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of published cohorts using Master Regulator (MR) conservation analysis revealed that most patients were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated, including in two cognate allografts and one cognate patient-derived xenograft (PDX). OncoLoop is a highly generalizable framework that can be extended to other cancers and potentially other diseases.

Significance StatementOncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround co-clinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available. Host phenology can drive the evolution of intermediate virulence strategies in some parasites AO_SCPLOWBSTRACTC_SCPLOWThe traditional mechanistic trade-offs resulting in a negative correlation between transmission and virulence are the foundation of nearly all current theory on the evolution of parasite virulence. Several ecological factors have been shown to modulate the optimal virulence strategies predicted from mechanistic trade-off models, but these ecological factors have not yet been shown to be sufficient to explain the intermediate virulence strategies observed in any natural system. The timing of seasonal activity, or phenology, is a common factor that influences the types and impact of many ecological interactions but is difficult to incorporate into virulence evolution studies. We develop a mathematical model of a disease system with seasonal host activity to study the evolutionary consequences of host phenology on the virulence of obligate-killer parasite. Results from this model demonstrated that seasonal host activity is sufficient to drive the evolution of intermediate parasite virulence in some types of natural disease systems, even when a traditional mechanistic trade-off between transmission and virulence is not assumed in the modeling framework. The optimal virulence strategy in these systems can be determined by both the duration of the host activity period as well as the variation in the host emergence timing. Parasites with low virulence strategies are favored in environments with long host activity periods and in environments in which hosts emerge synchronously. The results demonstrate that host phenology can be sufficient to select for intermediate optimal virulence strategies, providing an alternative mechanism to account for virulence evolution in some natural systems. Single amino-acid mutation in a Drosophila melanogaster ribosomal protein: an insight in uL11 transcriptional activity The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co- localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. RNA- seq analysis from wing imaginal discs shows enrichment in the GO categories "glutathione metabolism" for up-regulated genes in both uL11K3A and uL11K3Y mutants and "regulation of transcription" for down-regulated genes in uL11K3A only. Analysis of the cis-regulatory sequences of these genes suggests that uL11 might regulate transcription of target genes in concert with the couple of transcription factors Mad/Med that mediate response to the Bone Morphogenetic Protein (BMP) signaling pathway. Coronavirus-associated molecular mimicry through homology to a SARS-CoV-2 peptide could be leading to susceptibility in patients with HLA-A*02:01 and HLA-A*24:02 serotypes This study aims to predict autoimmunity-related pathological mechanisms that possess risk for individuals with specific human leukocyte antigen (HLA) serotypes and shared by certain coronaviruses including SARS-CoV-2, based on homology to a SARS-CoV-2 peptide. With the given aim, 1-) coronavirus-associated sequences, which are homologous to the 15mer SARS-CoV-2 peptide CFLGYFCTCYFGLFC, are obtained. 2-) Human peptides that have at least 7 residue matches with those coronavirus sequences, and the SARS-CoV-2 15mer, are found. 3-) Epitope pairs, which are sourced by those aligned coronavirus and human sequences are identified. 