Liberation from fibrogenesis or tumorigenesis via cellular senescence by a novel small molecule BackgroundUncontrolled proliferative diseases such as fibrosis or cancer can be fatal. We previously found that a compound containing a chromone scaffold, ONG41008, had potent anti-fibrotic effects in diseased human lung myofibroblasts but not normal human lung fibroblasts.

MethodsWe investigated the effects of ONG41008 on tumor cells, and compared these effects with those in pathogenic myofibrotic cells and normal fibroblasts cells.

FindingsStimulation of A549 lung carcinoma epithelial cells with ONG41008 resulted in cellular senescence, indicating that dysregulated cell proliferation is common to fibrotic cells and tumor cells. Replicative senescence of A549 cells resulted in multinucleation, which was followed by oncogene-induced senescence. There was significant upregulation of expression and nuclear translocation of p-TP53 and p16 in ONG41008-treated A549 cells, and all cells died after 72 hr. Similar effects occurred after ONG41008 treatment in several human aggressive cancer cell lines such as PANC1, MCF7, PC3, or primary non-small cell lung carcinoma cells. Unlike cisplatin, ONG41008 was not toxic to normal human lung fibroblasts or primary prostate epithelial cells, suggesting ONG41008 can distinguish the intracellular microenvironment between normal cells and aged or diseased cells. This effect might occur as a result of the increased NAD/NADH ratio or increased lactate dehydrogenase levels in aged or diseased cells.

InterpretationTo our best knowledge, this is the first study to show that a small molecule can arrest uncontrolled proliferation during fibrogenesis or tumorigenesis in vitro. ONG41008 could be a potential drug for a broad range of fibrotic or tumorigenic diseases.

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSThe notion that aging is a disease and that diseases occur as a consequence of aging was first put forward by David Sinclair and colleagues (Aging Cell;14(4):497-510). There are biological systems that provide evidence for this notion; for example, somatic cells can revert to embryonic cells, producing younger somatic cells. This phenomenon underlies induced pluripotent stem cells. Another example is that some types of jellyfish can live forever. These examples suggest that a counter-aging program exists in animals. Human diseases are the manifestations of cell aging generated by the accumulation of somatic mutations. Aged and pathogenic cells are senescent, so a drug that specifically targeted senescent cells might initiate a cellular program that could ameliorate age-associated disease. Indeed, the kinase inhibitor dasatinib induces cellular senescence (Clin Ther 2007 29:2289-2308). In 2017, two drugs that target senescent cells were identified: quercetin and fisetin. These drugs selectively kill senescent cells, and are referred to as senolytic drugs or senotherapeutics (Aging 2017 8;9(3):955-963). Although it is established that senescent cells accumulate in cancer and idiopathic pulmonary fibrosis (Nat Commun 2017 23;8 :14532), the effect of senolytic drugs in these diseases is largely unknown.

Added value of this studyThis study characterized a novel drug, termed ONG41008, which was found to have both senogenic and senolytic effects in cell-based assays. ONG41008 induced senescence in myofibroblasts and several cancer cell lines representative of aggressive human cancers, which was followed by cell death. Importantly, ONG41008 exhibited essentially no toxicity on normal human lung fibroblasts or primary prostate epithelial cells.

Implication of all the evidenceBased on our results, we believe that ONG41008 is a potent inducer of cellular senescence (replicative senescence and oncogene-induced senescence) and causes arrest of uncontrolled, pathogenic proliferation of myofibroblasts or cancer cells.
CD155 and EndoA1 mediate growth and tissue invasion downstream of MAP4K4 in medulloblastoma cells The composition of the plasma membrane (PM)-associated proteome of tumor cells determines cell-cell and cell-matrix interactions and the response to environmental cues. Whether the PM-associated proteome impacts the phenotype of Medulloblastoma (MB) tumor cells and how it adapts in response to growth factor cues is poorly understood. Using a spatial proteomics approach, we observed that hepatocyte growth factor (HGF)-induced activation of the receptor tyrosine kinase c-MET in MB cells changes the abundance of transmembrane and membrane-associated proteins. The depletion of MAP4K4, a pro-migratory effector kinase downstream of c-MET, leads to a specific decrease of the adhesion and immunomodulatory receptor CD155 and of components of the fast-endophilin-mediated endocytosis (FEME) machinery in the PM-associated proteome of HGF-activated MB cells. The decreased surface expression of CD155 or of the FEME effector Endophilin A1 reduces growth and invasiveness of MB tumor cells in the tissue context. These data thus describe a novel function of MAP4K4 in the control of the PM-associated proteome of tumor cells and identified two downstream effector mechanisms controlling proliferation and invasiveness of MB cells.

