Deficiency of the paternally inherited gene Magel2 alters the development of separation-induced vocalization and maternal behavior in mice The behavior of offspring results from the combined expression of maternal and paternal genes. Genomic imprinting silences some genes in a parent-of-origin specific manner, a process that, among all animals, occurs only in mammals. How genomic imprinting affects the behavior of mammalian offspring, however, remains poorly understood. Here, we studied how the loss of the paternally inherited gene Magel2 in mouse pups affects the emission of separation-induced ultrasonic vocalizations (USV). Using quantitative analysis of more than one hundred thousand USVs, we characterized the rate of vocalizations as well as their spectral features from postnatal days 6 to 12 (P6-P12), a critical phase of mouse development during which pups fully depend on the mother for survival. Our analyses show that Magel2 deficient offspring emit separation-induced vocalizations at lower rates and with altered spectral features. We also show that dams display altered behavior towards their own Magel2 deficient offspring. In a test to compare the retrieval of two pups, dams retrieve wildtype control pups first and faster than Magel2 deficient offspring. These results suggest that the loss of Magel2 impairs the expression of separation-induced vocalization in pups as well as maternal behavior, both of which support the pups growth and development.
Switch-like and persistent memory formation in individual Drosophila larvae Associative learning allows animals to use past experience to predict future events. The circuits underlying memory formation support immediate and sustained changes in function, often in response to a single example. Larval Drosophila is a genetic model for memory formation that can be accessed at molecular, synaptic, cellular, and circuit levels, often simultaneously, but existing behavioral assays for larval learning and memory do not address individual animals, and it has been difficult to form long lasting memories, especially those requiring synaptic re-organization. We demonstrate a new assay for learning and memory capable of tracking the changing preferences of individual larvae. We use this assay to explore how activation of a pair of reward neurons changes the response to the innately aversive gas Carbon Dioxide (CO2). We confirm that when coupled to odor presentation in appropriate temporal sequence, optogenetic reward reduces avoidance of CO2. We find that learning is switch-like: all-or-none and quantized in two states. Memories can be extinguished by repeated unrewarded exposure to CO2 but are stabilized against extinction by repeated training or overnight consolidation. Finally, we demonstrate long-lasting protein synthesis dependent and independent memory formation.
Excitation and emission spectra of in silico red-shift predicted iLOV mutants iLOV is a flavin mononucleotide-binding fluorescent protein used for in vivo cellular imaging similar to the green fluorescent protein. To expand the range of applications of iLOV, spectrally tuned red-shifted variants are desirable to have reduced phototoxicity and better tissue penetration. In this report, we experimentally tested two iLOV mutants, iLOVL470T/Q489K and iLOVV392K/F410V/A426S, which were previously computationally proposed by Khrenova et al. (DOI: 10.1021/acs.jpcb.7b07533) to have red-shifted excitation and emission spectra. We found that mutants bearing the V392K mutation lost the ability to bind FMN. While iLOVL470T/Q489K is about 20% brighter compared to WT iLOV in vitro, it exhibits a blue shift in contrast to QM/MM predictions. Additionally, both mutants are expressed at low levels and have undetectable fluorescence in living cells, which prevents their utilization in imaging applications. Our results suggest that rational fluorescent protein design efforts can benefit from in silico protein stability and ligand affinity calculations. LabGym: quantification of user-defined animal behaviors using learning-based holistic assessment Quantifying animal behavior is important for many branches of biological research. Current computational tools for quantifying behaviors typically rely on a few pre-defined, simplified features to identify a behavior. However, such an approach underuses the behavioral information and restricts the tools applicability to limited behavior types or animal species. Here we report a new tool, LabGym, for quantifying animal behaviors without such limitations. Combining a novel approach for effective evaluation of animal motion with customizable convolutional recurrent networks for capturing spatiotemporal details, LabGym provides holistic behavioral assessment and accurately identify user-defined animal behaviors without restrictions on behavior types or animal species. It then calculates various parameters to provide quantitative measurements of the intensity and dynamics of each behavior. LabGym requires neither any intermediate step that causes information loss nor programming knowledge from users for post-hoc analysis. It tracks multiple animals simultaneously in various experimental settings for high-throughput and versatile analysis, and provides users a