title: The propensity to sign-track is associated with externalizing behaviour and distinct patterns of reward-related brain activation in youth

abstract: Externalizing behaviours in childhood often predict impulse control disorders in adulthood; however, the underlying biobehavioural risk factors are incompletely understood. In animals, the propensity to signtrack, or the degree to which incentive motivational value is attributed to reward cues, is associated with externalizing-type behaviours and deficits in executive control. Using a Pavlovian conditioned approach paradigm, we quantified sign-tracking in healthy 9-12-year-olds. We also measured parent-reported externalizing behaviours and anticipatory neural activations to outcome-predicting cues using the monetary incentive delay fMRI task. Sign-tracking was associated with attentional and inhibitory control deficits and the degree of amygdala, but not cortical, activation during reward anticipation. These findings support the hypothesis that youth with a propensity to sign-track are prone to externalizing tendencies, with an over-reliance on subcortical cue-reactive brain systems. This research highlights sign-tracking as a promising experimental approach delineating the behavioural and neural circuitry of individuals at risk for externalizing disorders.


title: Complex and critical roles for the AtrR transcription factor in control of cyp51A expression in Aspergillus fumigatus

abstract:
Aspergillus fumigatus is the major filamentous fungal pathogen in humans. The gold standard treatment of A. fumigatus is based on azole drug use but the appearance of azole-resistant isolates is increasing at an alarming rate. The cyp51A gene encodes the enzymatic target of azole drugs and azole-resistant alleles of cyp51A often have an unusual genetic structure containing a duplication of a 34 or 46 bp region in the promoter causing enhanced gene transcription. These tandem repeats are called TR34/TR46 and produce duplicated binding sites for the SrbA and AtrR transcription factors. Using site-directed mutagenesis, we demonstrate that both the SrbA (sterol response element: SRE) and AtrR binding sites (AtrR response element: ATRE) are required for normal cyp51A gene expression. Loss of either the SRE or ATRE from the distal 34 bp repeat of the TR34 promoter (further 5 from the transcription start site) caused loss of expression of cyp51A and decreased voriconazole resistance. Surprisingly, loss of these same binding sites from the proximal 34 or 46 bp repeat led to increased cyp51A expression and voriconazole resistance. These data indicate that these duplicated regions in the cyp51A promoter function differently. Our findings suggest that the proximal 34 or 46 bp repeat in cyp51A recruits a corepressor that requires multiple factors to act while the distal repeat is free of this repression and provides the elevated cyp51A expression caused by these promoter duplications.

ImportanceAspergillus fumigatus is the most common human filamentous fungal pathogen. Azole drugs are the current therapy of choice for A. fumigatus but the prevalence of azole resistance is increasing. The main genetic alteration causing azole resistance is a change in the cyp51A gene that encodes the target of these drugs. Azole-resistant cyp51A alleles routinely contain duplications in their promoter regions that cause increased gene transcription. Here, we demonstrate that clinical isolates containing a 34 or 46 bp duplication in the cyp51A promoter required the presence of the transcription factor-encoding atrR gene to exhibit elevated azole resistance. Elimination of transcription factor binding sites in the cyp51A gene have differential actions on expression of the resulting mutant allele. These data dissect the molecular inputs to cyp51A transcription and reveal a complicated function of the promoter of this gene that is critical in azole resistance.

category: microbiology


title: Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering

abstract: Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional condensates, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harboring distinct ecDNA species (EGFR, MYC, PDGFR). We found no evidence that ecDNA cluster with one another or closely interact with transcriptional condensates. Cells with EGFR-containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy was similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with the cellular transcriptional machinery.

category: genetics


title: Hyperactivation of YAP/TAZ drives alterations in mesangial cells through stabilization of N-MYC in diabetic nephropathy

abstract: Mesangial cells (MCs) in the kidney are central to maintaining glomerular integrity, and their impairment leads to major glomerular diseases including diabetic nephropathy (DN). Although high blood glucose elicits abnormal alterations in MCs, the underlying molecular mechanism is poorly understood. Here, we show that YAP and TAZ, the final effectors of the Hippo pathway, are highly increased in MCs of patients with DN and of Zucker diabetic fatty rats. Moreover, high glucose directly induces activation of YAP/TAZ through the canonical Hippo pathway in cultured MCs. Hyperactivation of YAP/TAZ in mouse model MCs recapitulates the hallmarks of DN, including excessive proliferation of MCs and extracellular matrix deposition, endothelial cell impairment, glomerular sclerosis, albuminuria, and reduced glomerular filtration rate. Mechanistically, activated YAP/TAZ bind and stabilize N-Myc protein, one of the Myc family of oncogenes. N-Myc stabilization leads to aberrant enhancement of its transcriptional activity and eventually to MC impairments and DN pathogenesis. Together, these findings shed light on how high blood glucose in diabetes mellitus leads to DN and support a rationale that lowering blood glucose in diabetes mellitus could delay DN pathogenesis.

category: pathology


title: Distinct genetic determinants and mechanisms of SARS-CoV-2 resistance to remdesivir

abstract:
The nucleoside analog remdesivir (RDV) is an FDA-approved antiviral for the treatment of SARS- CoV-2 infections, and as such it is critical to understand potential genetic determinants and barriers to RDV resistance. In this study, SARS-CoV-2 was subjected to 13 passages in cell culture with increasing concentrations of GS-441524, the parent nucleoside of RDV. At passage 13 the RDV resistance of the lineages ranged from 2.7-to 10.4-fold increase in EC50. Sequence analysis of the three lineage populations identified non-synonymous mutations in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp): V166A, N198S, S759A, V792I and C799F/R. Two of the three lineages encoded the S759A substitution at the RdRp Ser759-Asp-Asp active motif. In one lineage, the V792I substitution emerged first then combined with S759A. Introduction of the S759A and V792I substitutions at homologous nsp12 positions in viable isogenic clones of the betacoronavirus murine hepatitis virus (MHV) demonstrated their transferability across CoVs, up to 38-fold RDV resistance in combination, and a significant replication defect associated with their introduction. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a [~]10- fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, while nsp12-V792I diminished the UTP concentration needed to overcome the template-dependent inhibition associated with RDV. The in vitro selected substitutions here identified were rare or not detected in the >6 million publicly available nsp12-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in various clinical settings.

One Sentence SummarySARS-CoV-2 develops in vitro resistance to remdesivir by distinct and complementary mutations and mechanisms in the viral polymerase


title: Altered meningeal immunity contributing to the autism-like behavior of BTBR T+ Itpr3tf/J mice.

abstract:
Autism spectrum disorder (ASD) is a complicated neurodevelopmental disorder, which is categorized by deficiency of social contact and communication, and stereotyped forms of performance. Meningeal immunity conditions the immune reflection and immune defense in the meningeal area involving meningeal lymphatic organization, glymphatic structure, immune cells, and cytokines. The development of meningeal immunity dysfunction might be the leading cause for many neural diseases including ASD. The inbred mouse strain BTBRT+Itpr3tf/J (BTBR) shows multiple ASD-like behavioral phenotypes, thus making this strain a widely used animal model for ASD. In our previous study, we reported an altered peripheral immune profile in BTBR mice. Herein, we are investigating immunological and neural interactions associated with the aberrant behavior of BTBR mice. BTBR mice have an increased level of immune cell deposition in the meninges along with a higher level of CD4+ T cells expressing CD25 and of B and myeloid cells expressing more MHCII than C57BL/6 (B6) mice, which have normal behaviors. BTBR mice also have higher levels of autoantibodies to dsDNA, Aquaporin-4, NMDAR1, Pentraxin/SAP and Caspr2 than B6 mice, which may affect neural functions. Interestingly, the T regulatory (Treg) cell population and their function was significantly reduced in the meninges and brain draining lymph nodes, which may explain the increased level of activated B and T cells in the meninges of BTBR mice. A low level of Treg cells, less IL-10 production by Treg, and activated T and B cells in meninges together with higher autoantibody levels might contribute to the development of autism-like behavior through neuroinflammation, which is known to be increased in BTBR mice.

HighlightsO_LIBTBR mice have higher level of immune cell deposition in the meninges compared to C57BL/6 (B6) mice.
C_LIO_LIMeningeal T cells and B cells of BTBR mice express a higher level of CD25 and MHCII, respectively, than those of B6 mice.
C_LIO_LIBTBR mice have a higher level of serum autoantibodies to dsDNA and brain antigens (Aquaporin-4, NMDAR1, Pentraxin/SAP and Caspr2) than B6 mice.
C_LIO_LIT regulatory (Treg) cell population was reduced in the meninges and brain draining lymph nodes of BTBR mice with lower cytokine production of IL-10.
C_LIO_LIFewer Treg cells and more activated meningeal T and B cells together with higher autoantibody levels might contribute to the development of the autism-like behavior of BTBR mice.
C_LI

category: immunology


title: Quantitative evaluation of warming and hemodilution as two techniques to increase the flow rate of blood during simulated massive transfusion of packed red blood cells

abstract: Massive bleeding is a leading cause of deaths in trauma, surgeries, wartime injuries, and childbirth. In these situations of ongoing blood loss, it is critical for patients to be rapidly resuscitated. Currently, patients who require major transfusions are given cold and concentrated blood from the blood bank. This blood does not flow fast due to its viscosity. Equipment for rapid resuscitation such as rapid transfusers are very costly and are not accessible everywhere. Additionally, specialized training is required to operate these machines. Therefore, it is necessary to determine simple, quick, and effective ways to increase the flow rate of blood. The flow rate can be increased by warming and hemodilution. The purpose of this study was to quantify the effects of these simple and quick methods since they can be implemented anywhere with easily accessible equipment. In this experiment, expired packed red blood cells were warmed and diluted. The time the blood took to flow through the tubing was measured and a flow rate was calculated for each trial. The research hypothesis stated that diluted blood at 39{degrees}C would have the fastest flow rate. The research hypothesis was supported and there was a 366% in flow rate by warming and diluting blood. There was a 232% increase by simply diluting the blood and a 170% increase by warming. Six t-tests were conducted for this data and they were all significant at a level of 0.001. These results indicate the significant effects of warming and diluting blood and this research could improve patient care during emergencies and massive blood loss.