4-) Epitope pairs that are predicted to bind strongly not only to the same HLA allele with each other but also to the same HLA allele as those of the respective alignment of the SARS-CoV-2 peptide are selected. Following are the identified proteins or peptides (with HLA-A*02:01 or HLA-A*24:02 epitopes), as described in 1-to-4: Immunoglobulin heavy chain junction regions, CRB1 isoform I precursor, slit homolog 2 protein, hCG1995581, hCG2028737, phospholipid phosphatase-related protein type 2. Among those, CRB1 isoform I precursor sequence with the predicted HLA-A*24:02 epitope aligns with the highest number of different sequences. Results imply autoimmunity risk in COVID-19 patients with HLA-A*02:01 and HLA-A*24:02 serotypes, through molecular mimicry, as a shared pathogenicity risk that can be prevalent upon getting infected with certain coronaviruses. These can pave way to improved risk groups assessment and autoimmunity treatment options, for COVID-19 and its associated diseases. Also, the approach in this study can be used to predict prospective pathologies of the transmissible variants in susceptible humans. Relative contributions of sex hormones, sex chromosomes, and gonads to sex differences in tissue gene regulation Sex differences in physiology and disease in mammals result from the effects of three classes of factors that are inherently unequal in males and females: reversible (activational) effects of gonadal hormones, permanent (organizational) effects of gonadal hormones, and cell-autonomous effects of sex chromosomes, as well as genes driven by these classes of factors. Often, these factors act together to cause sex differences in specific phenotypes, but the relative contribution of each and the interactions among them remain unclear. Here, we used the Four Core Genotypes (FCG) mouse model with or without hormone replacement to distinguish the effects of each class of sex-biasing factors on transcriptome regulation in liver and adipose tissues. We found that the activational hormone levels have the strongest influence on gene expression, followed by the organizational gonadal sex effect and, lastly, sex chromosomal effect, along with interactions among the three factors. Tissue specificity was prominent, with a major impact of estradiol on adipose tissue gene regulation, and of testosterone on the liver transcriptome. The networks affected by the three sex-biasing factors include development, immunity and metabolism, and tissue-specific regulators were identified for these networks. Furthermore, the genes affected by individual sex-biasing factors and interactions among factors are associated with human disease traits such as coronary artery disease, diabetes, and inflammatory bowel disease. Our study offers a tissue-specific account of the individual and interactive contributions of major sex-biasing factors to gene regulation that have broad impact on systemic metabolic, endocrine, and immune functions. 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. In vitro culture of bovine fibroblasts using select serum-free media supplemented with Chlorella vulgaris extract Standard cell culture practices require addition of animal-derived serum to culture media to achieve adequate cell growth. Typically, 5-10% by volume of fetal bovine serum (FBS) is used, which accounts for a vast majority of the cost of media while also imposing environmental and ethical concerns associated with the use of animal serum. Here we tested the efficacy of culturing cells by replacing serum in the media with algae extract and select additives. Using LC-MS, we compared molecular signatures of FBS to Chlorella algae extracts and identified NAD(H)/NADP(H) as common and relatively abundant features in their characteristic profiles. Bovine fibroblasts, cultured in serum-free media supplemented with C. vulgaris extract and just two growth factors plus insulin, showed significant growth with enhanced viability compared to control cells cultured without serum, albeit still lower than that of controls cultured with 10% FBS. Moreover, C. vulgaris extract enhanced cell viability beyond that of cells cultured with the two growth factors and insulin alone. These results suggest that key components in serum which are essential for cell growth may also be present in C. vulgaris extract, demonstrating that it may be used at least as a partial alternative to serum for cell culture applications. 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. Protein sequence sampling and prediction from structural data The increasing integration between protein engineering and machine learning has led to many interesting results. A problem still to solve is to evaluate the likelihood that a sequence will fold into a target structure. This problem can be also viewed as sequence prediction from a known structure.