way to generate visualizable behavioral datasets that are valuable resources for research in neuroscience, behavioral science, and computer science. We demonstrate its efficacy in capturing subtle behavioral changes in animals ranging from soft-bodied invertebrates to mammals. Viral spillover risk in High Arctic increases in a glacierised watershed While many viruses have a single natural host, host restriction can be incomplete, hereby leading to spillovers to other host species. However, such spillover risks are difficult to quantify. As climate change is rapidly transforming environments, it is becoming critical to quantify the potential for spillovers. To address this issue, we resorted to an unbiased metagenomics approach, and focused on two environments, soil and lake sediments from Lake Hazen, the largest High Arctic freshwater lake in the world. We used DNA and RNA sequencing to reconstruct the lakes virosphere and its range of eukaryotic hosts, and estimated the spillover risk by measuring the congruence between the viral and the eukaryotic host phylogenetic trees. We show that spillover risk is higher in lake sediments than in soil and increased with runoff from glacier melt, a proxy for climate change. Should climate change also shift species range of potential viral vectors and reservoirs northwards, the High Arctic could become fertile ground for emerging pandemics. Cellular-level phenotyping of tumor-immune microenvironment (TiME) in patients in vivo reveals distinct inflammation and endothelial anergy signatures Immunotherapies have shown unprecedented clinical benefits in several malignancies1-3. However, clinical responses remain variable and unpredictable, indicating the need to develop predictive platforms that can improve patient stratification4. Phenotyping of tumors into hot, altered, or cold5 based on assessment of only T-cell infiltration in static tumor biopsies provides suboptimal prediction of immunotherapy response6,7. In vivo dynamic mechanisms within the tumor microenvironment such as tumor angiogenesis and leukocyte trafficking5,8,9 also play a central role in modulating anti-tumor immunity and therefore immunotherapy response. Here, we report novel tumor immune microenvironment (TiME) phenotyping in vivo in patients with non-invasive spatially-resolved cellular-level imaging based on endogenous contrast. Investigating skin cancers as a model, with reflectance confocal microscopy (RCM) imaging10, we determined four major phenotypes with variable prevalence of vasculature (Vasc) and inflammation (Inf) features: VaschiInfhi, VaschiInflo, VascloInfhi and Vascmed/hiInflo. The VaschiInfhi phenotype correlates with high immune activation, exhaustion, and vascular signatures while VaschiInflo with endothelial anergy and immune exclusion. Automated quantification of TiME features demonstrates moderate-high accuracy and correlation with corresponding gene expression. Prospectively analyzed response to topical immunotherapy show highest response in VascloInfhi, and reveals the added value of vascular features in predicting treatment response. Our novel in vivo cellular-level imaging and phenotyping approach can potentially advance our fundamental understanding of TiME, develop robust predictors for immunotherapy outcomes and identify novel targetable pathways in future. Cluster-specific gene markers enhance Shigella and Enteroinvasive Escherichia coli in silico serotyping Shigella and enteroinvasive Escherichia coli (EIEC) cause human bacillary dysentery with similar invasion mechanisms and share similar physiological, biochemical and genetic characteristics. The ability to differentiate Shigella and EIEC from each other is important for clinical diagnostic and epidemiologic investigations. The existing genetic signatures may not discriminate between Shigella and EIEC. However, phylogenetically, Shigella and EIEC strains are composed of multiple clusters and are different forms of E. coli. In this study, we identified 10 Shigella clusters, 7 EIEC clusters and 53 sporadic types of EIEC by examining over 17,000 publicly available Shigella/EIEC genomes. We compared Shigella and EIEC accessory genomes to identify the cluster-specific gene markers or marker sets for the 17 clusters and 53 sporadic types. The gene markers showed 99.63% accuracy and more than 97.02% specificity. In addition, we developed a freely available in silico serotyping pipeline named Shigella EIEC Cluster Enhanced Serotype Finder (ShigEiFinder) by incorporating the cluster-specific gene markers and established Shigella/EIEC serotype specific O antigen genes and modification genes into typing. ShigEiFinder can process either paired end Illumina sequencing reads or assembled genomes and almost perfectly differentiated Shigella from EIEC with 99.70% and 99.81% cluster assignment accuracy for the assembled genomes and mapped reads respectively. ShigEiFinder was able to serotype over 59 Shigella serotypes and 22 EIEC serotypes and provided a high specificity with 99.40% for assembled genomes and 99.38% for mapped reads for serotyping. The cluster markers and our new serotyping tool, ShigEiFinder (https://github.com/LanLab/ShigEiFinder), will be useful for epidemiologic and diagnostic investigations.

