Comparative toxicogenomics of glyphosate and Roundup herbicides by mammalian stem cell-based genotoxicity assays and molecular profiling in Sprague-Dawley rats Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remains controversial. As GBHs are more cytotoxic that glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs namely MON 52276 (EU), MON 76473 (UK) and MON 76207 (USA) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (both at 0.5, 50, 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased mir-30 while miR-10 levels were increased. DNA methylation profiling of liver revealed 5,727 and 4,496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate.
Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ-9-tetrahydrocannabinol In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development.
First evidence of in vitro cytotoxic effects of marine microlitter on Merluccius merluccius and Mullus barbatus, two Mediterranean commercial fish species Marine litter is composed mainly of plastics and is recognized as a serious threats to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exist on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 {+/-} 0.02 microlitter items m-3 was found in water samples. FT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter.

HighlightsO_LI0.30 {+/-} 0.02 microlitter items m-3 were found at the surface of coastal Northern Tyrrhenian sea
C_LIO_LI14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual were retrieved from the stomach of hakes and mullets
C_LIO_LIThe ingested microlitter contained plastic items
C_LIO_LIMicrolitter was validated as a carrier of bacteria, fungi and flagellates
C_LIO_LISplenic cells exposed to two microlitter conditions for 72 hours suffered cytotoxicity
C_LI Global analysis of human-to-mouse contact-dependent intercellular mRNA and lncRNA transfer in cell culture Full-length mRNAs can transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the transferome) is unknown. Here, we analyzed whole transcriptome mRNA and lncRNA transfer between heterogeneous human-mouse cell populations in in vitro co-culture using RNA-sequencing. Our data indicate that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells (MCF7). These results were validated by both quantitative RT-PCR and in situ hybridization, and analysis shows that typically <1% of endogenous mRNAs and lncRNAs undergo transfer. Non-selective expression-dependent RNA transfer was further validated using synthetic RNA reporters. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/470233v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@13a7176org.highwire.dtl.DTLVardef@1ef1c75org.highwire.dtl.DTLVardef@1355eeforg.highwire.dtl.DTLVardef@ebc275_HPS_FORMAT_FIGEXP  M_FIG C_FIG Rad51 filaments assembled in the absence of the complex formed by the Rad51 paralogs Rad55 and Rad57 are outcompeted by translesion DNA polymerases on UV-induced ssDNA gaps The bypass of DNA lesions that block replicative polymerases during DNA replication relies on DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway relies on specialized DNA polymerases that incorporate nucleotides in front of base lesions, potentially inducing mutagenesis. Two error-free pathways can bypass the lesions: the template switching pathway, which uses the sister chromatid as a template, and the homologous recombination pathway (HR), which also can use the homologous chromosome as template. The balance between error-prone and error-free pathways controls the mutagenesis level. Therefore, it is crucial to precisely characterize factors that influence the pathway choice to better understand genetic stability at replication forks. In yeast, the complex formed by the Rad51 paralogs Rad55 and Rad57 promotes HR and template-switching at stalled replication forks. At DNA double-strand breaks (DSBs), this complex promotes Rad51 filament formation and stability, notably by counteracting the Srs2 anti-recombinase. To explore the role of the Rad55-Rad57 complex in error-free pathways, we monitored the genetic interactions between Rad55-Rad57, the translesion polymerases Pol{zeta} or Pol{eta}, and Srs2 following UV radiation that induces mostly single-strand DNA gaps. We found that the Rad55-Rad57 complex was involved in three ways. First, it protects Rad51 filaments from Srs2, as it does at DSBs. Second, it promotes Rad51 filament stability independently of Srs2. Finally, we observed that UV-induced HR is almost abolished in Rad55-Rad57 deficient cells, and is partially restored upon Pol{zeta} or Pol{eta} depletion. Hence, we propose that the Rad55-Rad57 complex is essential to promote Rad51 filament stability on single-strand DNA gaps, notably to counteract the error-prone TLS polymerases and mutagenesis.

