Miracle-dz/UniFAR-FTUs huggingface dataset:
Adipose mTORC2 is essential for arborization of sensory neurons in white adipose tissue and whole-body energy homeostasis

Adipose tissue, via sympathetic and sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue is poorly characterized. Taking advantage of recent progress in whole-mount three-dimensional imaging of adipose tissue, we identified a neuronal network of calcitonin gene-related protein (CGRP)-positive sensory neurons in white adipose tissue (WAT). Furthermore, we show that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, mediates sensory innervation in WAT. Based on visualization of neuronal networks, mTORC2-deficient WAT displayed reduced arborization of (CGRP)-positive sensory neurons, while sympathetic neurons were unaffected. This selective loss of sensory innervation followed reduced expression of growth-associated protein 43 (GAP43) in CGRP-positive sensory neurons. Finally, we found that loss of sensory innervation in WAT correlated with systemic insulin resistance. Our findings suggest that adipose mTORC2 is necessary for sensory innervation in WAT which likely contributes to WAT-to-CNS communication.
Multisensory integration in neurons of the medial pulvinar of macaque monkey

The pulvinar is a heterogeneous thalamic nucleus, which is well developed in primates. One of its subdivisions, the medial pulvinar, is connected to many cortical areas, including the visual, auditory and somatosensory cortices, as well as with multisensory areas and premotor areas. However, except for the visual modality, little is known about its sensory functions. A hypothesis is that, as a region of convergence of information from different sensory modalities, the medial pulvinar plays a role in multisensory integration. To test this hypothesis, two macaque monkeys were trained to a fixation task and the responses of single-units to visual, auditory and auditory-visual stimuli were examined. Analysis revealed auditory, visual and multisensory neurons in the medial pulvinar. It also revealed multisensory integration in this structure, mainly subadditive and suppressive. These findings show that the medial pulvinar is involved in multisensory integration.
Stage-specific gene and transcript dynamics in human male germ cells

Cell differentiation processes are highly dependent on cell stage-specific gene expression, including timely production of alternatively spliced transcripts. One of the most transcriptionally rich tissues is the testis, where the process of spermatogenesis, or generation of male gametes, takes place. To date, germ cell-specific transcriptome dynamics remain understudied due to limited transcript information emerging from short-read sequencing technologies. To fully characterize the transcriptional profiles of human male germ cells and to understand how the human spermatogenic transcriptome is regulated, we compared whole transcriptomes of men with different types of germ cells missing from their testis. Specifically, we compared the transcriptomes of testis lacking germ cells (Sertoli cell-only phenotype; SCO; n=3), with an arrest at the stage of spermatogonia (SPG; n=4), spermatocytes (SPC; n=3), and round spermatids (SPD; n=3), with the transcriptomes of testis with normal and complete spermatogenesis (Normal; n=3). We found between 839 and 4,138 differentially expressed genes (DEGs, log2 fold change ≥ 1) per group comparison, with the most prevalent changes observed between SPG and SPC arrest samples, corresponding to the entry into meiosis. We detected highly germ cell-type specific marker genes among the topmost DEGs of each group comparison. Moreover, applying state-of-the-art bioinformatic analysis we were able to evaluate differential transcript usage (DTU) during human spermatogenesis and observed between 1,062 and 2,153 genes with alternatively spliced transcripts per group comparison. Intriguingly, DEGs and DTU genes showed minimal overlap (< 8%), suggesting that stage-specific splicing is an additional layer of gene regulation in the germline. By generating the most complete human testicular germ cell transcriptome to date, we unravel extensive dynamics in gene expression and alternative splicing during human spermatogenesis.
Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

The formation of a clathrin-coated vesicle is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment “hook” to an “anchor” protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation – initiation, invagination, and even fission – were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin alone is sufficient for intracellular clathrin-coated vesicle formation.
Comparison of microbial communities from diverse biological matrices using mock community as an in situ positive control