In the current work, we propose improvements in the recent architecture of Geometric Vector Perceptrons [1] in order to optimize the sampling of sequences from a known backbone structure. The proposed model differs from the original in that there is: (i) no updating in the vectorial embedding, only in the scalar one, (ii) only one layer of decoding. The first aspect improves the accuracy of the model and reduces the use of memory, the second allows for training of the model with several tasks without incurring data leakage.

We treat the trained classifier as an Energy-Based Model and sample sequences by sampling amino acids in a non-autoreggresive manner in the empty positions of the sequence using energy-guided criteria and followed by random mutation optimization. We improve the median identity of samples from 40.2% to 44.7%.

An additional question worth investigating is whether sampled and original sequences fold into similar structures independent of their identity. We chose proteins in our test set whose sampled sequences show low identity (under 30%) but for which our model predicted favorable energies. We used AlphaFold [2, 3] and observed that the predicted structures for sampled sequences highly resemble the predicted structures for original sequences, with an average TM-score of 0.84. Structures of the Omicron spike trimer with ACE2 and an anti-Omicron antibody: mechanisms for the high infectivity, immune evasion and antibody drug discovery The Omicron variant of SARS-CoV-2 has rapidly become the dominant infective strain and the focus efforts against the ongoing COVID-19 pandemic. Here we report an extensive set of structures of the Omicron spike trimer by its own or in complex with ACE2 and an anti-Omicron antibody. These structures reveal that most Omicron mutations are located on the surface of the spike protein, which confer stronger ACE2 binding by nearly 10 folds but become inactive epitopes resistant to many therapeutic antibodies. Importantly, both RBD and the closed conformation of the Omicron spike trimer are thermodynamically unstable, with the melting temperature of the Omicron RBD decreased by as much as 7{degrees}C, making the spiker trimer prone to random open conformations. An unusual RBD-RBD interaction in the ACE2-spike complex unique to Omicron is observed to support the open conformation and ACE2 binding, serving the basis for the higher infectivity of Omicron. A broad-spectrum therapeutic antibody JMB2002, which has completed Phase 1 clinical trial, is found to interact with the same two RBDs to inhibit ACE2 binding, in a mode that is distinguished from all previous antibodies, thus providing the structural basis for the potent inhibition of Omicron by this antibody. Together with biochemical data, our structures provide crucial insights into higher infectivity, antibody evasion and inhibition of Omicron. Overexpression-based detection of translatable circular RNAs is vulnerable to coexistent linear RNA byproducts In RNA field, the demarcation between coding and non-coding has been negotiated by the recent discovery of occasionally translated circular RNAs (circRNAs). Although absent of 5 cap structure, circRNAs can be translated cap-independently. Complementary intron-mediated overexpression is one of the most utilized methodologies for circRNA research but not without bearing echoing skepticism for its poorly defined mechanism and latent coexistent side products. In this study, leveraging such circRNA overexpression system, we have interrogated the protein-coding potential of 30 human circRNAs containing infinite open reading frames in HEK293T cells. Surprisingly, pervasive translation signals are detected by immunoblotting. However, intensive mutagenesis reveals that numerous translation signals are generated independently of circRNA synthesis. We have developed a dual tag strategy to isolate translation noise and directly demonstrate that the fallacious translation signals originate from cryptically spliced linear transcripts. The concomitant linear RNA byproducts, presumably concatemers, can be translated to allow pseudo rolling circle translation signals, and can involve backsplicing junction (BSJ) to disqualify the BSJ-based evidence for circRNA translation. We also find non-AUG start codons may engage in the translation initiation of circRNAs. Taken together, our systematic evaluation sheds light on heterogeneous translational outputs from circRNA overexpression vector and comes with a caveat that ectopic overexpression technique necessitates extremely rigorous control setup in circRNA translation and functional investigation. Common variants in breast cancer risk loci predispose to distinct tumor subtypes BackgroundGenome-wide association studies (GWAS) have identified multiple common breast cancer susceptibility variants. Many of these variants have differential associations by estrogen receptor (ER), but how these variants relate with other tumor features and intrinsic molecular subtypes is unclear.

MethodsAmong 106,571 invasive breast cancer cases and 95,762 controls of European ancestry with data on 173 breast cancer variants identified in previous GWAS, we used novel two-stage polytomous logistic regression models to evaluate variants in relation to multiple tumor features (ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and grade) adjusting for each other, and to intrinsic-like subtypes.

ResultsEighty-five of 173 variants were associated with at least one tumor feature (false discovery rate <5%), most commonly ER and grade, followed by PR and HER2. Models for intrinsic-like subtypes found nearly all of these variants (83 of 85) associated at P<0.05 with risk for at least one luminal-like subtype, and approximately half (41 of 85) of the variants were associated with risk of at least one non-luminal subtype, including 32 variants associated with triple-negative (TN) disease. Ten variants were associated with risk of all subtypes in different magnitude. Five variants were associated with risk of luminal A-like and TN subtypes in opposite directions.