Author summaryProcessive and accurate DNA polymerases replicate genomic DNA during the S phase of each cell cycle. DNA base lesions on template DNA block these polymerases and result in an accumulation of single-stranded DNA gaps behind moving replication forks. These gaps are filled-in by error-prone and error-free pathways. In this work, we show that the complex made by the Rad51 paralogs Rad55 and Rad57 is essential for the error-free homologous recombination gap-filling pathway when DNA replication is stalled by UV-induced DNA lesions, but not for DNA double strand break repair. Interestingly, we found that homologous recombination is efficiently outcompeted by error-prone translesion DNA polymerases in Rad55-Rad57-deficient cells. We propose that the Rad55-Rad57 complex is essential for Rad51 filament stability at UV-induced DNA gaps to promote efficient error-free homologous recombination. Furthermore, our study in yeast predicts that inhibitors of error-prone DNA polymerases might selectively target cancer cells in which RAD51 paralogs are mutated. Outcome-selective reinstatement is predominantly context-independent, and associated with c-Fos activation in the posterior dorsomedial striatum Research from human and animal studies has found that responding that has been successfully reduced following treatment can return upon exposure to certain contexts. An individual in recovery from alcohol use disorder, for example, might relapse to drinking upon visiting their favourite bar. However, most of these data have been derived from experiments involving a single (active) response, and the context-dependence of returned responding in situations involving choice between multiple actions and outcomes is less well-understood. We thus investigated how outcome-selective reinstatement - a procedure involving choice between multiple actions - was affected by altering the physical context in rats. In Experiment 1, rats were trained over 6 days to press a left lever for one food outcome (pellets or sucrose) and a right lever for the other outcome. Then, rats received an extinction session in either the same context (A) as lever press training, or in a different context (B). Rats were tested immediately (5 minutes) after extinction in Context A or B such that there were four groups in total: AAA, ABB, ABA, and AAB. Reinstatement testing consisted of one food outcome being delivered  freely (i.e. unearned by lever pressing and unsignalled by cues) to the food magazine every 4 minutes in the following order: Sucrose, Pellet, Pellet, Sucrose. Selective reinstatement was considered intact if pellet delivery increased pressing selectively on the pellet lever, and sucrose delivery selectively increased pressing on the sucrose lever. This result (Reinstated > Nonreinstated) was observed for rats in group AAA and ABB, but not rats in groups ABA and AAB. Experiment 2 was conducted identically, except that rats received two extinction sessions over two days and tested one day later. This time, all groups demonstrated intact outcome-selective reinstatement regardless of context. Analysis of c-Fos expression in several brain regions revealed that only c-Fos expression in the posterior dorsomedial striatum (pDMS) was related to intact reinstatement performance. Overall, these results suggest that outcome-selective reinstatement is predominantly context-independent, and that intact reinstatement is related to neuronal activity in the pDMS.

HighlightsO_LIOutcome-selective reinstatement is predominantly context-independent
C_LIO_LIOutcome-selective reinstatement is entirely context-independent after multiple extinction sessions
C_LIO_LIOutcome-selective reinstatement increases c-Fos expression in dorsomedial striatum
C_LIO_LIc-Fos expression in orbitofrontal cortex and dorsal hippocampus is unaffected by selective reinstatement.