category: physiology


title: Interplay between rule learning and rule switching in a perceptual categorization task

abstract: To adapt to a changing world, we must be able to switch between rules already learned and, at other times, learn rules anew. Often we must do both at the same time, switching between known rules while also constantly re-estimating them. Here, we show these two processes, rule switching and rule learning, rely on distinct but intertwined computations, namely fast inference and slower incremental learning. To this end, we studied how monkeys switched between three rules. Each rule was compositional, requiring the animal to discriminate one of two features of a stimulus and then respond with an associated eye movement along one of two different response axes. By modeling behavior we found the animals learned the axis of response using fast inference (rule switching) while continuously re-estimating the stimulus-response associations within an axis (rule learning). Our results shed light on the computational interactions between rule switching and rule learning, and make testable neural predictions for these interactions.

category: neuroscience


title: The effects of sequencing depth on the assembly of coding and noncoding transcripts in the human genome

abstract:
BackgroundInvestigating the functions and activities of genes requires proper annotation of the transcribed units. However, transcript assembly efforts have produced a surprisingly large variation in the number of transcripts, and especially so for noncoding transcripts. The heterogeneity of the assembled transcript sets might be partially explained by sequencing depth.

ResultsHere, we used real and simulated short-read sequencing data as well as long-read data to systematically investigate the impact of sequencing depths on the accuracy of assembled transcripts. We assembled and analyzed transcripts from 671 human short-read data sets and four long-read data sets. At the first level, there is a positive correlation between the number of reads and the number of recovered transcripts. However, the effect of the sequencing depth varied based on cell or tissue type, the type of read considered and the nature and expression levels of the transcripts. The detection of coding transcripts saturated rapidly for both short-read and long-reads, however, there was no sign of saturation for noncoding transcripts at any sequencing depth. Increasing long-read sequencing depth specifically benefited transcripts containing transposable elements. Finally, we show how single-cell RNA-seq can be guided by transcripts assembled from bulk long-read samples, and demonstrate that noncoding transcripts are expressed at similar levels to coding transcripts but are expressed in fewer cells.

ConclusionsThis study shows the impact of sequencing depth on transcript assembly. Sequencing read depth has a relatively minor impact on coding transcript assembly, but a major effect on the assembly of noncoding transcripts. This study highlights important factors to consider when deciding the sequencing read depths to be used for transcript assembly.

category: bioinformatics


title: A Phase-separating Thiol-ene Photoresin for Volumetric Bioprinting of Macroporous Hydrogels

abstract: Macroporous hydrogels facilitate solute transport and cell-cell communication in 3D, but materials allowing for in situ pore formation and 3D-printing in aqueous solutions are scarce. Here, a phase-separating thiol-ene photoresin is developed for light-assisted 3D-printing of hierarchical macroporous hydrogels that support 3D cell growth. The resin consists of norbornene-functionalized polyvinyl alcohol, di-thiol crosslinker and dextran sulfate, which can rapidly form a hydrogel with interconnected pores by photopolymerization-induced phase separation (PIPS). The pore size is tunable in the range of 2-40 m as quantified by fast Fourier transformation of confocal imaging data and depends on light intensity, polymer composition and molecular charge. After 3D photoencapsulation, high cell viability (>90%) is achieved and cell spreading in the macroporous hydrogels is significantly higher than in conventional nanoporous hydrogels. The incorporation of a thermo-reversible gelatin network enables its printability for tomographic volumetric bioprinting in the presence of human mesenchymal stem cells. A cm-scale cell-laden hydrogel construct with vasculature-like channels and interconnected pores can be 3D-printed in 12 seconds. The materials are cell-compatible, low-cost, easy-to-make and highly efficient for PIPS and light-based 3D-printing, which is unachievable with conventional 3D-printable hydrogels. Such materials are promising for future photofabrication of complex tissues with a hierarchical porosity within seconds.

category: bioengineering


title: Pectin cell wall remodeling through PLL12 and callose deposition through polar CALS7 are necessary for long-distance phloem transport

abstract: In plants, the phloem distributes photosynthetic products for metabolism and storage over long distances. It relies on specialized cells, the sieve elements, which are enucleated and interconnected through large so-called sieve pores in their adjoining cell walls. Reverse genetics identified PECTATE LYASE LIKE 12 (PLL12) as critical for plant growth and development. Using genetic complementation, we establish that PLL12 enzyme activity is required exclusively late during cell differentiation. Physiological assays suggested a role specifically in phloem long distance transport while short distance symplastic transport does not require PLL12. Exploiting CALLOSE SYNTHASE 7 (CALS7) as a polar fluorescent marker, we identified structural defects in sieve pores in pll12. Due to the serial arrangement of sieve elements in the phloem, such defects should affect a plants ability specifically for symplastic transport over long distances.

category: plant biology


title: The NAD+ precursor NMN activates dSarm to trigger axon degeneration in Drosophila

abstract: Axon degeneration contributes to the disruption of neuronal circuit function in diseased and injured nervous systems. Severed axons degenerate following the activation of an evolutionarily conserved signaling pathway, which culminates in the activation of SARM1 in mammals to execute the pathological depletion of the metabolite NAD+. SARM1 NADase activity is activated by the NAD+ precursor nicotinamide mononucleotide (NMN). In mammals, keeping NMN levels low potently preserves axons after injury, however, it remains unclear whether NMN is also a key mediator of axon degeneration, and dSarm activation, in flies. Here, we demonstrate that lowering NMN levels in Drosophila through the expression of a newly generated prokaryotic NMN-Deamidase (NMN-D) preserves severed axons for months and keeps them circuit-integrated for weeks. NMN-D alters the NAD+ metabolic flux by lowering NMN, while NAD+ remains unchanged in vivo. Increased NMN synthesis, by the expression of mouse nicotinamide phosphoribosyltransferase (mNAMPT), leads to faster axon degeneration after injury. We also show that NMN-induced activation of dSarm mediates axon degeneration in vivo. Finally, NMN-D delays neurodegeneration caused by loss of the sole NMN-consuming and NAD+-synthesizing enzyme dNmnat. Our results reveal a critical role for NMN in neurodegeneration in Drosophila, which extends beyond axonal injury. The potent neuroprotection by reducing NMN levels is similar or even stronger than the interference with other essential mediators of axon degeneration in Drosophila.


title: Clemizole and Trazodone are Effective Antiseizure Treatments in a Zebrafish Model of STXBP1 Disorder

abstract:
ObjectiveCRISPR-Cas9-generated zebrafish carrying a 12 base-pair deletion in stxbpb1b, a paralog sharing 79% amino acid sequence identity with human, exhibit spontaneous electrographic seizures during larval stages of development. Zebrafish stxbp1b mutants provide an efficient preclinical platform to test antiseizure therapeutics. The present study was designed to test prototype antiepileptic drugs approved for clinical use and two recently identified repurposed drugs with antiseizure activity.

MethodsLarval homozygous stxbp1b zebrafish (4 days post-fertilization) were agarose-embedded and monitored for electrographic seizure activity using a local field recording electrode placed in midbrain. Frequency of ictal-like events was evaluated at baseline and following 45 min of continuous drug exposure (1 mM, bath application). Analysis was performed on coded files by an experimenter blinded to drug treatment and genotype.

ResultsPhenytoin, valproate, ethosuximide, levetiracetam, and diazepam had no effect on ictal-like event frequency in stxbp1b mutant zebrafish. Clemizole and trazodone decreased ictal-like event frequency in stxbp1b mutant zebrafish by 80% and 83%, respectively. These results suggest that repurposed drugs with serotonin receptor binding affinities could be effective antiseizure treatments.

SignificanceClemizole and trazodone were identified in a larval zebrafish model for Dravet syndrome. Based primarily on these preclinical zebrafish studies, compassionate-use and double-blind clinical trials with both drugs have progressed. The present study extends this approach to a preclinical zebrafish model representing STXBP1-related disorders, and suggests that future clinical studies may be warranted.


title: Development of 3D breast cancer models with human T cells expressing engineered MAIT cell receptors

abstract: Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune-cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we developed proof-of-concept 3D tumor models with increasing complexity to study the cytotoxic responses of CD8+ T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast (HDF) tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3-day culture period, demonstrating that the engineered TCR recognized MR1 expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as IFN{gamma}, IL-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune-cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune-cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.


title: mTOR-regulated Mitochondrial Metabolism Limits Mycobacterium-induced Cytotoxicity

abstract: Necrosis of macrophages in the tuberculous granuloma represents a major pathogenic event in tuberculosis. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused specifically by ESAT-6, the principal secreted substrate of the specialized mycobacterial secretion system ESX-1, a key virulence mediator. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis, albeit humanitys most lethal pathogen, is successful in only a minority of infected individuals.


title: An Acyclic Phosphonate Prodrug of HPMPC is Effective Against VZV in Skin Organ Culture and Mice

abstract: Varicella zoster virus (VZV) causes chicken pox and shingles and is prevalent worldwide. Acyclovir and penciclovir (and its prodrugs) are first-line treatments for VZV infections, but they are not highly potent against VZV and resistance may arise in immunocompromised people on long-term therapy. HPMPC (cidofovir) is active against VZV, but cidofovir is not approved for treating VZV diseases, is nephrotoxic, and is not orally bioavailable. Here, we present the synthesis and evaluation of USC-373, a phosphonate prodrug of HPMPC with activity against VZV and other DNA viruses. In cultured fibroblasts, it was potent against VZV Ellen laboratory strain and was not overtly toxic, with EC50 of 4 nM and CC50 of 0.20 M, producing a selectivity index of 50. In ARPE-19 cells, USC-373 was effective against VZV-ORF57-Luc wild type strain and the acyclovir-resistant isogenic strain. In human skin organ culture, USC-373 formulated in cocoa butter and applied topically prevented VZV-ORF57-Luc spread without toxicity. In NuSkin mice with human skin xenografts, one daily dose of 3 mg/kg was effective by the subcutaneous route, and one daily dose of 10 mg/kg was effective by the oral route. Remarkably, a 10 mg/kg oral dose given every other day was also effective. USC-373 was well tolerated and mice did not lose weight or show signs of distress. The prodrug modifications of USC-373 increase the potency and oral bioavailability compared to its parent nucleoside analog, HPMPC.