Metataxonomy has become the standard for characterizing the diversity and composition of microbial communities associated with multicellular organisms and their environment. Understanding the interactions between the microbiotas within the same ecosystem is essential for fully understanding the role of microorganisms in evolutionary and ecological processes; however, such comparative studies across diverse biological samples are rare. In particular, currently available protocols assume a uniform DNA extraction, amplification and sequencing efficiency for all sample types and taxa. The addition of a mock community (MC) to biological samples before the DNA extraction step could aid identification of technical biases, but the impact of MC on diversity estimates is unknown. Here, standardized aliquots of bovine faecal samples with high or low biomass were extracted with high or low doses of MC, characterized using standard Illumina technology for metataxonomics, and analysed with custom bioinformatic pipelines. We showed that a MC was an informative in situ positive control provided an estimate of 16S rRNA sample gene copies (which allowed a more direct measure of community size), and detected sample outliers. However, we also demonstrated that if the recommended dose of MC is added to a sample with low biomass, diversity estimates were distorted. Using our results, we recommend MC doses for a range of sample types, including rhizosphere soil, whole invertebrates, and vertebrate faecal samples. Importance The simultaneous processing of the sample microbiota with a known number of readily identifiable MC cells (co-extracted with the sample cells) or SNA (co-amplified with the sample DNAs) can be a valuable in situ positive control. However, guidelines regarding their application are very limited and do not consider the effect of these controls on sample diversity estimates. We demonstrate that a MC co-extracted with the study sample provides several advantages, such as highlighting bias in DNA extraction of gram positive, identifying potential sample outliers and inferring the number of 16S rRNA gene copies in the sample. However, the incorporation of a MC requires prior knowledge of sample biomass, as high ratios of MC to sample microbiota lead to biased sample diversity estimates. Practical advice on determining the appropriate MC dose for a wide range of sample types, including rhizosphere soil, whole invertebrates and mammalian faecal samples are provided.
A mRNA Vaccine Encoding for a RBD 60-mer Nanoparticle Elicits Neutralizing Antibodies and Protective Immunity Against the SARS-CoV-2 Delta Variant in Transgenic K18-hACE2 Mice

Two years into the COVID-19 pandemic there is still a need for vaccines to effectively control the spread of novel SARS-CoV-2 variants and associated cases of severe disease. Here we report a messenger RNA vaccine directly encoding for a nanoparticle displaying 60 receptor binding domains (RBDs) of SARS-CoV-2 that acts as a highly effective antigen. A construct encoding the RBD of the delta variant elicits robust neutralizing antibody response with neutralizing titers an order of magnitude above currently approved mRNA vaccines. The construct also provides protective immunity against the delta variant in a widely used transgenic mouse model. We ultimately find that the proposed mRNA RBD nanoparticle-based vaccine provides a flexible platform for rapid development and will likely be of great value in combatting current and future SARS-CoV-2 variants of concern.
Investigating the “two-hit hypothesis”: Effects of prenatal maternal immune activation and adolescent cannabis use on neurodevelopment in mice

Background Prenatal exposure to maternal immune activation (MIA) and chronic adolescent cannabis use are both risk factors for neuropsychiatric disorders. However, exposure to a single risk factor may not result in major mental illness, indicating that multiple exposures may be required for illness onset. Here, we examine whether combined exposure to prenatal MIA and adolescent delta-9-tetrahydrocannabinol (THC), the main psychoactive component of cannabis, lead to enduring neuroanatomical and behavioural changes in adulthood. Methods Mice were prenatally exposed to viral mimetic, poly I:C (5mg/kg), or vehicle at gestational day (GD)9, and postnatally exposed to chronic THC (5mg/kg, intraperitoneal) or vehicle during adolescence (postnatal day [PND]28-45). Longitudinal magnetic resonance imaging (MRI) was performed pre-treatment, PND25, post-treatment, PND50, and in adulthood, PND85, followed by behavioural tests for anxiety-like, social, and sensorimotor gating. Postmortem assessment of cannabinoid (CB)1 and 2 receptor expressing cells was performed in altered regions identified by MRI (anterior cingulate and somatosensory cortices, striatum, and hippocampus). Results Subtle deviations in neurodevelopmental trajectory and subthreshold anxiety-like behaviours were observed in mice exposed to both risk factors. Sex-dependent effects were observed in patterns of shared brain-behaviour covariation, indicative of potential sex differences in response to MIA and THC. Density of CB1 and CB2 receptor positive cells was significantly decreased in all mice exposed to MIA, THC, or both. Conclusions These findings suggest that there may be a cumulative effect of risk factor exposure on gross neuroanatomical development, and that the endocannabinoid system may be sensitive to both prenatal MIA, adolescent THC, or the combination.
Molecular Characterisation Of Urine And Vaginal Samples And Their Antibiotic Susceptibility Pattern Associated With β-Lactamases Resistance Gene