Impact statementThe differentiation of Shigella strains from enteroinvasive E. coli (EIEC) is important for clinical diagnosis and public health epidemiologic investigations. The similarities between Shigella and EIEC strains make this differentiation very difficult as both share common ancestries within E. coli. However, Shigella and EIEC are phylogenetically separated into multiple clusters, making high resolution separation using cluster specific genomic markers possible. In this study, we identified 17 Shigella or EIEC clusters including five that were newly identified through examination of over 17,000 publicly available Shigella and EIEC genomes. We further identified an individual or a set of cluster-specific gene markers for each cluster using comparative genomic analysis. These markers can then be used to classify isolates into clusters and were used to develop an in silico pipeline, ShigEiFinder (https://github.com/LanLab/ShigEiFinder) for accurate differentiation, cluster typing and serotyping of Shigella and EIEC from Illumina sequencing reads or assembled genomes. This study will have broad application from understanding the evolution of Shigella/EIEC to diagnosis and epidemiology.

Data summarySequencing data have been deposited at the National Center for Biotechnology Information under BioProject number PRJNA692536.

RepositoriesRaw sequence data are available from NCBI under the BioProject number PRJNA692536. H3K36 methylation and DNA-binding both promote Ioc4 recruitment and Isw1b remodeller function The Isw1b chromatin-remodelling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain, including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeller. The Ioc4-PWWP domain preferentially binds H3K36me3-containing nucleosomes. Its ability to bind DNA is required for nucleosome binding. It is also furthered by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 and DNA promote the interaction of full-length Ioc4 with nucleosomes in vitro and they are necessary for its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain promotes efficient remodelling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing the production of non-coding RNAs. Interfacial tension driven open droplet microfluidics We developed an open channel droplet microfluidic system that autonomously generates droplets at low Ca (~10-4-10-3) by leveraging competing hydrostatic and capillary pressure. With only our open channel polytetrafluoroethylene (PTFE) device, pipettes, and commercially available carrier fluid, we produce hundreds of microliter droplets; tubing, electronics, or pumps are not required, making droplet technology feasible for research labs without external flow generators. Furthermore, we demonstrated conceptual applications that showcase the process of droplet generation, splitting, transport, incubation, mixing, and sorting in our system. Unlike conventional droplet microfluidics, the open nature of the device enables the use of physical tools such as tweezers and styli to directly access the system; with this, we developed a new method of droplet sorting and transfer that capitalizes on the Cheerios effect, the aggregation of buoyant objects along a liquid interface. Our platform offers enhanced usability, direct access to the droplet contents, easy manufacturability, compact footprint, and high customizability. This design is a first step in exploring the space of power-free open droplet microfluidic systems and provide design rules for similar channel designs. Coordinated changes in gene expression, H1 variant distribution and genome 3D conformation in response to H1 depletion Up to seven members of the histone H1 family may contribute to chromatin compaction and its regulation in human somatic cells. In breast cancer cells, knock-down of multiple H1 variants deregulates many genes, promotes the appearance of genome-wide accessibility sites and triggers an interferon response via activation of heterochromatic repeats. However, how these changes in the expression profile relate to the re-distribution of H1 variants as well as to genome conformational changes have not been yet studied. Here, we combined ChIP-seq of five endogenous H1 variants with Chromosome Conformation Capture analysis in wild-type and H1 knock-down T47D cells. The results indicate that H1 variants coexist in the genome in two large groups depending on the local DNA GC content and that their distribution is robust with respect to multiple H1 depletion. Despite the small changes in H1 variants distribution, knock-down of H1 translated into more isolated but de-compacted chromatin structures at the scale of Topologically Associating Domains or TADs. Such changes in TAD structure correlated with a coordinated gene expression response of their resident genes. This is the first report describing simultaneous profiling of five endogenous H1 variants within a cell line and giving functional evidence of genome topology alterations upon H1 depletion in human cancer cells. Designing single-cell experiments to harvest fluctuation information while rejecting measurement noise Measurement error is a complicating factor that reduces or distorts the information contained in any experiment. This problem becomes even more serious in the context of experiments that are