title: Hedgehogs are the major amplifying hosts of severe fever with thrombocytopenia syndrome virus

abstract: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne bandavirus mainly transmitted by Haemaphysalis longicornis in East Asia, mostly in rural areas. To date, the amplifying host involved in the natural transmission of SFTSV remains unidentified. Our epidemiological field survey conducted in endemic areas in China showed that hedgehogs were widely distributed, had heavy tick infestations, and had high SFTSV seroprevalence and RNA prevalence. After experimental infection of Erinaceus amurensis and Atelerix albiventris hedgehogs with SFTSV, robust but transitory viremias were detected, which lasted for around nine to eleven days. The infected hedgehogs experienced light weight loss and histopathology of the spleen showed hemorrhagic necrosis and lymphopenia, with infected hedgehogs recovering after viral clearance. Remarkably, SFTSV transmission cycle between hedgehogs and nymph/adult H. longicornis was easily accomplished under laboratory condition with 100% efficiency. Furthermore, naive H. longicornis ticks could be infected by SFTSV-positive ticks co-feeding on naive hedgehogs, with transstadial transmission of SFTSV also confirmed. We also found that SFTSV viremia remained high in hedgehogs during hibernation, suggesting that this mechanism might contribute to the persistence of SFTSV from one year to the next. Of concern, we recently found evidence of the natural circulation of SFTSV in the urban area of Beijing City in China involving H. longicornis ticks and E. amurensis hedgehogs. Our study suggests that the hedgehogs are the major wildlife amplifying hosts of SFTSV and that urban outbreaks of SFTSV might occur in the future.


title: Meta-analysis reveals the global picture of antibiotic resistance gene prevalence across environments

abstract: The environment is an important component in the emergence and transmission of antimicrobial resistance (AMR). Despite that, current AMR monitoring initiatives lack comprehensive reference data for the vast majority of environments. To enable monitoring to detect deviations from the normal background resistance levels in the environment, it is necessary to establish the typical baseline of AMR in a variety of settings. In an attempt to establish this baseline level, we here performed a comprehensive literature survey across 2516 scientific papers, 150 of which contained relevant qPCR data on antibiotic resistance genes (ARGs) in environments associated with potential routes of AMR dissemination. The collected data include 1487 samples distributed across 30 different countries and 17 environmental types, in a time span from 2005 to 2018. More than 330 different genes were identified from the collected studies. Most surveyed environments contained a diverse set of ARGs, but generally at low abundances. We used linear mixed models and overrepresentation analysis to identify time trends and associations between ARGs and specific environments. Altogether these data represent a comprehensive overview of the occurrence and levels of ARGs in different environments, providing background data for risk assessment models within current and future AMR monitoring frameworks.


title: Liver Protein Expression in NASH Mice on a High-Fat Diet

abstract: Male MS-NASH mice were maintained on a high-fat diet for 16 weeks with and without red algae-derived minerals. Obeticholic acid (OCA) was used as a comparator in the same strain and diet. C57BL/6 mice maintained on a standard (low-fat) rodent chow diet were used as a control. At the end of the in-life portion of the study, body weight, liver weight, liver enzyme levels and liver histology were assessed. Samples obtained from individual livers were subjected to Tandem Mass Tag labeling / mass spectroscopy for protein profile determination. As compared to mice maintained on the low-fat diet, all high-fat-fed mice had increased whole body and liver weight, increased liver enzyme (aminotransferases) levels and widespread steatosis / ballooning hepatocyte degeneration. Histological evidence for liver inflammation and collagen deposition was also present, but changes were to a lesser extent. A moderate reduction in ballooning degeneration and collagen deposition was observed with mineral supplementation. Control mice on the high-fat diet alone demonstrated multiple protein changes associated with dysregulated fat and carbohydrate metabolism, lipotoxicity and oxidative stress. Cholesterol metabolism and bile acid formation were especially sensitive to diet. In mice receiving multi-mineral supplementation along with the high-fat diet, there was reduced liver toxicity as evidenced by a decrease in levels of several cytochrome P450 enzymes and other oxidantgenerating moieties. Additionally, elevated expression of several keratins was also detected in mineral-supplemented mice. The protein changes observed with mineral supplementation were not seen with OCA. Our previous studies have shown that mice maintained on a high-fat diet for up to 18 months develop end-stage liver injury including hepatocellular carcinoma. Mineral-supplemented mice were substantially protected against tumor formation and other end-state consequences of high-fat feeding. The present study identifies early (16-week) protein changes occurring in the livers of the high-fat diet-fed mice, and how the expression of these proteins is influenced by mineral supplementation. These findings help elucidate early protein changes that contribute to end-stage liver injury and potential mechanisms by which dietary minerals may mitigate such damage.

category: molecular biology


title: Aberrant mechanosensitive signaling underlies activation of vascular endothelial xanthine oxidoreductase that promotes aortic aneurysm formation in Marfan syndrome

abstract: Marfan syndrome (MFS) is an inherited connective tissue disorder caused by mutations in the FBN1 gene encoding fibrillin-1, a matrix component of extracellular microfibrils. The main cause of morbidity and mortality in MFS is thoracic aortic aneurysm and dissection, but the underlying mechanisms remain undetermined. We found a significant increase in reactive oxygen species (ROS) generation in ascending aorta of MFS patients and MFS mice harboring the Fbn1 mutation (C1039G), which was associated with up-regulation of xanthine oxidoreductase (XOR) protein in aortic endothelial cells (ECs). Mechanosensitive signaling involving focal adhesion kinase (FAK)-p38 mitogen-activated protein kinase (MAPK) and early growth response-1 (Egr- 1) was aberrantly activated in ascending aorta of Fbn1C1039G/+ mice, and mechanical stress on human aortic ECs up-regulated XOR expression through FAK-p38 MAPK activation and Egr-1 up-regulation. Inhibition of XOR function by ECs-specific disruption of Xdh gene or by systemic administration of XOR inhibitor febuxostat in Fbn1C1039G/+ mice suppressed ROS generation, FAK-p38 MAPK activation, and Egr-1 up-regulation, leading to attenuation of aortic aneurysm formation. These findings unveil aberrant mechanosensitive signaling in vascular ECs triggering endothelial XOR activation and ROS generation as a culprit underlying the pathogenesis of aortic aneurysm formation in MFS, and highlight a drug repositioning approach using a uric acid lowering drug febuxostat as a potential therapy for MFS.


title: LKB1 acts as a critical brake for the glucagon-mediated fasting response

abstract: As important as the fasting response is for survival, an inability to shut it down once nutrients become available can lead to exacerbated disease and severe wasting. The liver is central to transitions between feeding and fasting states, with glucagon being a key initiator of the hepatic fasting response. However, the precise mechanisms controlling fasting are not well defined. One potential mediator of these transitions is Liver Kinase B1 (LKB1) given its role in nutrient sensing. Here, we show LKB1 knockout mice have a severe wasting and prolonged fasting phenotype despite increased food intake. By applying RNA sequencing and intravital microscopy we show that loss of LKB1 leads to a dramatic reprogramming of the hepatic lobule through robust upregulation of periportal genes and functions. This is likely mediated through the opposing effect LKB1 has on glucagon pathways and gene expression. Conclusion: our findings show that LKB1 acts as a brake to the glucagon-mediated fasting response resulting in "periportalization" of the hepatic lobule and whole-body metabolic inefficiency. These findings reveal a new mechanism by which hepatic metabolic compartmentalization is regulated by nutrient-sensing.


title: The grapevine leaves that are (or were) not: constraints on leaf development and choosing to see what seemingly is not

abstract: The shapes of leaves have been extensively measured and modeled. Using conventional statistical approaches there exist powerful methods to discern between groups and classify shapes. Embedded in calculated morphospaces is information that allows us to visualize theoretical leaves. These unmeasured leaves, that may or may not exist, are never considered in their relation to empirically observable leaves nor how the negative morphospace can inform us about the forces responsible for shaping leaf morphology. Using landmark data for 8465 grapevine leaves, we model leaf shape using an allometric indicator of leaf size, the ratio of vein to blade areas. The borders of the observable morphospace are restricted by the constraint of the subregions of the leaf reducing to nothing. Assuming that self-intersecting polygonal regions in leaves are a developmental constraint, we define a region of morphospace that represents leaf shapes that are possible but not observed. We end with a discussion of how we choose not to observe what seemingly does not exist and the ramifications of this perspective to survivorship bias in the sciences in general.