C_LI Size dependent regeneration capacity of functionalized Capra ear derived micro-tissue scaffolds for treatment of cartilage defects Cartilage regeneration remains a great challenge in orthopedic treatment owing to their avascular in nature and lack of self-healing ability. Various clinical treatment options are widely used including osteochondral graft transplantation, micro-fracture, blood clot formation and tissue debridement to recover the damaged cartilage. In this circumstance, cartilage defect recovery via tissue engineered functional micro tissue delivery is becoming an emerging trend in musculoskeletal therapeutics. In this study, functional micro-scaffolds (MS) were generated from Capra ear cartilage, and were separated into size-wise groups. The scaffolds were decellularized via NaOH treatment. The cell adhesion study indicated that Capra adipose tissue derived mesenchymal stem cells (ADMSCs) adhesion is more in ~100 m MSs in comparison to 150-300 m MSs. It may be assumed that the cells are compatible to grow on fibrous surface area (100 m) in comparison to dense surface (150-300 m). Further, 100 m MSs were transformed into functional micro tissues (FMTs) in presence of high density ADMSCs in a hanging droplet culture system. The FMTs was transferred to F127 block polymer hydrogel for 3D culture. After 21 d cultures, the FMT clusters were evaluated for quantitative gene expression. To assess the in vivo cartilage defect regeneration potential, FMTs were delivered to rabbit auricular cartilage defect for 15, 30 and 60 d studies. The H&E-stained histological analysis showed that the cartilage defect is almost healed in 60 d study in comparison to 15 and 30 d study. Confronting population models with experimental microcosm data: from trajectory matching to state-space models O_LIPopulation and community ecology traditionally has a very strong theoretical foundation with well-known models, such as the logistic and its many variations, and many modification of the classical Lotka-Volterra predator-prey and interspecific competition models. More and more, these classical models are confronted to data via fitting to empirical time-series for purposes of projections or for estimating model parameters of interest. However, the interface between mathematical population or community models and data, provided by a statistical model, is far from trivial.
C_LIO_LIIn order to help empiricists, especially researchers working with experimental laboratory populations in micro- and mesocosms, make informed decisions, we here ask which error structure one should use when fitting classical deterministic ODE models to empirical data, from single species to community dynamics and trophic interactions. We use both realistically simulated data and empirical data from microcosms to answer this question in a Bayesian framework.
C_LIO_LIWe find that many model parameters can be estimated precisely with an appropriate choice of error structure using pure observation error or state-space models, if observation errors are not too high. However, Allee effect models are typically hard to identify and state-space models should be preferred with when model complexity increases.
C_LIO_LIOur work shows that, at least in the context of experimental laboratory populations, deterministic models can be used to describe stochastic population dynamics that include process variability and observation error. Also, many scenarios do not require a complex state-space model formulation and simpler trajectory matching is sufficient for accurate parameter estimates. Finally, we provide a comprehensive tutorial for fitting these models in R.
C_LI Emergence of a novel mobile RND-type efflux pump gene cluster, tmexC3D2-toprJ1b, in Pseudomonas species Tigecycline exhibits promising activity against multidrug-resistant Gram-negative bacteria (MDR-GNB), whose infections are difficult to treat with antimicrobials. However, mobile tigecycline resistance genes, such as tmexCD-toprJ, have emerged in Enterobacterales isolated in China, Vietnam, and possibly other countries in the world. In this study, we investigated tigecycline-nonsusceptible GNB in Japan. Eight tigecycline- and carbapenem-nonsusceptible isolates of Pseudomonas alcaligenes were obtained from sewage water from a medical institution in Japan in 2020. Whole genome analysis of all P. alcaligenes isolates was performed using short-read sequencing, and the isolate KAM426 was further analyzed using long-read sequencing. For important antimicrobial resistance genes, analysis of surrounding structures and comparison with similar sequences in the public genome database were performed. We identified a novel hybrid type of tmexCD-toprJ gene cluster, tmexC3D2-toprJ1b consisting of tmexC3, tmexC2, and toprJ1b, in phylogenetically clonal isolates of P. alcaligenes. The complete genome sequence of KAM426 revealed that this isolate co-harbors tmexC3D2-toprJ1b and two copies of the carbapenemase gene blaIMP-1 on the chromosome. tmexC3D2-toprJ1b in KAM426 was flanked by the IS5/IS1182 family transposase gene, suggesting that the gene cluster was acquired by horizontal gene transfer (HGT). tmexC3D2-toprJ1b