Graphical abstractc-MET activation upon HGF stimulation induces c-MET internalization and induces downstream MAP4K4 activity. (1) MAP4K4 is required downstream of activated c-MET for the maintenance of surface presentation of CD155 in activated cells. CD155 expression is required for MB cell migration, invasion and proliferation in the tissue context. (2) MAP4K4 is required downstream of activated c-MET to maintain membrane depolarization, possibly by regulating the surface localization of several ion channels and transporters. (3) MAP4K4 is required downstream of activated c-MET cause PM-proximal localization of FEME effector CIP4, FBP17 and CIN85. The FEME effector endophilin A is necessary for MB cell migration, invasion and dissemination.
The "Alu-ome" shapes the epigenetic environment of regulatory elements controlling cellular defense Promoters and enhancers are sites of transcription initiation (TSSs) and carry active histone modifications, including H3K4me1, H3K4me3, and H3K27ac. Yet, the principles governing the boundaries of such regulatory elements are still poorly characterized. Alu elements are good candidates for a boundary function, being highly abundant in gene-rich regions, while essentially excluded from regulatory elements. Here, we show that the interval from the TSS to the first upstream Alu accommodates all H3K4me3 and most H3K27ac marks, while excluding DNA methylation. Remarkably, the average length of this intervals greatly varies in-between tissues, being longer in stem-and shorter in immune-cells. The very shortest TSS-to-Alu intervals were observed at promoters active in T cells, particularly at immune genes, correlating with RNA polymerase II transcription through the first Alu and accumulation of H3K4me1 signal on this first Alu. Finally, DNA methylation at first-Alus was found to evolved with age, regressing from young to middle-aged, then recovering later in life. Thus, the first Alus upstream of TSSs appear as dynamic boundaries marking the transition from DNA methylation to active histone modifications at regulatory elements, while also participating in the recording of immune gene transcriptional events by positioning H3K4me1-modified nucleosomes. Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression BackgroundChimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures.

MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model.

ResultsIn comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model.

ConclusionsThis proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors. Polycystins are upregulated in fibrotic kidneys and promote renal fibrosis Autosomal polycystic kidney disease (ADPKD) is caused by mutations in PKD1or PKD2 gene, which encodes polycystin-1 or polycystin-2 respectively. Paradoxically, polycystins are up-regulated in human ADPKD kidneys and overexpression of polycystin-1 even induced renal cyst formation in mice, suggesting that up-regulation of polycystin is pathogenic. In this study, we determined the role of polycystin in renal fibrosis and explored its underlying mechanisms. We found that the expression of polycystin-1 or polycystin-2 was positively correlated with the expression of collagen-I in the kidney of three different fibrotic mouse models. Pharmaceutical inhibition or genetic knockout of polycystin-2 reduced the expression of pro-fibrotic markers in fibrotic kidneys of two animal models. Moreover, conditional knockout of Pkd1 gene attenuated renal fibrosis in two mouse models. Enhancer of Zeste homolog 2 (EZH2) is a methyltransferase which plays important role in renal fibrosis. We found that EZH2 was positively correlated with the expression of polycystins in fibrotic kidneys. Conditional knockout of the Ezh2 attenuated the anti-fibrotic responses induced by Pkd1 deletion in fibrotic kidneys.