title: Multiphasic hepatitis B virus kinetic patterns in humanized chimeric mice can be explained via stochastic agent-based modeling of intracellular virion production cycles

abstract: Serum hepatitis B virus (HBV) kinetics in urokinase-type plasminogen activator/severe combined immunodeficient (uPA-SCID) mice reconstituted with humanized livers from inoculation to steady state is highly dynamic despite the absence of an adaptive immune response. We developed a stochastic agent-based model that includes virion production cycles in individual infected human hepatocytes. The model was calibrated using a genetic algorithm approach with the serum HBV kinetics observed in mice inoculated with 108 HBV genome equivalents and fit the data well when the following viral production parameters were assumed: (1) An eclipse phase lasting 5-50 hours and (2) a post-eclipse phase production rate that is based on increasing production cycles initially starting with a long production cycle of 1 virion per 20 hours that gradually reaches 1 virion per hour after approximately 3-4 days before virion production increases dramatically to reach to a steady state production rate of 4 virions per hour per cell. The model was then validated by showing it could accurately simulate the viral kinetics observed with lower HBV inoculation doses (104-107 genome equivalents) in which similar, but delayed patterns were observed. Together, modeling suggests that it is the cyclic nature of the virus lifecycle combined with an initial slow but increasing rate of HBV production from each cell that plays a role in generating the observed multiphasic HBV kinetic patterns in humanized mice.

category: systems biology


title: ScRNA-seq Expression of APOC2 and IFI27 Identifies Four Families of Alveolar Macrophage Superclusters in Cystic Fibrosis and Healthy BALF

abstract: Alveolar macrophages (AMs) reside on the luminal surface of the airways and alveoli, ensuring proper gas exchange by ingesting cellular debris and pathogens, and regulating inflammatory responses. Therefore, understanding the heterogeneity and diverse roles played by AMs and recruited monocytes is critical for treating airway diseases. We performed single-cell RNA sequencing on 113,213 bronchoalveolar lavage cells from four healthy and three uninflamed cystic fibrosis subjects and identified four AM superclusters (families) based on the expression of IFI27 and APOC2 genes. These 4 AM families have at least eight distinct functional members (subclusters) named after their differentially expressed gene(s): IGF1, CCL18, CXCL5, Cholesterol, Chemokine, Metallothionein, Interferon and small-cluster AMs. Interestingly, the Chemokine cluster further divides with each subcluster selectively expressing a unique combination of chemokines. One of the most striking observations, besides the heterogeneity, is the conservation of AM family members in relatively equal ratio across all AM superclusters and individuals. Finally, transcriptional data and TotalSeq technology were used to investigate cell surface markers that distinguish resident AMs from recruited monocytes. Overall, functional specializations of the AM subclusters suggest that there are AM niches with defined programming states, highlighting a clear division of labor.


title: ScRNA-seq Expression of IFI27 and APOC2 Identifies Four Alveolar Macrophage Superclusters in Healthy BALF


title: Pinpointing the tumor-specific T-cells via TCR clusters

abstract: Adoptive T cell transfer (ACT) is a promising approach to cancer immunotherapy, but its efficiency fundamentally depends on the extent of tumor-specific T-cell enrichment within the graft. This can be estimated via activation with identifiable neoantigens, tumor-associated antigens (TAAs), or living or lyzed tumor cells, but these approaches remain laborious, time-consuming, and functionally limited, hampering clinical development of ACT. Here, we demonstrate that homology cluster analysis of T cell receptor (TCR) repertoires efficiently identifies tumor-reactive TCRs allowing to: 1) detect their presence within the pool of tumor-infiltrating lymphocytes (TILs); 2) optimize TIL culturing conditions, with IL-2low/IL-21/anti-PD-1 combination showing increased efficiency; 3) investigate surface marker-based enrichment for tumor-targeting T cells in freshly-isolated TILs (enrichment confirmed for CD4+ and CD8+ PD-1+/CD39+ subsets), or re-stimulated TILs (informs on enrichment in 4-1BB-sorted cells). We believe that this approach to the rapid assessment of tumor-specific TCR enrichment should accelerate T cell therapy development.


title: A flexible network of lipid droplet associated proteins support embryonic integrity of C. elegans

abstract: In addition to coordinating the storage and mobilization of neutral fat, lipid droplets (LDs) are conserved organelles that can accommodate additional cargos in order to support animal development. However, it is unclear if each type of cargo is matched with a specific subset of LDs. Is the identity of each subset rigidly maintained? Here, we report that SEIP-1/seipin defines a subset of oocyte LDs that are required for proper eggshell formation in C. elegans. Using a photoconvertible fluorescent protein-based imaging assay, we found that SEIP-1 positive LDs were selectively depleted after fertilization, coincident of the formation of a lipid-rich permeability barrier of the eggshell. Loss of SEIP-1 function caused impenetrant embryonic arrest, which could be suppressed by depleting a LD-structural protein PLIN-1/perilipin. The embryonic development of seip-1; plin-1 mutant in turn depended on another LD-associated protein, RAB-18/Rab18. Our results are consistent with the hypothesis that SEIP-1 dependent and independent mechanisms act redundantly to ensure the packaging and export of lipid-rich permeability barrier constituents via LDs. The identity of these LDs, as defined by their associated proteins, exhibits unexpected plasticity that ultimately ensures the survival of embryos ex utero.

category: cell biology


title: Condensation of a nuclear mRNA export factor regulates mRNA transport during stress

abstract:
Unidirectional mRNA transport from the nucleus to the cytoplasm via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this critical process and its regulation is lacking. Here, we use single RNA imaging to show that cells use selective mRNA retention to re-wire mRNA export during stress. We demonstrate that upon glucose withdrawal the essential export factor Nab2 forms RNA-dependent condensates in the nucleus blocking bulk mRNA export while selectively allowing the transport of stress-induced mRNAs. This is accompanied by reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore, and experimental Dbp5 depletion triggers Nab2 condensation and nuclear mRNA accumulation. Condensation can be recapitulated in vitro, with Nab2 forming RNA-dependent liquid droplets, which fail to form in the presence of active Dbp5. Our results suggest that cells use condensation to selectively regulate mRNA export and to control gene expression during stress.

HighlightsO_LIThe nuclear Poly(A)-binding protein Nab2 forms condensates upon acute cellular depletion of the DEAD-box ATPase Dbp5
C_LIO_LINab2 dimerization but not its intrinsically disordered regions (IDRs) are essential for condensation in vitro and in vivo
C_LIO_LIGlucose stress leads to global mRNA retention in the nucleus and depends on Nab2 condensation
C_LIO_LINab2 condensation confers selectivity of RNA retention during glucose stress
C_LIO_LIProper Nab2 condensation is essential for survival after prolonged stress
C_LI


title: A lymphatic-stem cell interactome regulates intestinal stem cell activity

abstract: Barrier epithelia depend on resident stem cells for homeostasis, defense and repair. Intestinal stem cells (ISCs) of the small and large intestines respond to their local microenvironments (niches) to fulfill a continuous demand for tissue turnover, yet the complexity of their niches is still unfolding. Here, we report an extensive lymphatic network that intimately associates with ISCs within these niches. Devising a lymphatic:organoid coculture system, we show that lymphatic-secreted factors maintain ISCs while inhibiting precocious differentiation. Employing a new deconvolution algorithm, BayesPrism, to pair single-cell and spatial transcriptomics, we cartograph the lymphatic ligand:ISC receptor interactomes at high resolution. We unearth crypt lymphatics as a major source of WNT-signaling factors (WNT2, R-SPONDIN-3) known to drive ISC behavior, and REELIN, a hitherto unappreciated ISC regulator secreted by crypt lymphatics. Together, our studies expose lymphatics as a central hub for niche factors that govern the regenerative potential of ISCs.


title: Thyroid hormones deficiency impairs male germ cell development: a cross talk between hypothalamic-pituitary-thyroid, and - gonadal axes in zebrafish

abstract: In vertebrates, thyroid hormones, including thyroxine (T4) and triiodothyronine (T3), are critical players in controlling different physiological processes such as development, growth, metabolism among others. There is evidence in mammals that thyroid hormones are also an important component of the hormonal system that controls reproduction, although studies in fish remain poorly investigated. Here we tested this hypothesis by investigating the effects of methimazole-induced hypothyroidism on the testicular function in adult D. rerio. Treatment of fish with methimazole, in vivo, significantly affected the progression of zebrafish spermatogenesis by inducing the accumulation of pre-meiotic cells, delaying cell differentiation and meiosis, as well as reducing the number of spermatozoa. The observed impairment of spermatogenesis by methimazole was correlated with significant changes in transcript levels for several genes involved in the control of reproduction. Using an in vitro approach, we also demonstrated that in addition to affecting the components of the brain-pituitary-peripheral axis, T3 also exerts direct action at the level of the testis. These results support the hypothesis that thyroid hormones are an essential component of multifactorial control of reproduction and testicular function in zebrafish and possibly other vertebrates.


title: Targeted degradation of PCNA outperforms stoichiometric inhibition to result in programed cell death

abstract: Targeted protein degradation has emerged as a powerful technology - both as a biological tool and for broadening the therapeutic proteome. As tools to probe this approach on historically intractable targets, we have previously advanced  biodegraders -- targeted degradation fusion constructs composed of mini-proteins/peptides linked to modified E3 ligase receptors. Herein, we gain deeper insights into the utility and potential of biodegraders, through a detailed study on Con1-SPOP, a biodegrader which rapidly degrades the potential cancer target, proliferating cell nuclear antigen (PCNA). In a variety of settings, the active biodegrader (Con1-SPOP) proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut). Specifically, in addition to more potent anti-proliferative effects in both 2D cell culture and 3D spheroids, PCNA degradation uniquely induced DNA damage, cell apoptosis and necrosis. Global proteomic profiling of a stable cell-line expressing Con1-SPOP under doxycycline (Dox) induction revealed that impaired mitotic division and mitochondria dysfunction is a direct consequence of PCNA degradation, effects not seen with the stoichiometric inhibitor protein. To evaluate the therapeutic potential of biodegraders, we showed that Dox-induced Con1-SPOP achieved complete tumor-growth inhibition in a xenograft model. To explore application of biodegraders as a novel therapeutic modality, modified mRNA encoding Con1-SPOP was synthesized and encapsulated into lipid nanoparticles (LNPs). The approach successfully delivered mRNA in vitro to deplete endogenous PCNA within hours of application and with nanomolar potency. Overall, our results demonstrate the utility of biodegraders as biological tools and highlight target-degradation as a more efficacious approach versus stoichiometric inhibition. Finally, once in vivo delivery and expression are optimized, biodegraders may be leveraged as an exciting therapeutic modality.