seemed to have spread to other Pseudomonas species such as Pseudomonas aeruginosa via HGT mediated by mobile gene elements such as a plasmid. This study identified tmexCD-toprJ-like tigecycline resistance genes in Japan for the first time and suggests that diverse tmexCD-toprJ-like gene clusters, including tmexC3D2-toprJ1b, have spread among MDR-GNB worldwide. Further epidemiological genomic studies in clinical and environmental settings are needed. Integrative small and long RNA-omics analysis of human healing and non-healing wounds discovers cooperating microRNAs as therapeutic targets MicroRNAs (miR) are important posttranscriptional regulators and exhibit a high potential to be utilized in diagnosis and therapy. However, our insufficient knowledge of the miR-mediated gene regulation in human skin wound healing severely hinders the identification of clinically relevant miRs. Here, we performed paired small RNA and long RNA sequencing in human tissue samples, including matched skin and acute wounds collected at each healing stage and chronic non-healing venous ulcers (VU). With integrative small and long RNA-omics analysis, we developed a compendium (https://www.xulandenlab.com/humanwounds-mirna-mrna), which will be an open, comprehensive resource to broadly aid wound healing research. With this first clinical, wound-centric resource of miRs and mRNAs, we identified 17 pathologically relevant miRs that exhibited abnormal VU expression and displayed their targets enriched explicitly in the VU gene signature. Intermeshing regulatory networks controlled by these miRs revealed their high cooperativity in contributing to chronic wound pathology characterized by persistent inflammation and proliferative phase initiation failure. Furthermore, we demonstrated that miR-34a, miR-424, and miR-516, upregulated in VU, cooperatively suppressed keratinocyte growth while promoting inflammatory response. Collectively, our study opens the possibility of developing innovative wound treatment that targets pathologically relevant cooperating miRs to attain higher therapeutic efficacy and specificity. The polyglutamine amyloid nucleus in living cells is a monomer with competing dimensions of order A long-standing goal of the study of amyloids has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of that event has made it inaccessible to classical biochemistry, structural biology, and computational approaches. Here, we addressed that limitation by measuring the dependence of amyloid formation on concentration and conformational templates in living cells, whose volumes are sufficiently small to resolve the outcomes of independent nucleation events. We characterized numerous rationally designed sequence variants of polyglutamine (polyQ), a polypeptide that precipitates Huntingtons and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. This effort uncovered a pattern of approximately twelve Qs, only for polypeptides exceeding the clinical length threshold, that allow for amyloid nucleation to occur spontaneously within single polypeptides. Nucleation was inhibited by intermolecular phase separation. Using atomistic molecular dynamics simulations, we found that the pattern encodes a minimal steric zipper of interdigitated side chains. Lateral growth of the steric zipper competed with axial growth to produce "pre-amyloid" oligomers. By illuminating the structural mechanism of polyQ amyloid formation in cells, our findings reveal a potential molecular etiology for polyQ diseases, and may provide a roadmap for the design of new therapies. Prenatal environmental conditions underlie alternative reproductive tactics that drive the formation of a mixed-kin cooperative society Although animal societies often evolve due to limited natal dispersal that results in kin clustering and facilitates cooperation among relatives, many species form cooperative groups with low and variable kin structure. Groups in such mixed-kin societies often comprise residents and immigrants of the same sex that compete for breeding opportunities. To understand how such mixed-kin societies form despite their potential for social conflict, we investigated the environmental causes and subsequent fitness consequences of dispersal decisions in male cooperatively breeding superb starlings (Lamprotornis superbus) that live in a climatically unpredictable environment. We show that the two alternative reproductive tactics--natal dispersal or philopatry--exhibit reproductive tradeoffs resulting in equal lifetime inclusive fitness. The tactic an individual adopts is governed by the environment its parents experience prior to laying rather than the environment it experiences during its juvenile stage. When individuals adopt the tactic not predicted by early life environmental conditions, their fitness is reduced. Ultimately, climate-driven oscillating selection may help stabilize mixed-kin animal societies, despite their reduced kin structure and potential for social conflict. Measuring transcription factor binding and gene expression using barcoded self-reporting transposon calling cards and transcriptomes Calling cards technology using self-reporting transposons enables the identification of DNA-protein interactions through RNA sequencing. Although immensely powerful, current implementations of calling cards in bulk experiments on populations of cells are technically cumbersome and require many replicates to identify independent insertions into the same genomic locus. Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself. An additional barcode incorporated during reverse transcription enables simultaneous transcriptome measurement in a facile and affordable protocol. We demonstrate that barcoded self-reporting transposons recover in vitro binding sites for four basic helix-loop-helix transcription factors with important roles in cell fate specification: ASCL1, MYOD1, NEUROD2, and NGN1. Further, simultaneous calling cards and transcriptional profiling during transcription factor overexpression identified both binding sites and gene expression changes for two of these factors. Lastly, we demonstrated barcoded calling cards can record binding in vivo in the mouse brain. In sum, RNA-based identification of transcription factor binding sites and gene expression through barcoded self-reporting transposon calling cards and transcriptomes is an efficient and powerful method to infer gene regulatory networks in a population of cells. Trial and error: a hierarchical modeling approach to test-retest assessment The concept of test-retest reliability indexes the consistency of a measurement across time. High reliability is critical for any scientific study, but specifically for the study of individual differences. Evidence of poor reliability of commonly used behavioral and functional neuroimaging tasks is mounting. Reports on low reliability of task-based fMRI have called into question the adequacy of using even the most common, well-characterized cognitive tasks with robust population-level effects, to measure individual differences. Here, we lay out a hierarchical framework that estimates reliability as a correlation divorced from trial-level variability, and show that reliability estimates tend to be higher compared to the conventional framework that adopts condition-level modeling and ignores across-trial variability. We examine how estimates from the two frameworks diverge and assess how different factors (e.g., trial and subject sample sizes, relative magnitude of cross-trial variability) impact reliability estimates. We also show that, under specific circumstances, the two statistical frameworks converge. Results from the two approaches are approximately equivalent if (a) the trial sample size is sufficiently large, or (b) cross-trial variability is in the same order of magnitude as, or less than, cross-subject variability. As empirical data indicate that cross-trial variability is large in most tasks, this work highlights that a large number of trials (e.g., greater than 100) may be required to achieve precise reliability estimates. We reference the tools TRR and 3dLMEr for the community to apply trial-level models to behavior and neuroimaging data and discuss how to make these new measurements most useful for current studies. T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor It is well known that Kaiso protein encoded by ZBTB33 gene is a transcription repressor and that Kaiso-P120ctn interaction increases the shift of Kaiso from the nucleus into the cytoplasm. However, the regulatory mechanisms of Kaiso compartmentalization are far from clear. Here, we reported that AKT1 could phosphorylate 606-threonine residue (T606) within the RSSTIP motif of Kaiso in the cytoplasm. The T606-phosphorylated Kaiso (pT606-Kaiso) could directly bind to 14-3-3 family proteins and the depletion of T606 phosphorylation by T606A mutation abolished most of the Kaiso-14-3-3 binding. In addition, the Kaiso-P120ctn interaction was essential for the pT606-Kaiso accumulation in the cytoplasm. Notably, enforced 14-3-3{sigma} (SFN) overexpression could increase the pT606-Kaiso accumulation in the cytoplasm and de-repress the transcription of Kaiso target gene CDH1. Decreased amounts of both pT606-Kaiso and CDH1 proteins were frequently observed in human gastric cancer tissues relative to paired normal controls. The mRNA levels of 14-3-3{sigma} and Kaiso target gene CDH1 were positively and significantly correlated with each other in bioinformatics analyses using publicly available RNA-seq datasets for human normal tissues (n=11688, r=0.60, p<0.001) in the GTEx project and for cancer cell lines (n=1156, r=0.41, p<0.001) in the CCLE project. Furthermore, Kaiso T606A mutant (unable to be phosphorylated) significantly increased the migration and invasion of cancer cells in vitro as well as boosted the growth of these cells in vivo. In conclusion, Kaiso could be phosphorylated by AKT1 at the T606 and the pT606-Kaiso accumulates in the cytoplasm through binding to 14-3-3/P120ctn that de-represses the expression of Kaiso target gene CDH1 in normal tissues. Decreased Kaiso phosphorylation may contribute to the development of gastrointestinal cancer. The status of Kaiso phosphorylation is a determinant factor for the role of Kaiso in the development of cancer. Comparison of hybrid learning and remote education in the implementation of the 'Adopt a Microorganism' methodology 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. 