In conclusion, polycystins are up-regulated in injured kidneys and polycystins promote renal fibrosis through up-regulation of EZH2 in fibrotic kidneys, suggesting that renal fibrosis in polycystic kidneys is not the direct effect of PKD gene mutations.

SignificanceMutations of polycystins cause autosomal polycystic kidney disease (ADPKD) which is associated with increased renal fibrosis in cystic kidneys. However, the role of polycystin in renal fibrosis remains unclear. Here, we showed that the expression of polycystins is positively correlated with the development of renal fibrosis in injured kidneys. Pharmaceutical inhibition or genetic deletion of polycystin-2 attenuated renal fibrosis in fibrotic kidneys. Moreover, genetic deletion of polycystin-1 inhibited renal fibrosis in two different animal models. Finally, we showed that ablation of the methyltransferase EZH2 mitigated the anti-fibrotic effect of polycystin-1 deletion in fibrotic kidneys. We conclude that polycystin promotes through EZH2 and renal fibrosis in cystic kidney is not the direct effect of PKD gene mutations. Evaluation of methods for estimating coalescence times using ancestral recombination graphs The ancestral recombination graph (ARG) is a structure that describes the joint genealogies of sampled DNA sequences along the genome. Recent computational methods have made impressive progress towards scalably estimating whole-genome genealogies. In addition to inferring the ARG, some of these methods can also provide ARGs sampled from a defined posterior distribution. Obtaining good samples of ARGs is crucial for quantifying statistical uncertainty and for estimating population genetic parameters such as effective population size, mutation rate, and allele age. Here, we use standard neutral coalescent simulations to benchmark the estimates of pairwise coalescence times from three popular ARG inference programs: ARGweaver, Relate, and tsinfer+tsdate. We compare 1) the true coalescence times to the inferred times at each locus; 2) the distribution of coalescence times across all loci to the expected exponential distribution; 3) whether the sampled coalescence times have the properties expected of a valid posterior distribution. We find that inferred coalescence times at each locus are most accurate in ARGweaver, and often more accurate in Relate than in tsinfer+tsdate. However, all three methods tend to overestimate small coalescence times and underestimate large ones. Lastly, the posterior distribution of ARGweaver is closer to the expected posterior distribution than Relates, but this higher accuracy comes at a substantial trade-off in scalability. The best choice of method will depend on the number and length of input sequences and on the goal of downstream analyses, and we provide guidelines for the best practices. Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose) In Vitro and In Vivo AO_SCPLOWBSTRACTC_SCPLOWADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally-occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts) - optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems. Spike mimicry of thrombopoietin may induce thrombocytopenia in COVID-19 SARS-CoV-2 causes COVID-19, a disease curiously resulting in varied symptoms and outcomes, ranging from asymptomatic to fatal. Autoimmunity due to cross-reacting antibodies resulting from molecular mimicry between viral antigens and host proteins may provide an explanation. We computationally investigated molecular mimicry between SARS-CoV-2 Spike and known epitopes. We discovered molecular mimicry hotspots in Spike and highlight two examples with tentative autoimmune potential and implications for understanding COVID-19 complications. We show that a TQLPP motif in Spike and thrombopoietin shares similar antibody binding properties. Antibodies cross-reacting with thrombopoietin may induce thrombocytopenia, a condition observed in COVID-19 patients. Another motif, ELDKY, is shared in multiple human proteins such as PRKG1 and tropomyosin. Antibodies cross-reacting with PRKG1 and tropomyosin may cause known COVID-19 complications such as blood-clotting disorders and cardiac disease, respectively. Our findings illuminate COVID-19 pathogenesis and highlight the importance of considering autoimmune potential when developing therapeutic interventions to reduce adverse reactions. Feed efficiency of lactating Holstein cows was not as repeatable across diets as within diet over subsequent lactation stages BackgroundImproving feed efficiency has become a common target for dairy farmers to meet the requirement of producing more milk with fewer resources. To improve feed efficiency, a prerequisite is to ensure that the cows identified as most or least efficient will remain as such, independently of diet composition. Therefore, the current research analysed the ability of lactating dairy cows to maintain their feed efficiency while changing the energy density of the diet by changing its concentration in starch and fibre. A total of 60 lactating Holstein cows, including 33 primiparous cows, were first fed a high starch diet-low fibre (diet S+F-), then switched over to a low starch diet-high fibre (diet S-F+). To know if diet affect feed efficiency, we compared the ability of feed efficiency to be maintained within a diet over subsequent lactation stages, known as repeatability of feed efficiency, with its ability to be maintained across diets, known as reproducibility of feed efficiency. To do so we used two indicators: the estimation of the error of repeatability/reproducibility, which is commonly used in metrology, and the coefficient of correlation of concordance (CCC), which is used in biology. The effect of diet change could also lead to a change in cows sorting behaviour which could potentially affect feed efficiency if for example the most efficient cows select more concentrate than the least efficient. We therefore analysed the relationship between the differences in individual feed refusals composition and the differences in feed efficiency. To do so, the composition of each feed refusal was described with its near infra-red (NIR) spectroscopy and was performed on each individual feed ingredient, diet and refusals and used as composition variable. The variability of the NIR spectra of the refusals was described with its principal components thanks to a principal component analysis (PCA). The Pearson correlation was estimated to check the relationship between feed efficiency and refusals composition, i.e. sorting behaviour.