title: Single-cell genome-wide association reveals a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus

abstract: Diversity in the human genome is one factor that confers resistance and susceptibility to infectious diseases. This is observed most dramatically during pandemics, where individuals exhibit large differences in risk and clinical outcomes against a pathogen infecting large portions of the worlds populations. Here, we developed scHi-HOST (single cell High-throughput Human in vitrO Susceptibility Testing), a method for rapidly identifying genetic variants that confer resistance and susceptibility to pathogens. scHi-HOST leverages scRNA-seq (single-cell RNA-sequencing) to simultaneously assign genetic identity to individual cells in mixed infections of cell lines of European, African, and Asian origin, reveal associated genetic variants for viral entry and replication, and identify expression quantitative trait loci (eQTLs). Applying scHi-HOST to influenza A virus (IAV), we identified eQTLs at baseline and in genes that are induced by IAV infection. Integration of scHi-HOST with a human IAV challenge study (Prometheus) revealed that a missense variant in ERAP1 (Endoplasmic reticulum aminopeptidase 1; rs27895) was associated with IAV burden in cells and human volunteers. Functional studies using RNA interference, ERAP1 inhibitor, and overexpression of alternative alleles demonstrated that ERAP1 is exploited by IAV to promote infection. Specifically, the nonsynonymous substitution, which results in a glycine to aspartate substitution at ERAP1 residue 348, would disrupt the substrate binding pocket of ERAP1, likely resulting in a significantly altered preference for substrates, poorer catalytic efficiency, or both. Finally, rs27895 exhibits substantial population differentiation, with the higher frequency of the minor T allele in two African populations likely contributing to the greater permissivity of cells from these populations to IAV infection. scHi-HOST is an important resource for understanding susceptibility to influenza and is a broadly applicable method for decoding human genetics of infectious disease.


title: Spatial and Temporal Regulation of Parent-of-Origin Allelic Expression in the Endosperm

abstract: Genomic imprinting promotes differential expression of parental alleles in the endosperm of flowering plants, and is regulated by epigenetic modification such as DNA methylation and histone tail modifications in chromatin. After fertilization, the endosperm develops through a syncytial stage before it cellularizes and becomes a nutrient source for the growing embryo. Both in early and late endosperm development regional compartmentalization has been shown, and different transcriptional domains suggest divergent spatial and temporal regional functions. The analysis of the role of parent-of-origin allelic expression in the endosperm as a whole and also investigation of domain specific functions has been hampered by the availability of the tissue for high-throughput transcriptome analyses and contamination from surrounding tissue. Here we have used Fluorescence-Activated Nuclear Sorting (FANS) of nuclear targeted eGFP fluorescent genetic markers to capture parental specific allelic expression from different developmental stages and specific endosperm domains. This RNASeq approach allows us to successfully identify differential genomic imprinting with temporal and spatial resolution. In a systematic approach we report temporal regulation of imprinted genes in the endosperm as well as region specific imprinting in endosperm domains. Our data identifies loci that are spatially differentially imprinted in one domain of the endosperm while biparentally expressed in other domains. This suggests that regulation of genomic imprinting is dynamic and challenges the canonical mechanisms for genomic imprinting.


title: Group dynamics and habitat use of the Giant Otter, Pteronura brasiliensis (Zimmermann, 1780), in seasonally flooded forest in the Araguaia River, Central Brazil: A 10-years study.

abstract: We carried out monthly surveys of the giant otter population between 2010 and 2020 in a study area comprised of 1,500 hectares of igapo flooded forest with oxbow lakes in the Cantao region of central Brazil. We recorded 16-32 resident adults in the study area each year, distributed in 4-8 groups. Resident groups exhibited extensive home range overlap, with each group using several lakes and larger lakes used in rotation by up to six groups. Dens and campsites were also shared by multiple groups, but lakes were used by only one group at a time, and encounters between groups were very rare. 24 adult otters were observed to join an existing group. Some individuals changed groups multiple times. Resident adult turnover was high. Each year an average of 36% of resident adults were new immigrants, and 72% of groups left the area within two years. Resident groups had, on average, one litter every three years, and annual cub production showed high variability and a negative correlation to the number of new immigrants in the area. No pairs of giant otters reproduced successfully during the study. Groups of three otters formed through the recruitment of an adult individual by an existing pair and reproduced as successfully as larger groups. Group dynamics and territorial behavior in the Cantao flooded forest ecosystem, where optimal giant otter habitat is continuous in all directions, were found to be different from that reported in areas composed of patchy (isolated oxbow lakes) or linear (rivers) habitat. This suggest that giant otter social and territorial behavior is plastic and adapts to the spatial characteristics of the habitat.

category: animal behavior and cognition


title: Establishment of physiologically relevant oxygen gradients in microfluidic organ chips

abstract: In vitro models of human organs must accurately reconstitute oxygen concentrations and gradients that are observed in vivo to mimic gene expression, metabolism, and host-microbiome interactions. Here we describe a simple strategy to achieve physiologically relevant oxygen tension in a two-channel human small intestine-on-a-chip (Intestine Chip) lined with primary human duodenal epithelium and intestinal microvascular endothelium in parallel channels separated by a porous membrane while both channels are perfused with oxygenated medium. This strategy was developed using computer simulations that predicted lowering the oxygen permeability of poly-dimethlysiloxane (PDMS) chips in specified locations using a gas impermeable film will allow the cells to naturally decrease the oxygen concentration through aerobic respiration and reach steady-state oxygen levels < 36 mm Hg (< 5%) within the epithelial lumen. The approach was experimentally confirmed using chips with embedded oxygen sensors that maintained this stable oxygen gradient. Furthermore, Intestine Chips cultured with this approach supported formation of a villus epithelium interfaced with a continuous endothelium and maintained intestinal barrier integrity for 72 h. This strategy recapitulates in vivo functionality in an efficient, inexpensive, and scalable format that improves the robustness and translatability of Organ Chip technology for studies on microbiome as well as oxygen sensitivity.


title: Extracellular Matrix Levels Modulate Outgrowths Dynamics in Ovarian Cancer.

abstract: Ovarian carcinoma (OC) form outgrowths that extend from the outer surface of an afflicted organ into the peritoneum. OC outgrowth formation is poorly understood because there is a limited availability of OC cell culture models to examine the behavior of cell assemblies that form outgrowths. Prompted by immuno-chemical evaluation of extracellular matrix (ECM) components, laminin {gamma}1 and collagens, in human tissues representing untreated and chemotherapy-recovered OC, we developed laminin and collagen-rich ECM-reconstituted cell culture models amenable to studies of cell assemblies that can form outgrowths. We demonstrate that ECM promotes outgrowth formation in fallopian tube non-ciliated epithelial cells (FNE) expressing mutant p53R175H and various OC cell lines. Outgrowths were initiated by cell assemblies that had undergone outward translocation and, upon mechanical detachment, could intercalate into mesothelial cell monolayers. Electron microcopy, optical coherence tomography (OCT) and small-amplitude oscillatory shear experiments revealed that elevating ECM levels increased ECM fibrous network thickness and led to high shear elasticity of ECM environment. These physical characteristics were associated with suppression of outgrowths. Culture environment with low ECM content mimicked viscoelasticity and fibrous networks of ascites and supported cell proliferation, cell translocation and outgrowth formation. These results highlight the importance of ECM microenvironments in modulating OC growth and could provide additional explanation of why primary and recurrent ovarian tumors form outgrowths that protrude into the peritoneal cavity containing ascites.

category: cancer biology


title: Cannabinoid receptor 1 is required for neurodevelopment of striosome-dendron bouquets

abstract: Cannabinoid receptor 1 (CB1R) has strong effects on neurogenesis and axon pathfinding in the prenatal brain. Endocannabinoids that activate CB1R are abundant in the early postnatal brain and in mothers milk, but few studies have investigated their function in newborns. We examined postnatal CB1R expression in the major striatonigral circuit from striosomes of the striatum to the dopamine-containing neurons of the substantia nigra. CB1R enrichment was first detectable between postnatal days 5 and 7, and this timing coincided with the formation of  striosome-dendron bouquets, the elaborate anatomical structures by which striosomal neurons control dopaminergic cell activity through inhibitory synapses. In Cnr1-/- knockouts lacking CB1R expression, striosome-dendron bouquets were markedly disorganized by postnatal day 11 and at adulthood, suggesting a postnatal pathfinding connectivity function for CB1R in connecting striosomal axons and dopaminergic neurons analogous to CB1Rs prenatal function in other brain regions. Our finding that CB1R plays a major role in postnatal wiring of the striatonigral dopamine system, with lasting consequences at least in mice, points to a crucial need to determine whether lactating mothers use of CB1R agonists (e.g., in marijuana) or antagonists (e.g., type 2 diabetes therapies) can disrupt brain development in nursing offspring.


title: Neuronal membrane proteasomes homeostatically regulate neural circuit activity in vivo and are required for learning-induced behavioral plasticity

abstract: Protein degradation is critical for brain function through processes that remain poorly understood. Here we investigated the in vivo function of a recently reported neuronal membrane-associated proteasome (NMP) in the brain of Xenopus laevis tadpoles. We demonstrated that NMPs are present in the tadpole brain with biochemistry and electron microscopy, and showed that they actively degrade neuronal activity-induced nascent proteins. Using in vivo calcium imaging in the optic tectum, we showed that acute inhibition of NMP function rapidly increased spontaneous neuronal activity, resulting in hyper-synchronization among tectal neurons. At the circuit level, inhibiting NMPs abolished learning-dependent improvement in a visuomotor behavior paradigm in live animals. Our data provide the first in vivo characterization of NMP functions in the vertebrate nervous system and suggest that NMP-mediated degradation of activity-induced nascent proteins may serve as a homeostatic modulatory mechanism in neurons that is critical for regulating neuronal activity and experience-dependent circuit plasticity.