specifically designed to quantify single-cell gene expression heterogeneity, where important RNA and protein copy numbers are themselves subject to the inherently random fluctuations of biochemical reactions. It is not clear how measurement noise should be managed, in addition to other experiment design variables such as sampling size and frequency, to ensure that collected data will provide useful insights on gene expression mechanisms of interest. To address these fundamental single-cell experiment design challenges, we propose a computational framework that makes explicit use of measurement error modeling and Fisher Information Matrix-based criteria to decide between experimental methods. This unified approach not only quantifies how different noise characteristics affect uncertainty in parameter estimation, but also enables a systematic approach to design hybrid experiments that combine different measurement modalities. The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. Unsupervised clustering analysis of SARS-Cov-2population structure reveals six major subtypes atearly stage across the world Identifying the population structure of the newly emerged coronavirus SARS-CoV-2 has significant potential to inform public health management and diagnosis. As SARS-CoV-2 sequencing data accrued, grouping them into clusters is important for organizing the landscape of the population structure of the virus. Due to the limited prior information on the newly emerged coronavirus, we utilized four different clustering algorithms to group 16,873 SARS-CoV-2 strains, which automatically enables the identification of spatial structure for SARS-CoV-2. A total of six distinct genomic clusters were identified using mutation profiles as input features. Comparison of the clustering results reveals that the four algorithms produced highly consistent results, but the state-of-the-art unsupervised deep learning clustering algorithm performed best and produced the smallest intra-cluster pairwise genetic distances. The varied proportions of the six clusters within different continents revealed specific geographical distributions. In particular, our analysis found that Oceania was the only continent on which the strains were dispersively distributed into six clusters. In summary, this study provides a concrete framework for the use of clustering methods to study the global population structure of SARS-CoV-2. In addition, clustering methods can be used for future studies of variant population structures in specific regions of these fast-growing viruses. First detection of herpesvirus and prevalence of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. Water levels primarily drive variation in photosynthesis and nutrient use of scrub Red Mangroves in the southeastern Florida Everglades We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 {micro}mol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades. 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. The obstacles and potential clues of prime editing applications in tomato, a dicot plant Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement. Admp regulates tail bending by controlling ventral epidermal cell polarity via phosphorylated myosin translocation. The tails transient but pronounced ventral bending is found in many chordate embryos and provides an interesting model of how tissue interactions contribute to the overall shape (Lu et al., 2020). Here, we identify one of the upstream regulators of tail bending. Admp knock-down embryo (Admp MO) completely inhibited ventral tail bending. Admp MO showed specific inhibition of the cell-cell intercalation ventral epidermis. In the early stages of the intercalation, each ventral epidermal cell exhibited a boat-shaped morphology (boat cell) with a narrow apical surface in which phosphorylated myosin (pMLC) accumulated. In the Admp MO, the number of the boat cells reduced and pMLC was observed at the basal side in the Admp MO embryo. Finally, laser cutter experiments showed a pMLC-dependent relaxation of the ventral tail bending, which was observed when each ventral midline epidermal cell was cut along the apico-basal axis. Based on these results, we propose a novel function for Admp, in which Admp controls the pMLC translocation to the apical side of the ventral epidermal cells and leads the tail bending by ventral resistance to the notochord extension.

Summary StatementAdmp is an upstream regulator of the tail bending in the protochordate Ciona tailbud embryo, regulating tissue polarity of the ventral midline epidermis by the translocation of the phosphorylated myosin. 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. Non-targeted multimodal metabolomics and lipidomics data from ovine rumen fluid fractions BackgroundMetabolomics is a powerful and sensitive approach for investigating low molecular weight metabolite profiles present in rumen biofluids to identify potential roles of metabolites in rumen microbiome and understanding host-level regulatory mechanisms associated with animal production.

FindingsRumen samples from sheep grazed on a mixed ryegrass and clover pasture diet, were fractionated based on molecular weight and analysed using metabolomics and lipidomics to detect the small molecules present in ovine rumen fluid fractions.