ResultsThe error of reproducibility of feed efficiency across diets was 2.95 MJ/d. This error was significantly larger than the errors of repeatability estimated within diet, which were 2.01 MJ/d within diet S-F+ and 2.40 MJ/d within diet S+F-. The CCC was 0.64 between feed efficiency estimated within diet S+F- and feed efficiency estimated within diet S-F+. This CCC was smaller than the one observed for feed efficiency estimated within diet between two subsequent lactation stages (CCC = 0.72 within diet S+F- and 0.85 within diet S-F+). Feed efficiency was poorly correlated to the first two principal components, which explained 90% of the total variability of the NIR spectra of the individual refusals. This suggests that feed sorting behaviour did not explain differences in feed efficiency.

ConclusionsFeed efficiency was significantly less reproducible across diets than repeatable within the same diet over subsequent lactation stages, but cows ranking for feed efficiency was not significantly affected by diet change. This loss in repeatability across diets could be due to a more pronounced feed sorting subsequent to the change in diet composition. However, the differences in sorting behaviour between cows were not associated to feed efficiency differences in this trial neither with the S+F- diet nor with the S-F+ diet. Those results have to be confirmed on diets having different forage to concentrate ratios to ensure that the least and most efficient cows will not change. PIN-FORMED1 polarity in the shoot is insensitive to the polarity of neighbouring cells In plants, epidermal cells are planar-polarized along an axis marked by the asymmetric localization patterns of several proteins including PIN-FORMED1 (PIN1), which facilitates the directional efflux of the plant hormone auxin to pattern phyllotaxis (Heisler et al., 2010; Mansfield et al., 2018; Reinhardt et al., 2003). While PIN1 polarity is known to be regulated non-cell autonomously via the MONOPTEROS (MP) transcription factor, how this occurs has not been determined (Bhatia et al., 2016). Here we use mosaic expression of the serine threonine kinase PINOID (PID) to test whether PIN1 polarizes according to the polarity of neighbouring cells. Our findings reveal that PIN1 is insensitive to the polarity of PIN1 in neighbouring cells arguing against auxin flux or extracellular auxin concentrations acting as a polarity cue, in contrast to previous model proposals (Abley et al., 2016; Stoma et al., 2008). RIG-I immunotherapy overcomes radioresistance in p53-positive malignant melanoma Radiation therapy induces cytotoxic DNA damage, which results in cell-cycle arrest and activation of cell-intrinsic death pathways, but its application has been limited by the radioresistance of tumors, such as in malignant melanoma. RIG-I is a cytosolic immune receptor expressed in all somatic cells, including tumor cells, with a key role in sensing viral RNA. RIG-I specific oligonucleotide ligands elicit a robust cell-intrinsic antiviral response and immunogenic cell death in tumor cells and are being tested in clinical trials. Nonetheless, their potential to overcome radioresistance has not yet been explored. Here, we demonstrate that activation of RIG-I enhances the extent and immunogenicity of irradiation-induced tumor cell death in human and murine melanoma cell lines in vitro and improved survival in the murine B16 melanoma model. Pathway analysis of transcriptomic data revealed a central role for p53 downstream of the combination treatment, which was corroborated using p53-/- B16 cells. In vivo, the effect of irradiation on immune-cell activation and inhibition of tumor growth was absent in mice carrying p53-/- B16 tumors, while the response to RIG-I stimulation in those mice was maintained. Our results identify p53 as pivotal for the synergistic antitumoral effect of RIG-I and irradiation, resulting in potent induction of immunogenic tumor-cell death. Thus, the administration of RIG-I ligands in combination with radiotherapy is a promising therapeutic approach to treating radioresistant tumors with a functional p53 pathway, such as malignant melanoma. Amino acids targeted based metabolomics study in non-segmental Vitiligo: a pilot study IntroductionVitiligo is an asymptomatic disorder that results from the loss of pigments (melanin), causing skin or mucosal depigmentation and impairs beauty.