title: Evolution of neural activity in circuits bridging sensory and abstract knowledge

abstract: The ability to associate sensory stimuli with abstract classes is critical for survival. How are these associations implemented in brain circuits? And what governs how neural activity evolves during abstract knowledge acquisition? To investigate these questions, we consider a circuit model that learns to map sensory input into abstract classes via gradient descent synaptic plasticity. We focus on typical neuroscience tasks (simple, and context-dependent, categorization), and study how both synaptic connectivity and neural activity evolve during learning. To make contact with the current generation of experiments we analyze activity via standard measures such as selectivity, correlations, and tuning symmetry. We find that the model is able to recapitulate experimental observations, including seemingly disparate ones. We determine how, in the model, the behaviour of these measures depends on details of the circuit and the task. These dependencies make experimentally-testable predictions about the circuitry supporting abstract knowledge acquisition in the brain.


title: ACCURATE DECODING OF THE SPINAL CORD OUTPUT IN HUMANS WITH IMPLANTED HIGH-DENSITY ELECTRODE ARRAYS

abstract: Invasive electromyography opened a new window to explore motoneuron behaviour in vivo. However, the technique is limited by the small fraction of active motoneurons that can be concurrently detected, precluding a population analysis in natural tasks. Here, we developed a high-density intramuscular electrode for in vivo human recordings along with a fully automatic methodology that could detect the discharges of action potentials of up to 67 concurrently active motoneurons with 99% accuracy. These data revealed that motoneurons of the same pool receive common synaptic input at frequencies up to 75 Hz and that late recruited motoneurons inhibit the discharges of those recruited earlier. These results constitute an important step in the population coding analysis of the human motor system in vivo.


title: Non-identifiability and the Blessings of Misspecification in Models of Molecular Fitness and Phylogeny

abstract: Understanding the consequences of mutation for molecular fitness and function is a fundamental problem in biology. Recently, generative probabilistic models have emerged as a powerful tool for estimating fitness from evolutionary sequence data, with accuracy sufficient to predict both laboratory measurements of function and disease risk in humans, and to design novel functional proteins. Existing techniques rest on an assumed relationship between density estimation and fitness estimation, a relationship that we interrogate in this article. We prove that fitness is not identifiable from observational sequence data alone, placing fundamental limits on our ability to disentangle fitness landscapes from phylogenetic history. We show on real datasets that perfect density estimation in the limit of infinite data would, with high confidence, result in poor fitness estimation; current models perform accurate fitness estimation because of, not despite, misspecification. Our results challenge the conventional wisdom that bigger models trained on bigger datasets will inevitably lead to better fitness estimation, and suggest novel estimation strategies going forward.

category: evolutionary biology


title: Forced proximity promotes the formation of enduring cooperative relationships in vampire bats

abstract: Spatial assortment can be both a cause and consequence of cooperation. Proximity promotes cooperation when individuals preferentially help nearby partners, and conversely, cooperation drives proximity when individuals move towards more cooperative partners. However, these two causal directions are difficult to distinguish with observational data. Here, we experimentally test if forcing randomly selected pairs of equally familiar female common vampire bats (Desmodus rotundus) into close spatial proximity promotes the formation of enduring cooperative relationships. Over 114 days, we sampled 682 hours of interactions among 21 females captured from three distant sites to track daily allogrooming rates over time. We compared these rates before, during, and after a one-week period, during which we caged random triads of previously unfamiliar and unrelated vampire bats in close proximity. After the week of close proximity when all bats could again freely associate, the allogrooming rates of pairs forced into proximity increased more than those of the 126 control pairs. This work is the first to experimentally demonstrate the causal effect of repeated interactions on cooperative investments in vampire bats. Future work should determine the relative importance of mere association versus interactions (e.g. reciprocal allogrooming) in shaping social preferences.


title: Reversal Learning Phenotypes are Linked with Novel Genetic Loci in Diversity Outbred Mice

abstract: Impulsive behavior and impulsivity are heritable phenotypes that are strongly associated with risk for substance use disorders in human subjects. Consequently, identifying the neurogenetic mechanisms that influence impulsivity may also reveal novel biological insights into addiction vulnerability. Past studies from our laboratory using the BXD and Collaborative Cross (CC) recombinant inbred mouse panels have revealed that behavioral indicators of impulsivity measured in a reversal learning task are heritable and are genetically correlated with aspects of intravenous cocaine self-administration. Genome wide linkage studies in the BXD panel revealed a quantitative trait locus (QTL) on chromosome 10, but the specific genes affecting this trait remain elusive. To achieve greater precision in our mapping efforts, we have turned to Diversity Outbred (DO) mice. A total of 392 DO mice (230 males, 295 females) were successfully phenotyped using the same reversal learning test utilized in our earlier studies. Our primary indicator of impulsive responding, a measure that isolates the relative difficulty mice have with reaching performance criteria under reversal conditions, revealed a genome wide significant QTL on chromosome 7 (max LOD score = 8.73, p<0.05). A measure of premature responding akin to that implemented in the 5-choice serial reaction time task yielded a suggestive QTL on chromosome 17 (max LOD score = 9.14, p<0.1). Positional candidate genes were prioritized (2900076A07Rik, Wdr73 and Zscan2) based upon expression QTL data we collected in DO and CC mice and analyses using publicly available gene expression and phenotype databases. These findings may advance understanding of the genetics that drive impulsive behavior and enhance risk for substance use disorders.


title: MUCOSECRETORY LUNG DISEASE: DIFFERENT ASSEMBLIES OF JAG1 AND JAG2 DETERMINE TRACHEOBRONCHIAL CELL FATE

abstract:
Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. These cell types are derived from tracheobronchial stem/progenitor cells via a Notch dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and Jagged2 (JAG2) is unclear. This study used primary human bronchial air-liquid- interface cultures, gamma secretase inhibition, and neutralizing antibodies to show: 1) JAG1 and JAG2 were necessary for secretory progenitor cell fate determination; 2) JAG2 suppressed squamous differentiation; and 3) pausing of the ciliated cell differentiation process after Notch inhibition. Histological, cell fractionation, cell surface biotinylation, and ubiquitination analyses demonstrated that all cells were JAG1 positive but that little JAG1 was present on the cell surface. In contrast, JAG2 was expressed in a positive-negative pattern and was abundant on the cell surface. Glycogen synthase kinase 3 (GSK3) and tankyrase inhibition studies showed that GSK3 regulated JAG2 trafficking, and that this mechanism was WNT-independent. Collectively, these data indicate that variation in JAG2 trafficking creates regions of high, medium, and low ligand expression. Thus, distinct assemblies of JAG1 and JAG2 may regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/478334v2_ufig1.gif" ALT="Figure 1">
View larger version (15K):
org.highwire.dtl.DTLVardef@37bb51org.highwire.dtl.DTLVardef@eaeaf5org.highwire.dtl.DTLVardef@e791dcorg.highwire.dtl.DTLVardef@1193d73_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Different assemblies of JAG1 and JAG2 may determine Notch signal strength and cell fate within the tracheobronchial epithelium. A cell which interacts with JAG1+ cells (blue squares) receives a low Notch signal (light yellow square). A cell which interacts with a mixture of JAG1+ and JAG1+/JAG2+ cells (purple squares) receives a medium (med) Notch signal (medium yellow square). A cell which interacts with JAG1+/JAG2+ cells receives a high Notch signal (bright yellow square).

C_FIG


title: NCOA4 drives ferritin phase separation to facilitate macroferritinophagy and endosomal microferritinophagy

abstract: A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to lysosomes to release iron ions. Two transport pathways have been reported: macroautophagy and ESCRT-dependent endosomal microautophagy. Although the membrane dynamics of these pathways differ, both require NCOA4, which is thought to be an autophagy receptor for ferritin. However, the exact function of NCOA4 remains elusive. Here, we found that ferritin particles form liquid-like condensates in a NCOA4-dependent manner. Homodimerization of NCOA4 and interaction between FTH1 and NCOA4 (i.e., multivalent interactions between ferritin particles and NCOA4) were required for the formation of ferritin condensates. Disruption of these interactions impaired ferritin degradation. Time-lapse imaging and three-dimensional correlative light and electron microscopy revealed that these ferritin-NCOA4 condensates were directly engulfed by autophagosomes and endosomes. In contrast, TAX1BP1 was not required for the formation of ferritin-NCOA4 condensates but was required for their incorporation into autophagosomes and endosomes. These results suggest that NCOA4 acts not only as a canonical autophagy receptor but also as a driver to form ferritin condensates to facilitate the degradation of these condensates by macroautophagy (i.e., macroferritinophagy) and endosomal microautophagy (i.e., microferritinophagy).


title: Matrix topology guides collective cell migration in vivo

abstract:
Diverse modes of cell migration shape organisms in health and disease and much research has focused on the role of intracellular and extracellular components in different cell migration phenomena. What is less explored, however, is how the arrangement of the underlying extracellular matrix that many cells move upon in vivo influences migration.

Combining novel transgenic lines and image analysis pipelines, reveals that during zebrafish optic cup formation cells use cryptopodia-like protrusions to migrate collectively and actively over a topologically changing matrix. These changing topologies correspond to different cell-matrix interactions. Interference with matrix topology results in loss of cryptopodia and inefficient migration. Thus, matrix topology influences the efficiency of directed collective cell migration during eye morphogenesis, a concept likely conserved in other developmental and disease contexts.