ConclusionsUntargeted metabolomics provides a detailed snapshot of the ovine ruminal fluid metabolome that can be used as reference for future studies on ovine rumen fluid or as a comparator for other ruminant species. All data and metadata are available for download in the MetaboLights database. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Nuclear Progestin Receptor Mediated Linkage of Blood Coagulation and Ovulation Ovulation is a dramatic remodeling process that includes rupture of blood capillaries and clotting, but coagulation is not thought to directly regulate this process. Herein, we report remarkable increases of coagulation factors V (f5, [~]3145-fold) and tissue factor (f3a, [~]120-fold) in zebrafish ovarian follicle cells during ovulation. This increase was mediated through the nuclear progestin receptor (Pgr), which is essential for ovulation in zebrafish, and was totally abolished in ovarian follicular cells from pgr-/- mutants. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin (DHP) via Pgr. Similar regulation of human F5 promoter activity was induced via human PGRB, suggesting a conserved mechanism. Site-directed mutagenesis of the zebrafish f5 promoter further demonstrated a direct regulation of coagulation factors via progestin response elements. Moreover, a stark increase of erythrocytes occurred in capillaries meshed in wildtype preovulatory follicles but was absent in pgr-/- mutants. Interestingly, anticoagulants significantly inhibited ovulation both in vitro and in vivo, respectively. Furthermore, reduced fecundity was observed in f5+/- female zebrafish. Taken together, our study provides plausible evidence for steroid regulation of coagulation factors, and a new hypothesis for blood clotting triggered ovulation in vertebrates. A novel sodium signaling complex regulates uterine activity Depolarization of the myometrial smooth muscle cell (MSMC) resting membrane potential is necessary for the transition of the uterus from a quiescent state to a contractile state. The molecular mechanisms involved in this transition are not completely understood. Here, we report a novel coupled system between the Na+-activated K+ channel (SLO2.1) and the non-selective Na+ leak channel (NALCN) which determines the MSMC membrane potential. We show that SLO2.1 currents are activated by an inward Na+ leak current carried by the NALCN channel leading to MSMC hyperpolarization. These results show an unanticipated role for the Na+ leak currents in activating a negative feedback system countering the excitable effects of Na+ currents. This is a novel role for the NALCN channel in which Na+ acts as an intracellular signaling molecule. In fact, we report here that the net effect of Na+ entry through NALCN channels is a hyperpolarization of the MSMCs plasma membrane because of the activation of SLO2.1 K channel. Importantly, we also report that a decrease in NALCN/SLO2.1 activity triggers both Ca2+ entries through VDCCs, promoting myometrial contraction. Consistently, with a functional coupling, our data show that NALCN and SLO2.1 are in proximity to one another in human MSMCs. We propose that the spatial arrangement of SLO2.1 and NALCN permits these channels to functionally interact in order to regulate human MSMC membrane potential and cell excitability to modulate uterine contractile activity. Transmembrane helices are an overlooked and evolutionarily conserved source of major histocompatibility complex class I and II epitopes Cytolytic T cell responses are predicted to be biased towards membrane proteins. The peptide-binding grooves of most haplotypes of histocompatibility complex class I (MHC-I) are relatively hydrophobic, therefore peptide fragments derived from human transmembrane helices (TMHs) are predicted to be presented more often as would be expected based on their abundance in the proteome. However, the physiological reason of why membrane proteins might be over-presented is unclear. In this study, we show that the over-presentation of TMH-derived peptides is general, as it is predicted for bacteria and viruses and for both MHCI and MHC-II. Moreover, we show that TMHs are evolutionarily more conserved, because single nucleotide polymorphisms (SNPs) are present relatively less frequently in TMH-coding chromosomal regions compared to regions coding for extracellular and cytoplasmic protein regions. Thus, our findings suggest that both cytolytic and helper T cells respond more to membrane proteins, because these are evolutionary more conserved. We speculate that TMHs therefore are less prone to escape mutations that enable pathogens to evade T cell responses. Fatal neuroinvasion and SARS-CoV-2 tropism in K18-hACE2 mice is partially independent on hACE2 expression Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

IMPORTANCECOVID-19 is predominantly a respiratory disease caused by SARS-CoV-2 that has infected more than 191 million people with over 4 million fatalities (2021-07-20). The development of animal models recapitulating distinctive features of severe COVID-19 is critical to enhancing our understanding of SARS-CoV-2 pathogenesis and in the evaluation of vaccine and therapeutic efficacy. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. Here, we show lethality of this model is invariably associated with viral neuroinvasion linked with viral replication and assembly. Importantly, pneumonia albeit invariably present was generally moderate with the absence of culturable infectious virus at peak neuroinvasion. The dynamics of viral neuroinvasion and pneumonia were only partially dependent on hACE2. Overall, this study provides an in-depth sequential characterization of the K18-hACE2 model following SARS-CoV-2 infection, highlighting its significance to further study the mechanisms of SARS-CoV-2 neuropathogenesis. de novo gene birth as an inevitable consequence of adaptive evolution Phylostratigraphy suggests that new genes are continually born de novo from non-genic sequences, and the genes that persist found new lineages, contributing to the adaptive evolution of organisms. While recent evidence supports the view that de novo gene birth is frequent and widespread, the mechanisms underlying this process are yet to be discovered. Here we hypothesize and examine a potential general mechanism of gene birth driven by the accumulation of beneficial mutations at non-genic loci. To demonstrate this possibility, we model this mechanism within the boundaries set by current knowledge on mutation effects. Estimates from this analysis are in line with observations of recurrent and extensive gene birth in genomics studies. Thus, we propose that, rather than being inactive and silent, non-genic regions are likely to be dynamic storehouses of potential genes.