ObjectiveDue to the complexity of the pathogenesis of this disease and various theories including self-safety theory, oxidative stress, neurological theory and internal defects of melanocytes behind it, and finally, the vast role of amino acids in body metabolism and various activities of the body, amino acids targeted based metabolomics was set up to follow any fluctuation inside this disease.

MethodologyThe study of amino acid profiles in plasma of people with non-segmental vitiligo using a liquid chromatography equipped with fluorescent detector was performed to find remarkable biomarkers for the diagnosis and evaluation of disease severity of patients with vitiligo. Twenty-two amino acids derivatized with o-phthalaldehyde (OPA) and fluorylmethyloxycarbonyl chloride (FMOC), were precisely determined. Next, the concentrations of these twenty-two amino acids and their corresponding molar ratios were calculated in 37 patients (including 18 females and 19 males) and corresponding 34 healthy individuals (18 females and 16 males). Using R programing, the data were completely analyzed between the two groups of patients and healthy to find suitable and reliable biomarkers.

ResultsInterestingly, comparing the two groups, in the patient group, tyrosine, cysteine, the ratio of tyrosine to lysine and the ratio of cysteine to ornithine were increased while, arginine, lysine, ornithine and glycine ratios to cysteine have been decreased. These amino acids were selected for identification of patients with accuracy of detection of approximately 0.95 using the assessment of logistic regression.