One-Sentence SummaryDynamic cell-matrix interactions, crucial for successful collective rim cell migration, rely on extracellular matrix topologies during optic cup development in vivo.

category: developmental biology


title: RNA induces unique tau strains and stabilizes Alzheimer disease seeds

abstract:
Tau aggregation causes neurodegenerative tauopathies, and trans-cellular propagation of tau assemblies of unique structure, i.e. strains, may underlie the diversity of these disorders. Polyanions have been reported to induce tau aggregation in vitro, but the precise trigger to convert tau from an inert to a seed-competent form in disease states is unknown. RNA triggers tau fibril formation in vitro and has been observed in association with neurofibrillary tangles in human brain. We tested whether RNA exerts sequence-specific effects on tau assembly and strain formation. Three RNA homopolymers, polyA, polyU, and polyC all bound tau, but only polyA triggered seed and fibril formation.

PolyA:tau seeds and fibrils were sensitive to RNase. The origin of RNA influenced the ability of tau to adopt a structure that would form stable strains. Human RNA potently induced tau seed formation and created tau conformations that preferentially formed stable strains in a HEK293T cell model, whereas other inducers produced strains that sectored. Finally, we found that soluble, but not insoluble, seeds from Alzheimers disease (AD) brain were sensitive to RNase. Thus, RNA specifically induces stable tau strains, and may trigger the formation of dominant pathological assemblies that propagate in AD, and possibly other tauopathies.

category: biochemistry


title: A Mesenchymal Tumor Cell State Confers Increased Dependency on the BCL-XL Anti-apoptotic Protein in Kidney Cancer

abstract:
Genome-wide genetic screens have identified cellular dependencies in many cancers. Using the Broad Institutes Achilles shRNA screening dataset, we mined for targetable dependencies by cell lineage. Our studies identified a strong dependency on BCL2L1, which encodes the BCL-XL anti-apoptotic protein, in a subset of kidney cancer cells. Genetic and pharmacological inactivation of BCL-XL, but not the related anti-apoptotic proteins BCL-2, led to fitness defects in renal cancer cells, and also sensitized them to chemotherapeutics. Neither BCL-XL levels (absolute or normalized to BCL-2) nor the status of the VHL gene, which is frequently mutated in kidney cancer, predicted BCL-XL dependence. Transcriptional profiling, however, identified a  BCL-XL dependency mRNA signature, which included elevated mesenchymal gene expression in BCL-XL dependent cells. Promoting mesenchymal transition increased BCL-XL dependence; whereas, conversion to a more differentiated state overcame BCL-XL dependence in kidney cancer cells. The  BCL-XL dependency mRNA signature was observed in almost a third of human clear cell Renal Cell Carcinomas (ccRCCs), which were also associated with worse clinical outcomes. Finally, an orally bioavailable BCL-XL inhibitor, A-1331852, showed anti-tumor efficacy in vivo. Altogether, our studies uncovered an unexpected link between cancer cell state and dependence on the anti-apoptotic BCL-XL protein and justify further testing on BCL-XL blockade as a potential way to target a clinically aggressive subset of human kidney cancers.

One Sentence SummaryCell state, but not pVHL and/or HIF status, defines the dependency of kidney cancer cells on the BCL-XL anti-apoptotic protein.


title: Dynamic human liver proteome atlas reveals functional insights into disease pathways

abstract:
Deeper understanding of liver pathophysiology would benefit from a comprehensive quantitative proteome resource at cell-type resolution to predict outcome and design therapy. Here, we quantify more than 150,000 sequence-unique peptides aggregated into 10,000 proteins across total liver, the major liver cell types, time-course of primary cell cultures and liver disease states. Bioinformatic analysis reveals that half of hepatocyte protein mass is comprised of enzymes and 23% of mitochondrial proteins, twice the proportion of other liver cell types. Using primary cell cultures, we capture dynamic proteome remodeling from tissue states to cell line states, providing useful information for biological or pharmaceutical research. Our extensive data serves as spectral library to characterize a human cohort of non-alcoholic steatohepatitis and cirrhosis. Dramatic proteome changes in liver tissue include signatures of stellate cell activation resembling liver cirrhosis and providing functional insights. We built a web-based dashboard application for the interactively exploration of our resource.

HighlightsO_LICell-type resolved liver proteome with copy numbers for 10,500 proteins
C_LIO_LITime-course of human liver primary cells uncovers functional proteome shifts
C_LIO_LIA human cohort study reveals liver proteome changes in NASH and cirrhosis
C_LIO_LIAn interactive web portal integrates the results for easy exploration
C_LI


title: Rhythmic Cilium in SCN Neuron is a Gatekeeper for the Intrinsic Circadian Clock

abstract:
The internal circadian rhythm is controlled by the central pacemaker in the hypothalamic suprachiasmatic nucleus (SCN). SCN drives coherent and synchronized circadian oscillations via intercellular coupling, which are resistant to environmental perturbations. Here we report that primary cilium is a critical device for intercellular coupling among SCN neurons and acts as a gatekeeper to maintain the internal clock in mice. A subset of SCN neurons, namely neuromedin S-producing (NMS) neurons, exhibit cilia dynamics with a pronounced circadian rhythmicity. Genetic ablation of ciliogenesis in NMS neurons enables a rapid phase shift of the internal clock under experimental jet lag conditions. The circadian rhythms of individual neurons in cilia-deficient SCN slices lose their coherence following external perturbations. Rhythmic cilia dynamics drive oscillations of Sonic Hedgehog (Shh) signaling and oscillated expressions of multiple circadian genes in SCN neurons. Genetic and chemical inactivation of Shh signaling in NMS neurons phenocopies the effect of cilia ablation. Our findings establish ciliary signaling as a novel interneuronal coupling mechanism in the SCN and may lead to novel therapy of circadian disruption-linked diseases.

One-Sentence SummaryRhythmic cilium is a critical device for intercellular coupling among SCN neurons and acts as gatekeeper for the internal clock.


title: Structural basis for regulation of a tripartite toxin-antitoxin system by dual phosphorylation

abstract: Many bacteria encode multiple toxin-antitoxin (TA) systems targeting separate, but closely related, cellular functions. The toxin of the E. coli hipBA system, HipA, is a kinase that inhibits inhibits translation via phosphorylation of glutamyl-tRNA synthetase. Enteropathogenic E. coli (EPEC) O127:H6 encodes an additional, tripartite TA module, hipBST, for which the HipT toxin was shown to specifically target tryptophanyl-tRNA synthetase, TrpS. Surprisingly, the function as antitoxin has been taken over by the third protein, HipS, but the molecular details of how activity of HipT is controlled remain poorly understood. Here, we show that HipBST is markedly different from HipBA and that the unique HipS protein, which is homologous to the N-terminal subdomain of HipA, has evolved to function as antitoxin by breaking the kinase active site. We also show how auto-phosphorylation at two conserved sites in the kinase toxin serve to dually regulate binding of HipS and kinase activity. Finally, we demonstrate that the HipBST complex is dynamic and present a cohesive model for the regulation and activation of this type of three-component system.

category: microbiology


title: Staphylococcus aureus prophages are gatekeepers of exogenous lipid utilization to control membrane flexibility and bacterial fitness

abstract:
Lipids are essential components of bacterial membranes and phages are quasi-ubiquitous in bacteria. Both are crucial mediators of bacteria-host interactions. In the major pathogen Staphylococcus aureus, two common prophages, {phi}11.1 and {phi}13, are preferentially integrated in lipase genes, respectively encoding Geh, a triglyceride lipase, and Hlb, a sphingomyelinase. Here we report how prophage status, and consequently lipase availability, impacts S. aureus metabolism of environmental lipids, and overall bacterial fitness. The fatty acid kinase subunit FakB1, known to promote saturated fatty acid incorporation, also facilitates incorporation of unsaturated fatty acids released from triglycerides by Geh; this process occurs only when {phi}11.1 is absent. Geh and Hlb lipase products compete for FakB1, and interference by prophage integration changes the outcome of this competition. Our findings could explain the selective advantage of prophage conservation, as a means of modulating bacterial membrane composition and preventing toxicity of environmental lipids. {phi}13 lysogeny also impaired the bacterial capacity to escape antibiotic inhibitors of FASII (fatty acid synthesis). We suggest that testing new therapeutics against S. aureus should consider prophage status and environmental lipids for accurate prediction of efficiency.

ImportanceStaphylococcus aureus is a major human health challenge. Its lipases are crucial for virulence and immune response escape. Prophages inserted in lipases are common in S. aureus but the selective benefits of their maintenance is little understood. We demonstrate that two prophages, inserted in triglyceride and sphingomyelin lipase genes, interactively modulate fatty acid incorporation into bacterial membranes. The presence or absence of prophage determine how bacteria respond to a complex lipid environment such as the host, by affecting membrane properties, fitness, and adaptation to antibiotics inhibiting fatty acid synthesis. Our findings highlight the importance of assessing prophage status and lipid environments when evaluating infection risk and therapy efficiency.


title: Staphylococcus aureus prophages are gatekeepers of lipid utilization to control membrane flexibility, bacterial fitness, and antibiotic adaptation


title: Evolution of chlorhexidine susceptibility and of the EfrEF operon among Enterococcus faecalis from diverse environments, clones and time spans

abstract: Chlorhexidine (CHX) is widely used to control the spread of pathogens (e.g. human/animal clinical settings, ambulatory care, food industry). E. faecalis, a major nosocomial pathogen, is broadly distributed in diverse hosts and environments facilitating its exposure to CHX over the years. Nevertheless, CHX activity against E. faecalis is understudied. Our goal was to assess CHX activity and the variability of ChlR-EfrEF proteins (associated with CHX tolerance) among 673 field isolates and 1784 E. faecalis genomes from PATRIC database from different sources, time spans, clonal lineages and antibiotic resistance profiles. CHX minimum inhibitory concentrations (MICCHX) and minimum bactericidal concentrations (MBCCHX) against E. faecalis presented normal distributions (0.5-64 mg/L). However, more CHX tolerant isolates were detected in the food chain and recent human infections, suggesting an adaptability of E. faecalis populations in settings where CHX is heavily used. Heterogeneity in ChlR-EfrEF sequences was identified, with isolates harboring incomplete ChlR-EfrEF proteins, particularly the EfrE identified in the ST40 clonal lineage, showing low MICCHX ([≤]1mg/L). Distinct ST40-E. faecalis subpopulations carrying truncated and non-truncated EfrE were detected, the former being predominant in human isolates. This study provides a new insight about CHX susceptibility and ChlR-EfrEF variability within diverse E. faecalis populations. The MICCHX/MBCCHX of more tolerant E. faecalis (MICCHX=8mg/L; MBCCHX=64mg/L) remain lower than in-use concentrations of CHX (>500mg/L). However, CHX increasing use combined with concentration gradients occurring in diverse environments potentially selecting multidrug-resistant strains with different CHX susceptibilities, alert to the importance of monitoring the trends of E. faecalis CHX tolerance within a One-Health approach.