Infection or inflammation of the vagina is called vaginitis. The two most common causes of vaginitis are bacterial vaginitis and Candida vaginitis. The causative organisms may be sexually transmitted or endogenous, usually by Gardnerella vaginalis and Mycoplasma hominis in combination with other anaerobes. Urinary tract infections (UTI) refer to the presence of microbial pathogens within the urinary tract and are caused by a range of pathogens but most commonly by Escherichia coli, Klebsiella pneumoniae etc. Highly increase in antibiotic-resistant among uropathogens, including ESBL producing E.coli, AmpC β-lactamase or carbapenemase-producing Enterobacteriaceae (e.g., New Delhi metallo-β-lactamase [NDM]) are being reported among health care unit. Carbapenem-resistant Enterobacteriaceae (CRE) is a growing concern worldwide and a major challenge by the WHO 2014. The aim of this study was to determine the occurrence of CTX-M gene and TEM gene and the possible role of ESBL in resistance to antibiotic. A study was conducted on a total of 100 urine and vaginal samples from Dr. B. Lal clinical laboratory and was cultured on Nutrient Agar and MacConkey agar for the detection of etiologic agents. Bacteria isolation, identification & biochemical characterization was done. Kirby-Bauer Disk Diffusion (KBDS) method was used for detection of ESBL-producing strains. A disc of Cefotaxime (30 µg) and Clavulanic acid (30 µg) were placed at a distance of 20 mm on Muller Hilton agar plate along with Imipenem(30µg), Piperacillin/Tazobactam(10µg) and Ceftazidime(30µg). The zone of inhibition generally greater than 20mm was considered as ESBL producer. Total 14 isolates were identified as ESBL producer. In order to find out the plasmid profile of these isolates, plasmid DNA isolation was done. Isolates was found to have one or more than one plasmid gene responsible for the ESBL resistance. This finding concludes that CTX-M gene is most common gene in ESBL producing isolates. It is highly recommended that antibiotic prescription should be monitored according to guideline. The prevalence of ESBL is increasing day by day and necessary steps should be taken to prevent the spread and emergence of resistance.
Identification of a Novel Osteogenetic Oligodeoxynucleotide (osteoDN) That Promotes Osteoblast Differentiation in a TLR9-Independent Manner

Recent studies have revealed that oligodeoxynucleotides (ODNs) designed from genome sequences have the potential to regulate cell fate. Currently, ODNs that conduct cell differentiation are nanomolecular drug candidates for regenerative medicine. Herein, we demonstrate that iSN40, an 18-base ODN derived from the Lacticaseibacillus rhamnosus GG genome, promoted the differentiation and calcification of osteoblasts that play a central role during bone formation. In the murine osteoblast cell line MC3T3-E1, iSN40 enhanced alkaline phosphatase activity at the early stage of differentiation and facilitated calcium deposition at the late stage by inducing the expression of osteogenic genes such as Msx2, osterix, collagen type 1α, osteopontin, and osteocalcin. Intriguingly, the CpG motif within iSN40 was not required for its osteogenetic activity, indicating that iSN40 functions in a TLR9-independent manner. These data suggest that iSN40, serving as an osteogenetic ODN (osteoDN), as a drug seed that target osteoblasts for bone regeneration.
Floating sphere assay: A rapid qualitative method for microvolume analysis of gelation

A huge, unprecedented demand for gelatin coupled with its implications on global sustainability has resulted in the need to discover novel proteins with gelling attributes for applications in the food industry. Currently used gelation assays require large sample volumes and thus the screening for novel gelling proteins is a formidable technical challenge. In this paper, we report the ‘Floating Sphere Assay’ which is a simple, economical, and miniaturized assay to detect minimum gelling concentration with volumes as low as 50 μl. Results from the Floating Sphere Assay are consistent with currently used methods for gelation tests and accurately estimate the Minimum Gelling Concentrations (MGCs) of gelatin, κ-carrageenan and gellan gum. The assay was also able to differentiate the strengths of strong and weak gellan gum gels prepared at pH 3.5 and pH 7.0 respectively. The Floating Sphere Assay can be utilized in high-throughput screens for gelling proteins and can accelerate the discovery of gelatin substitutes. 2 Highlights We report the Floating Sphere Assay that can be used to assesses minimum gelling concentration of solutions with volumes as low as 50 μl. Observing whether a glass sphere placed on the surface of a test solution floats or sinks is diagnostic of gel formation Floating Sphere Assay can distinguish a strong gel from a weak gel Floating Sphere Assay is a rapid and cost-effective approach to screen for novel plant-based gelatin alternatives.
SARS-CoV-2 spike proteins uptake mediated by lipid raft ganglioside GM1 in human cerebrovascular cells