ConclusionThese results indicate a disruption of the production of melanin, increased immune activity and oxidative stress, which are also involved in the effects of vitiligo. Therefore, these amino acids can be used as biomarker for the evaluation of risk, prevention of complications in individuals at risk and monitoring of treatment process. Failure to account for behavioral variability significantly compromises accuracy in indirect population monitoring Indirect wildlife population surveying largely depends upon counts of artefacts of behavior (e.g., nests or dung). Likelihood to encounter these artefacts is derived from both artefact production and decay, and variability in artefact production behavior is considered to contribute minimally to inaccuracy in wildlife estimation. Here we demonstrate how ignoring behavioral variability contributes to significant population misestimation, using an example of an endangered ape, the bonobo (Pan paniscus). Until now, a single estimate of (i.e., nest construction) has been used to extrapolate bonobo densities, assumed to be broadly representative of bonobo sign production behavior. We estimated nest construction rate across seasons and social groups at the Kokolopori Bonobo Reserve, DRC and find that nest construction rates in bonobos to be highly variable across populations as well as seasonal. Failure to account for this variability led to potentially severe degradation in accuracy of bonobo population estimates of abundance, accounting for a likely overestimation of bonobo numbers by 34%, and at worst as high as 80%. With this example, we demonstrate that failure to account for inter- and intra-population behavioral variation compromises the ability to estimate both relative and absolute wildlife abundances. We argue that variation in sign production is but one of several potential ways that behavioral variability can affect conservation monitoring, should be measured across contexts whenever possible, and must be considered in population estimation confidence intervals. With increasing attention to behavioral variability as a potential tool for conservation, conservationists must also account for the impact that behavioral variability can play upon wildlife population estimation. Our results underline the importance of observational research to wildlife monitoring schemes as a critical but overlooked component of conservation management. We extensively discuss the various avenues through which behavioral variability is likely to impact wildlife monitoring accuracy and precision and propose potential avenues for accounting for behavioral variability in wildlife monitoring. Depletion of CD206+ Tumour Macrophages via a Peptide-Targeted Star-Shaped Polyglutamate Inhibits Tumourigenesis and Metastatic Dissemination in Breast Cancer Models Although many studies have explored the depletion of tumour-associated macrophages (TAMs) as a therapeutic strategy for solid tumours, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent ("OximUNO") that specifically targets CD206+ TAMs and demonstrated efficacy in triple negative breast cancer (TNBC) mouse models. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In TNBC models, a fluorescently labelled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumour lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (demonstrating immunostimulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent the first report of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate. Metabolic reconstitution by a gnotobiotic microbiota varies over the circadian cycle The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiome to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat-mass than germ-free or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure and respiratory quotient using a novel isolator-housed metabolic cage system which allows long-term measurements without contamination risk. This demonstrated that microbiota- released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and germ-free mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well-integrated into appetite control. We also observed no significant difference in energy expenditure per gram lean mass between the different microbiota groups, suggesting that cumulative very small differences in energy balance, or altered energy storage must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: in germ-free mice the respiratory exchange ratio was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied germ-free mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a germ-free-like drop in liver glycogen storage during the light cycle and both liver and plasma metabolomes of OligoMM12 mice clustered closely with germ-free mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle, and which are absent in the OligoMM12 consortium. Double trouble: having two hosts reduces infection prevalence in vectored trypanosomatids Trypanosomatids are a diverse family of protozoan parasites, some of which cause devastating human and livestock diseases. There are two distinct infection life-cycles in trypanosomatids; some species complete their entire life-cycle in a single host (monoxenous) while others infect two hosts (dixenous). Dixenous trypanosomatids are mostly vectored by insects, and the human trypanosomatid diseases are caused mainly by vectored parasites. While infection prevalence has been described for subsets of hosts and trypanosomatids, little is known about whether monoxenous and dixenous trypanosomatids differ in infection prevalence. Here, we use meta-analyses to synthesise all published evidence of trypanosomatid infection prevalence for the last two decades, encompassing 931 unique host-trypansomatid systems. In examining 581 studies that describe infection prevalence, we find, strikingly, that monoxenous species are two-fold more prevalent than dixenous species across all hosts. We also find that dixenous trypanosomatids have significantly lower infection prevalence in insects than their non-insect hosts. Within monoxenous trypanosomatids, genera infecting bees are most prevalent and infection prevalence does not vary between wild and managed bees. To our knowledge, these results reveal for the first time, a fundamental difference in infection prevalence according to host specificity where vectored species suffer from lower infection prevalence as a result of a  jack of all trades, master of none style trade-off between the vector and subsequent hosts. 