ConclusionThis report demonstrates a high level of complexity in the etiology heterogeneity of breast cancer susceptibility variants and can inform investigations of subtype-specific risk prediction. Structure-Functional-Selectivity Relationship Studies on A-86929 Analogs and Small biaryl Fragments toward Discovery of biased D1 agonists Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through the D1- (D1R and D5R) and D2-class (D2R, D3R and D4R) of seven transmembrane receptors, and activates both G-protein- and {beta}-arrestin-dependent signaling pathways, to mediate its physiological effects. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinsons disease, or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side-effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling towards the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side-effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity-relationship (SAR) studies have embarked on discovering {beta}-arrestin-biased dopamine agonists, focused on D2 partial agonists, non-catechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R {beta}-arrestin biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold. Previously described and novel analogs of A-86929 were synthesized and screened in vitro for structure-functional-selectivity relationships (SFSR) studies to identify chemical motifs responsible for {beta}-arrestin biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein biased but none of them were exclusively arrestin-biased. Additionally, various catechol aryl fragments were designed and synthesized. Other compounds surveyed included hydroxyl and chloro analogs of dopamine to test for hydrogen bonding and ionic interactions. Some of these small molecular probes displayed weak bias towards the {beta}-arrestin pathway. Continued in-depth SFSR studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate discovery of potent and efficacious arrestin-biased dopamine receptor ligands. Multiple brace root phenotypes promote anchorage and limit root lodging in maize Plant mechanical failure (lodging) causes global yield losses of 7-66% in cereal crops. We have previously shown that the above-ground nodal roots (brace roots) in maize are critical for anchorage. However, it is unknown how brace root phenotypes vary across genotypes and the functional consequence of this variation. This study quantifies the contribution of brace roots to anchorage, brace root traits, plant height, and root lodging susceptibility in 52 maize inbred lines. We show that the contribution of brace roots to anchorage and root lodging susceptibility varies among genotypes and this contribution can be explained by plant architectural variation. Additionally, supervised machine learning models were developed and show that multiple plant architectural phenotypes can predict the contribution of brace roots to anchorage and root lodging susceptibility. Together these data define the plant architectures that are important in lodging resistance and show that the contribution of brace roots to anchorage is a good proxy for root lodging susceptibility. Fatal COVID-19 and non-COVID-19 Acute Respiratory Distress Syndrome is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State Without Fibrosis ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. Active enhancers strengthen insulation by RNA-mediated CTCF binding at TAD boundaries Vertebrate genomes are partitioned into Topologically Associating Domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at TAD boundaries correlates with better insulation, however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer:promoter interactions and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters we show that active TAD enhancers but not promoters induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs. Effect of a low concentration in chloride, sodium and potassium on oocyte maturation, oocyte hydration, ovulation and egg quality in rainbow trout. Water salinity is an important environmental factor known to have detrimental effects on salmonid reproduction, mostly when migrating female broodfish are held in sea water. In contrast, data obtained in freshwater are scarce and the impact of low water salinity, and more specifically of low water concentrations in sodium, chloride and potassium, during reproduction in freshwater is currently unknown. For this reason, and because ion and water fluxes are critical for the final steps of the female gamete formation, including oocyte hydration and ovulation, the aim of the present study was to investigate the impact of low salinity water on final oocyte maturation, ovulation and, ultimately, on egg quality, using rainbow trout as a physiological model and relevant aquaculture species.

Fish from the same commercial strain were raised either in a site characterized by low concentrations of Na+, K+, and Cl- ions in the water or in a closely located control site exhibiting higher concentration in these elements. Egg quality and duration of final oocyte maturation were investigated using innovative phenotyping tools such as automatic assessment of egg viability using the VisEgg system and non-invasive echograph-based monitoring of final oocyte maturation duration, respectively. Oocyte hydration during final oocyte maturation and after ovulation was also investigated. Finally, molecular phenotyping was performed using real-time PCR-based monitoring of several key players of final oocyte maturation and ovulation associated with ion and water transport, inflammation, proteolytic activity, and coagulation. Oocyte hydration and gene expression data were analyzed in the light of the duration of final oocyte maturation.

Here we show that low water salinity (i.e., low water concentration in chloride, sodium and potassium) negatively influences final oocyte maturation, ovulation and, ultimately, egg quality. Low water salinity triggered delayed ovulation and lower oocyte viability. When investigating the impact of low water salinity on final oocyte maturation duration, individuals presenting the most severe phenotypes exhibited impaired oocyte hydration and abnormally reduced gene expression levels of several key players of the ovulatory process. While the under expression of water (i.e., aquaporins) and ion (i.e., solute carriers) transporters is consistent with impaired oocyte hydration, our observations also indicate that the overall ovulatory gene expression program is disrupted. Our results raise the question of the mechanisms underlying the negative influence of low salinity water on the dynamics of the preovulatory phase, on the control of the oocyte homeostasis, including hydration, and on the overall success of the maturation-ovulation process.

HighlightsO_LILow water salinity impairs final oocyte maturation and egg quality in rainbow trout
C_LIO_LILow water salinity induces delayed ovulation and impaired oocyte hydration
C_LIO_LILow water salinity induces a dysregulation of several key ovulatory genes
C_LIO_LIMonitoring of final oocyte maturation can be performed using ultrasound staging
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