While there is clinical evidence of neurological manifestation in coronavirus disease-19, it’s unclear whether this is due to differential severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake from blood by cells of the cerebrovasculature. SARS-CoV-2 and its spike protein (SP) interact with the endothelium but the roles of extracellular peptidase domain on angiotensin converting enzyme 2 receptors (ACE2) and ACE2 independent pathways (such as glycans) are not fully elucidated. In addition, for SARS-CoV-2 to enter the brain parenchyma from blood it has to cross several cell types, including the endothelium, pericytes and vascular smooth muscle. Since SARS-CoV-2 interacts with host cells via it SP at the entry point of it life cycle, we used fluorescently labelled SP (SP-555) (wild type and mutants) to model viral behaviour, in vitro, for these cell types (endothelial, pericytes and vascular smooth muscle) to explore pathways of viral entry into brain from blood. There was differential SP uptake by these cell types. The endothelial cells had the least uptake, which may limit SP uptake into brain from blood. Uptake was mediated by ACE2, but it was dependent on SP interaction with ganglioside GM1 in the lipid raft. Mutation sites, N501Yand E484K and D614G, as seen in variants of interest, were differentially taken up by these cell types. There was greater uptake but neutralization with anti-ACE2 and anti-GM1antibodies was less effective. Our data suggested that GM1/lipid raft is an important entry point of SARS-CoV-2 into these cells since inhibition of SP uptake with both anti-ACE2 and anti-GM1 together was similar to that with only anti-GM1, and both ACE2 and GM1 are within the lipid raft region of plasma membrane. Thus, GM1 is a potential SARS-CoV-2 and therapeutic target at the cerebrovasculature.
Toxoplasma gondii ROP1 subverts murine and human innate immune restriction

Toxoplasma gondii is an intracellular parasite that can infect many different host species and is a cause of significant human morbidity worldwide. T. gondii secretes a diverse array of effector proteins into the host cell which are critical for infection; however, the vast majority of these secreted proteins are uncharacterised. Here, we carried out a pooled CRISPR knockout screen in the T. gondii Prugniaud strain in vivo to identify secreted proteins that contribute to parasite immune evasion in the host. We identify 22 putative virulence factors and demonstrate that ROP1, the first-identified rhoptry protein of T. gondii, has a previously unrecognised role in parasite resistance to interferon gamma-mediated innate immune restriction. This function is conserved in the highly virulent RH strain of T. gondii and contributes to parasite growth in both murine and human macrophages. While ROP1 affects the morphology of rhoptries, from where the protein is secreted, it does not affect rhoptry secretion. ROP1 interacts with the host cell protein C1QBP, which appears to facilitate parasite immune evasion. In summary, we identify 22 secreted proteins which contribute to parasite growth in vivo and show that ROP1 is an important and previously overlooked effector in counteracting both murine and human innate immunity.
The Effect of Auditory Stimulation on the Nonlinear Dynamics of Heart Rate: The Impact of Emotional Valence and Arousal

Background Although it is known that sound exposure evokes changes in autonomic activity, the effects of noise and music on the nonlinear behavior of heart rate fluctuations remain poorly understood and controversial. This study aims to assess the influence of sound subjective emotional valence and arousal on the nonlinear characteristics of the autonomic nervous system during passive listening. Methods In this study, forty-two subjects listened to four sounds: (1) white noise, (2) road traffic noise, (3) excitatory music, and (4) a lullaby. The experiment consisted of two consecutive sessions: five minutes of rest, followed by five minutes of listening. RR intervals were recorded during both sessions. The following linear and nonlinear heart rate variability indices were computed: SDNN, RMSSD, F, HF, approximate (ApEn) and sample entropy (SampEn), correlation dimension (D2), Poincare plot indices (SD1, SD2), fractal scaling exponents (alpha1, alpha2), and recurrence plot indices (Lmean, Lmax, DET, LAM, Vmax, TT, ShanEn). Results Excitatory music was associated with significant decrease in SDNN and RMSSD, diminished HF, and a substantial reduction in LF. Excitatory music exposure induced significant increases in DET, SD1 and SD2, but changes in DFA, SampEn, and D2 were nonsignificant. Traffic noise, white noise, and the lullaby did not cause significant changes in the measures of heart rate variability. Conclusion Presentation of music evoking strong negative emotions elicits a prominent decrease in respiratory sinus arrhythmia. Poincare plot and recurrence plot measures possess high sensitivity to high arousal and unpleasant music. Contrary to previous studies, we did not find the effects of relaxing music on heart rate variability.
RSC and GRFs confer promoter directionality by limiting divergent noncoding transcription

The directionality of gene promoters - the ratio of protein-coding over divergent noncoding transcription - is highly variable and regulated. How promoter directionality is controlled remains poorly understood. Here, we show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription. At gene promoters that are highly directional, depletion of RSC leads to a relative increase in divergent noncoding transcription and thus a decrease in promoter directionality. We find that RSC facilitates nucleosome positioning upstream in promoters at the sites of divergent transcription. These highly directional promoters are also enriched for the binding of GRFs such as Reb1 and Abf1. Ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 protein suppresses divergent transcription. Our data suggest that RSC and GRFs play a pervasive role in limiting divergent transcription. We propose that any DNA binding factor, when stably associated with cryptic transcription start sites, form barriers for repressing divergent transcription. Our study provides an explanation as to why certain promoters are more directional than others.