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. Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution. Rossmann-toolbox: a deep learning-based protocol for the prediction and design of cofactor specificity in Rossmann-fold proteins The Rossmann fold enzymes are involved in essential biochemical pathways such as nucleotide and amino acid metabolism. Their functioning relies on interaction with cofactors, small nucleoside-based compounds specifically recognized by a conserved {beta}{beta} motif shared by all Rossmann fold proteins. While Rossmann methyltransferases recognize only a single cofactor type, the S-Adenosylmethionine (SAM), the oxidoreductases, depending on the family, bind nicotinamide (NAD, NADP) or flavin-based (FAD) cofactors. In this study, we show that despite its short length, the {beta}{beta} motif unambiguously defines the specificity towards the cofactor. Following this observation, we trained two complementary deep learning models for the prediction of the cofactor specificity based on the sequence and structural features of the {beta}{beta} motif. A benchmark on two independent test sets, one containing {beta}{beta} motifs bearing no resemblance to those of the training set, and the other comprising 38 experimentally confirmed cases of rational design of the cofactor specificity, revealed the nearly perfect performance of the two methods. The Rossmann-toolbox protocols can be accessed via the webserver at https://lbs.cent.uw.edu.pl/rossmann-toolbox and are available as a Python package at https://github.com/labstructbioinf/rossmann-toolbox.

Key pointsO_LIThe Rossmann fold encompasses a multitude of diverse enzymes involved in most of the essential cellular pathways
C_LIO_LIProteins belonging to the Rossmann fold co-evolved with their nucleoside-based cofactors and require them for the functioning
C_LIO_LIManipulating the cofactor specificity is an important step in the process of enzyme engineering
C_LIO_LIWe developed an end-to-end pipeline for the prediction and design of the cofactor specificity of the Rossmann fold proteins
C_LIO_LIOwing to the utilization of deep learning approaches the pipeline achieved nearly perfect accuracy
C_LI mTORC2 contributes to murine systemic autoimmunity The development of many systemic autoimmune diseases, including systemic lupus erythematosus, is associated with overactivation of the type I interferon (IFN) pathway, lymphopenia, and increased follicular helper T (Tfh) cell differentiation. However, the cellular and molecular mechanisms underlying these immunological perturbations remain incompletely understood. Here we show that the mechanistic target of rapamycin complex 2 (mTORC2) promotes Tfh differentiation and disrupts Treg homeostasis. Inactivation of mTORC2 in total T cells, but not in Tregs, greatly ameliorated the immunopathology in a systemic autoimmunity mouse model. This was associated with reduced Tfh differentiation, B cell activation, and reduced T cell glucose metabolism. Finally, we show that type I IFN can synergize with TCR ligation to activate mTORC2 in T cells, which partially contributes to T cell lymphopenia. These data indicate that mTORC2 may act as downstream of type I IFN, TCR, and costimulatory receptor ICOS, to promote glucose metabolism, Tfh differentiation, and T cell lymphopenia, but not to suppress Treg function in systemic autoimmunity. Our results suggest that mTORC2 might be a rational target for systemic autoimmunity treatment. Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma The algorithms commonly used to select the best stable reference gene in RT-qPCR data analysis have their limitations. We showed that simple selection of the reference gene or pair of genes with the lowest stability value from the pool of potential reference genes - a commonly used approach - is not sufficient to accurately and reliably normalize the target gene transcript and can lead to biologically incorrect conclusions. For reliable assessment of changes in a target gene expression level, we propose our innovative GenExpA software, which works in a manner independent of the experimental model and the normalizer used. GenExpA software selects the best reference by combining the NormFinder algorithm with progressive removal of the least stable gene from the candidate genes in a given experimental model and in the set of daughter models assigned to it. The reliability of references is validated based on the consistency of the statistical analyses of normalized target gene expression levels through all models, described by the coherence score (CS). The use of the CS value imparts a new quality to qPCR analysis because it clarifies how low the stability value of reference must be in order for biologically correct conclusions to be drawn. We tested our method on qPCR data for the B4GALT genes family in melanoma, which is characterized by a high mutation rate, and in melanocytes. GenExpA is available at https://github.com/DorotaHojaLukowicz/GenExpA or https://www.sciencemarket.pl/baza-programow-open-source#oferty.