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. Modelling and assessing additional transmission routes for porcine reproductive and respiratory syndrome virus: vehicle movements and feed ingredients Accounting for multiple modes of livestock disease dissemination in epidemiological models remains a challenge. We developed and calibrated a mathematical model for transmission of porcine reproductive and respiratory syndrome virus (PRRSV), tailored to fit nine modes of between-farm transmission pathways including: farm-to-farm proximity (local transmission), contact network of batches of pigs transferred between farms (pig movements), re-break probabilities for farms with previous PRRSV outbreaks, with the addition of four different contact networks of transportation vehicles (vehicles to transport pigs to farms, pigs to markets, feed and crew) and the amount of animal by-products within feed ingredients (e.g. animal fat or meat and bone meal). The model was calibrated on weekly PRRSV outbreaks data. We assessed the role of each transmission pathway considering the dynamics of specific types of production (i.e., sow, nursery). Although our results estimated that the networks formed by transportation vehicles were more densely connected than the network of pigs transported between-farms, pig movements and farm proximity were the main PRRSV transmission routes regardless of farm types. Among the four vehicle networks, vehicles transporting pigs to farms explained a large proportion of infections, sow = 20.9%; nursery = 15%; and finisher = 20.6%. The animal by-products showed a limited association with PRRSV outbreaks through descriptive analysis, and our model results showed that the contribution of animal fat contributed only 2.5% and meat and bone meal only 0.03% of the infected sow farms. Our work demonstrated the contribution of multiple routes of PRRSV dissemination, which has not been deeply explored before. It also provides strong evidence to support the need for cautious, measured PRRSV control strategies for transportation vehicles and further research for feed by-products modeling. Finally, this study provides valuable information and opportunities for the swine industry to focus effort on the most relevant modes of PRRSV between-farm transmission. A rationalized definition of general tumor suppressor microRNAs excludes miR-34a While several microRNAs (miRNAs) have been proposed to act as tumor suppressors, a consensual definition of tumor suppressing miRNAs is still missing. Similarly to coding genes, we propose that tumor suppressor miRNAs must show evidence of genetic or epigenetic inactivation in cancers, and exhibit an anti-tumorigenic (e.g., anti-proliferative) activity under endogenous expression levels. Here we observe that this definition excludes the most extensively studied tumor suppressor candidate miRNA, miR-34a. In analyzable cancer types, miR-34a does not appear to be down-regulated in primary tumors relatively to normal adjacent tissues. Deletion of miR-34a is occasionally found in human cancers, but it does not seem to be driven by an anti-tumorigenic activity of the miRNA, since it is not observed upon smaller, miR-34a-specific alterations. Its anti-proliferative action was observed upon large, supra-physiological transfection of synthetic miR-34a in cultured cells, and our data indicates that endogenous miR-34a levels do not have such an effect. Our results therefore argue against a general tumor suppressive function for miR-34a, providing an explanation to the lack of efficiency of synthetic miR-34a administration against solid tumors. 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. The first multi-tissue diel genome-scale metabolic model of a woody plant highlights the suberin biosynthesis pathways in Quercus suber In the last decade, genome-scale metabolic models have been increasingly used to study plant metabolic behavior at the tissue and multi-tissue level under different environmental conditions. Quercus suber, also known as the cork oak tree, is one of the most important forest communities of the Mediterranean/Iberian region. In this work, we present the genome-scale metabolic model of the Q. suber (iEC7871), the first of a woody plant. The metabolic model comprises 7871 genes, 6231 reactions, and 6481 metabolites across eight compartments. Transcriptomics data was integrated into the model to obtain tissue-specific models for the leaf, inner bark, and phellogen, with specific biomass compositions. The tissue-specific models were merged into a diel multi-tissue metabolic model to predict interactions among the three tissues at the light and dark phases. The metabolic models were also used to analyze the pathways associated with the synthesis of suberin monomers. Nevertheless, the models developed in this work can provide insights into other aspects of the metabolism of Q. suber, such as its secondary metabolism and cork formation. 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
C_LI 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. Individual Variability in the Innate Functional Organization of the Human Brain The adult brain is organized into distinct functional networks, forming the basis of information processing and determining individual differences in behavior. Is this network organization genetically determined and present at birth? And what is the individual variability in this organization in neonates? Here, we use unsupervised learning to uncover intrinsic functional brain organization using resting-state connectivity from a large cohort of neonates (Developing Human Connectome Project). We identified a set of symmetric, hierarchical, and replicable networks: sensorimotor, visual, default mode, ventral attention, and high-level vision. We quantified individual variability across neonates, and found the most individual variability in the ventral attention networks. Crucially, the variability of these networks were not driven by SNR differences or differences from adult networks (Yeo et al., 2011). Finally, differential gene expression provided a potential explanation for the emergence of these distinct networks and identified potential genes of interest for future developmental and individual variability research. Overall, we found neonatal connectomes (even at the voxel-level) can reveal broad individual- specific information processing units. The presence of individual differences in neonates and the framework for personalized parcellations demonstrated here has the potential to improve prediction of behavior and future outcomes from neonatal and infant brain data. The neurogenic fate of the hindbrain boundaries relies on Notch-dependent asymmetric cell divisions The generation of cell diversity in the central nervous system occurs during embryogenesis and requires a precise balance between cell proliferation, commitment to specific fates, and further neuronal differentiation. The cellular and molecular mechanisms regulating this balance in the embryonic brain are still poorly understood. Here we study how the neurogenic capacity in the embryonic hindbrain is spatiotemporally allocated, and how the fate and the growth of the hindbrain boundary cells are regulated. By generating a CRISPR-based knock-in zebrafish transgenic line to specifically label the hindbrain boundaries, we unveiled that boundary cells undergo a functional transition to become neurogenic during hindbrain segmentation concurrently as they maintain the progenitor cell pool. Boundary cells engaged in neurogenesis coinciding with the onset of Notch signaling, which triggered their asymmetrical cell division. Our findings reveal that distinct neurogenic phases take place during hindbrain growth and suggest that boundary cells contribute to refine the final number, identity, and proportion of neurons in the brain.