title: Cell wall's mesoscale organization in Deinococcus radiodurans

abstract: S-layers are highly ordered coats of proteins localized on the cell surface of many bacterial species. In these structures, one or more proteins form elementary units that self-assemble into a crystalline monolayer tiling the entire cell surface. Here, the cell envelope of the radiation-resistant bacterium Deinococcus radiodurans was studied by high-resolution cryo-electron microscopy finding the crystalline regularity of the S-layer extended into the layers below. The cell envelope appears to be highly packed and resulting from a three-dimensional crystalline distribution of protein complexes organized in close continuity but allowing different degrees of voidness in the entire thickness. These insights grade S-layers to mesoscale hubs behaving as structural and functional architraves essential for the entire cell body.


title: Mesoscale organization in the cell envelope of Deinococcus radiodurans


title: A snapshot of the root phenotyping landscape in 2021

abstract:
Root phenotyping describes methods for measuring root properties, or traits. While root phenotyping can be challenging, it is advancing quickly. In order for the field to move forward, it is essential to understand the current state and challenges of root phenotyping, as well as the pressing needs of the root biology community.

In this letter, we present and discuss the results of a survey that was created and disseminated by members of the Graduate Student and Postdoc Ambassador Program at the 11th symposium of the International Society of Root Research. This survey aimed to (1) provide an overview of the objectives, biological models and methodological approaches used in root phenotyping studies, and (2) identify the main limitations currently faced by plant scientists with regard to root phenotyping.

Our survey highlighted that (1) monocotyledonous crops dominate the root phenotyping landscape, (2) root phenotyping is mainly used to quantify morphological and architectural root traits, (3) 2D root scanning/imaging is the most widely used root phenotyping technique, (4) time-consuming tasks are an important barrier to root phenotyping, (5) there is a need for standardised, high-throughput methods to sample and phenotype roots, particularly under field conditions, and to improve our understanding of trait-function relationships.

category: scientific communication and education


title: Cell Centred Finite Element Model for Intestinal Organoids Shape Analysis: From tissue architecture to mechanics

abstract:
Organoids, established from stem cells owing to their self-renewal and differentiation capacities, are self-organised three-dimensional tissue culture recapitulating the original cell populations and their associated functions, as well as tissue architecture. The intestinal organoids established from adult stem cells isolated from the intestinal crypts, recreate 3D epithelial mini-intestines. They represent an excellent tool to study intestinal stem cell capacities and their ability to reconstitute a fully polarised and functional epithelium. These 3D cultures recapitulate in vitro the tissue characteristics, including architecture, either in physiological or disease conditions whether they are established from healthy or pathological tissue samples respectively. In this regard, their use for potential treatments screening carries the hopes for a future personalized medicine for which image-analysis such as HCS are increasingly being developed. Numerous numerical models have been developed to study the effects of organoid development on their shape. Most of them remain mainly restricted in their physical description due to the complex inter-relationship between cell physics, phenotypes and behaviors, exploding the number of variables in modeling formulation. Finite Element Method (FEM) is a numerical analysis method employed in mechanics to model deformation and evaluate residual stress of complex structures making it difficult to obtain analytical solutions. Considering epithelial architecture as a homogeneous material where each cell is an elemental equivalent part of the problem, FEM allows a direct link between tissue architecture deformations and local mechanical constraints. Here we formalise a new organoid cell centred FEM with a physical description borrowed from the engineering world. This model can allow a better understanding of the individual contribution of physical/mechanical properties of individual cells on general tissue architecture.

Author SummaryStem cell research has experienced a breakthrough with the development of powerful tools, namely, organoids. The possibility to recreate in vitro from adult stem cells a fully functional adult tissue with its actual 3D architecture made accessible poorly defined steps in tissue morphogenesis or pathophysiology. If recent efforts have focused on studies of tissue specific organoids functions, modelling a generic bio-mechanic model of organoid remains necessary to decipher the mechanical biodiversity of organoid structures.

category: biophysics


title: Chronic brain functional ultrasound imaging in freely moving rodents performing cognitive tasks

abstract: Functional ultrasound imaging (fUS) is an emerging imaging technique that indirectly measures neural activity via changes in blood volume. To date it has not been used to image chronically during cognitive tasks in freely moving animals. Performing those experiments faces a number of exceptional challenges: performing large durable craniotomies with chronic implants, designing behavioural experiments matching the hemodynamic timescale, stabilizing the ultrasound probe during freely moving behavior, accurately assessing motion artifacts and validating that the animal can perform cognitive tasks at high performance while tethered. In this study, we provide validated solutions for those technical challenges. In addition, we present standardized step-by-step reproducible protocols, procedures and data processing pipelines that open up the opportunity to perform fUS in freely moving rodents performing complex cognitive tasks. Moreover, we present proof-of-concept analysis of brain dynamics during a decision making task. We obtain stable recordings from which we can robustly decode task variables from fUS data over multiple months. Moreover, we find that brain wide imaging through hemodynamic response is nonlinearly related to cognitive variables, such as task difficulty, as compared to sensory responses previously explored.


title: Rapidly evolving viral motifs target biophysically constrained binding pockets of host proteins

abstract:
Evolutionary changes in the host-virus interactome can alter the course of infection, but the biophysical and regulatory constraints that shape interface evolution remain largely unexplored. Here, we focus on viral mimicry of short host-like peptide motifs that allow binding to host domains and modulation of cellular pathways. We observe that motifs from unrelated viruses preferentially target conserved, widely expressed and highly connected host proteins, enriched with regulatory and essential functions. The interface residues within these host domains are more conserved and bind a larger number of cellular proteins than similar motif-binding domains that are not known to interact with viruses.

In stark contrast, rapidly evolving viral-binding human proteins form few interactions with other cellular proteins, display high tissue specificity and their interface residues have few inter-residue contacts. Our results distinguish between highly conserved and rapidly evolving host-virus interfaces, and show how regulatory, functional and biophysical factors limit host capacity to evolve, allowing for efficient viral subversion of host machineries.


title: Avoidance of premature splicing in short introns explains the polypyrimidine tract

abstract: Among eukaryotes, the major spliceosomal pathway is highly conserved. Long introns may contain additional regulatory sequences, but those in short introns seem to be nearly exclusively related to splicing. While regulatory sequences involved in splicing are well-characterized, little is known about their evolution. At the 3 end of introns, the splice signal nearly universally contains the dimer AG, which consists of purines. The polypyrimidine tract upstream of the 3 splice signal is characterized by over-representation of the pyrimidine bases cytosine and thymine. We hypothesize that the over-representation of pyrimidines in the polypyrimidine tract is caused by avoidance of a premature splicing signal-AG should be most under-represented dimer. DNA-strand asymmetry patterns in fruit flies of the genus Drosophila confirm this prediction. In short introns of Drosophila, a region between the 5 splice signal and the branch point, which at least contains the nucleotides 8-30, is considered to evolve neutrally. Comparing this presumably neutrally evolving region to the polypyrimidine tract, we infer a moderate scaled selection strength below four against 3 splicing signals. Patterns of asymmetry in other eukaryotes indicate that avoidance of premature splicing similarly affects the nucleotide composition in their polypyrimidine tracts.


title: Single-nuclei transcriptomics of dog hippocampus reveals the distinct cellular mechanism of domestication

abstract: The process of dog domestication leads to dramatic differences in behavioral traits compared to grey wolves. A class of putative positively selected genes is related to learning and memory, for instance, long-term potentiation and long-term depression. In this study, we constructed a single-nuclei transcriptomic atlas of the dog hippocampus to illustrate its cell types, cell lineage, and molecular features. Using the transcriptomes of 105,057 single-nuclei from the hippocampus of a Beagle dog, we identified 26 cell clusters and a putative trajectory of oligodendrocyte development. Comparative analysis revealed a significant convergence between dog differentially expressed genes (DEGs) and putative positively selected genes (PSGs). 40 putative PSGs were DEGs in the glutamatergic neurons, especially in the cluster 14, which is related to the regulation of nervous system development. In summary, this study provided a blueprint to understand the cellular mechanism of dog domestication.


title: Genomic analyses identify key molecules and significant signaling pathways in AZIN1 regulated prostate cancer cells

abstract: Antizyme inhibitor 1 (AZIN1) is a critical target in prostate cancer, which regulates the adenosine-to-inosine (A to I) RNA editing during the cancer progression. However, the potential signaling pathways and functions remain unknown. Here, our objective is to figure out the functional molecules and signaling pathways by analyzing the RNA-seq data. The GSE189379 was produced by the Illumina HiSeq 2000 (Homo sapiens). The KEGG and GO analyses showed that focal adhesion and proteoglycans are the mainly affected processes in prostate cancer with the loss of AZIN1. Moreover, we identified ten key molecules including FN1, HRAS, CCND1, RAD51, PCNA, TYMS, CASP3, RRM2, BIRC5, and CCNE2. Therefore, this study provides novel knowledge of AZIN1 mediated prostate cancer.