HighlightsGenExpA - next-generation software for normalizer selection and validation

The GenExpA tool defines how low the stability value of the reference should be

The GenExpA tool determines the level of gene expression analysis robustness

The GenExpA tool increases the speed of analysis and reduces cost Sarcoplasmic reticulum calcium handling in young DBA/2J vs C57BL/10 mdx models of Duchenne muscular dystrophy The DBA/2J (D2) mdx mouse has emerged as a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. In gastrocnemius muscles, both D2- and C57 mdx mice exhibited signs of impaired Ca2+ uptake, however, this was more severe in D2 mdx mice. Maximal SERCA activity was lowered only in D2 mdx gastrocnemius muscles and not C57 mdx muscles. Furthermore, in the left ventricle and diaphragm, Ca2+ uptake was impaired in C57 mdx muscles with lowered rates of Ca2+ uptake compared with C57 WT mice, whereas in muscles from D2 mdx mice, rates of Ca2+ uptake were unattainable due to the severe impairments in their ability to transport Ca2+. Overall, our study demonstrates that SERCA function is drastically impaired in young D2 mdx mice. 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. 14-3-3ζ constrains insulin secretion by regulating mitochondrial function in pancreatic β-cells While critical for neurotransmitter synthesis in the brain, members of the 14-3-3 protein family are often assumed to have redundant, over-lapping roles due to their high sequence homology and ubiquitous expression. Despite this assumption, various mammalian 14-3-3 isoforms have now been implicated in regulating cellular and organismal metabolism; however, these functions were primarily observed in cell lines or from systemic knockout mouse models. To date, we have begun to define the contributions of 14-3-3{zeta} in adipocytes, but whether 14-3-3{zeta} has additional metabolic roles in other cell types, such as the pancreatic {beta}-cell, is unclear. We previously documented a pro-survival role of 14-3-3{zeta} in MIN6 insulinoma cells, as depletion of 14-3-3{zeta} induced cell death, but paradoxically, whole-body deletion of 14-3-3{zeta} in mice resulted in significantly enlarged {beta}-cell area with no effects on insulin secretion. To better understand the role of 14-3-3{zeta} in {beta}-cells, we generated {beta}-cell-specific 14-3-3{zeta} knockout ({beta}14-3-3{zeta}KO) mice, and while no differences in {beta}-cell mass were observed, {beta}14-3-3{zeta}KO mice displayed potentiated insulin secretion due to enhanced mitochondrial function and ATP synthesis. Deletion of 14-3-3{zeta} led to profound changes to the {beta}-cell transcriptome, where pathways associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute treatment of mouse islets and human islets with pan-14-3-3 inhibitors recapitulated the potentiation in glucose-stimulated insulin secretion (GSIS) and mitochondrial function, suggesting that 14-3-3{zeta} is a critical isoform in {beta}-cells that regulates GSIS. In dysfunctional db/db islets and islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3{zeta}, was inversely associated with insulin secretory capacity, and pan-14-3-3 protein inhibition was capable of enhancing GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3{zeta} and its related isoforms in insulin secretion and mitochondrial function in {beta}-cells. A deeper understanding of how 14-3-3{zeta} influences {beta}-cell function will further advance our knowledge of how insulin secretion from {beta}-cells is regulated. Sperm Histone H3 Lysine 4 tri-methylation serves as a metabolic sensor of paternal obesity and is associated with the inheritance of metabolic dysfunction ObjectiveParental environmental exposures can strongly influence descendant risks for adult disease. How paternal obesity changes the sperm chromatin leading to the acquisition of metabolic disease in offspring remains controversial and ill-defined. The objective of this study was to assess: (1) whether obesity induced by a high-fat diet alters sperm histone methylation; (2) whether