SUMMARYHindbrain boundary cells undergo a functional transition to become neurogenic concurrently as they are maintained as a progenitor cell pool. This involves a behavioral switch from symmetrically dividing progenitor cells to asymmetrically dividing progenitors, which depends on Notch-activity. Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand dependent population distributions Homo-oligomeric ligand-activated proteins are ubiquitous in biology. The functions of such molecules are commonly regulated by allosteric coupling between ligand binding sites. Understanding the basis for this regulation requires both quantifying the free energy {Delta}G transduced between sites, and the structural basis by which it is transduced. We consider allostery in three variants of the model ring-shaped homo-oligomeric trp RNA binding attenuation protein, TRAP. First, we developed nearest-neighbor statistical thermodynamic binding models comprising microscopic free energies for ligand binding to isolated sites {Delta}GN0, and for coupling between one or both adjacent sites, {Delta}GN1 and {Delta}GN2. Using the resulting partition function (PF) we explored the effects of these parameters on simulated population distributions for the 2N possible liganded states. We then experimentally monitored liganddependent population shifts using conventional spectroscopic and calorimetric methods, and using native mass spectrometry (MS). By resolving species with differing numbers of bound ligands by their mass, native MS revealed striking differences in their ligand-dependent population shifts. Fitting the populations to a binding polynomial derived from the PF yielded coupling free energy terms corresponding to orders of magnitude differences in cooperativity. Uniquely, this approach predicts which of the possible 2N liganded states are populated at different ligand concentrations, providing necessary insights into regulation. The combination of statistical thermodynamic modeling with native MS may provide the thermodynamic foundation for a meaningful understanding of the structure-thermodynamic linkage that drives cooperativity.

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org.highwire.dtl.DTLVardef@2ce1f3org.highwire.dtl.DTLVardef@a6541borg.highwire.dtl.DTLVardef@1dbd08aorg.highwire.dtl.DTLVardef@58297e_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOTOC Figure.C_FLOATNO Ligand (Trp) binding to multiple sites on homo-oligomeric ring-shaped proteins like TRAP alters their functional states. Homotropic cooperativity is expected to alter the activation pathway in response to cellular ligand concentration. In the presence of positive nearest-neighbor cooperativity, ligand binding is favored at adjacent sites, whereas in the absence of cooperativity, a random "Normal" distribution is expected.

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