paternal obesity can induce metabolic disturbances across generations; (3) whether there could be cumulative damage to the sperm epigenome leading to enhanced metabolic dysfunction in descendants; and (4) whether obesity-sensitive regions associate with embryonic epigenetic and transcriptomic profiles. Using a genetic mouse model of epigenetic inheritance, we investigated the role of histone H3 lysine 4 methylation (H3K4me3) in the paternal transmission of metabolic dysfunction. This transgenic mouse overexpresses the histone demethylase enzyme KDM1A in the developing germline and has an altered sperm epigenome at the level of histone H3K4 methylation. We hypothesized that challenging transgenic sires with a high-fat diet would further erode the sperm epigenome and lead to enhanced metabolic disturbances in the next generations.

MethodsTo assess whether paternal obesity can have inter- or transgenerational impacts, and if so, to identify potential mechanisms of this non-genetic inheritance, we used wildtype C57BL/6NCrl and transgenic males with a pre-existing altered sperm epigenome. To induce obesity, sires were fed either a control or high-fat diet (10% or 60% kcal fat, respectively) for 10-12 weeks, then bred to wildtype C57BL/6NCrl female fed a regular diet. F1 and F2 descendants were characterized for metabolic phenotypes by examining the effects of paternal obesity by sex, on body weight, fat mass distribution, the liver transcriptome, intraperitoneal glucose and insulin tolerance tests. To determine whether obesity altered the F0 sperm chromatin, native chromatin immunoprecipitation-sequencing targeting H3K4me3 was performed. To gain insight into mechanisms of paternal transmission, we compared our sperm H3K4me3 profiles with embryonic and placental chromatin states, histone modification and gene expression profiles.

ResultsObesity-induced alterations in H3K4me3 occurred at genes implicated in metabolic, inflammatory, and developmental processes. These processes were associated with offspring metabolic dysfunction and corresponded to genes enriched for H3K4me3 in embryos, and overlapped embryonic and placenta gene expression profiles. Transgenerational susceptibility to metabolic disease was only observed when obese F0 had a pre-existing modified sperm epigenome. This coincided with increased H3K4me3 alterations in sperm and more severe phenotypes affecting their offspring.

ConclusionsOur data suggest sperm H3K4me3 might serve as a metabolic sensor that connects paternal diet with offspring phenotypes via the placenta. This non-DNA based knowledge of inheritance has the potential to improve our understanding of how environment shapes heritability and may lead to novel routes for the prevention of disease. This study highlights the need to further study the connection between the sperm epigenome, placental development and childrens health. SUMM4 biochemically complex couples insulator function and DNA replication timingcontrol The mechanisms that establish DNA replication timing programs in eukaryotes remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under-replication (UR) of late-replicating intercalary heterochromatin (IH) in polytene chromosomes. We developed a proteomics technique termed MERCI to isolate a native complex SUMM4 comprising SUUR and chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates the ATPase activity of SUUR and is required for its normal spatiotemporal distribution in vivo. Both SUMM4 subunits mediate the activities of gypsy insulator disrupting enhancer-promoter interactions and establishing chromatin barriers. Furthermore, SuUR or mod(mdg4) mutations reverse UR of IH. Our findings uncover a critical role for architectural proteins in attenuating replication fork progression and suggest an alternative mechanism for DNA replication timing that does not depend on an asynchronous firing of replication origins.

One-Sentence SummaryA stable protein complex comprising an insulator factor and a SNF2-like ATPase imparts late replication of heterochromatin.