Biology subjects

Rao, Y.

Publications and source records attributed to Rao, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Microglia facilitate and stabilize the response to general anesthesia

General anesthesia leads to a loss of consciousness and an unarousable state in patients. Although general anesthetics are widely used in clinical practice, their underlying mechanisms remain elusive. The potential involvement of nonneuronal cells is unknown. Microglia are important immune cells in the central nervous system (CNS) that play critical roles in CNS function and dysfunction. We unintentionally observed delayed anesthesia induction and early anesthesia emergence in microglia-depleted mice. We found that microglial depletion differentially regulates neuronal activities by suppressing the neuronal network of anesthesia-activated brain regions and activating emergence-activated brain regions. Thus, microglia facilitate and stabilize the anesthesia status. This influence is not mediated by dendritic spine plasticity. Instead, it relies on the activation of microglial P2Y12 and subsequent calcium influx, which facilitates the general anesthesia response. Together, we elucidate the regulatory role of microglia in general anesthesia, extending our knowledge of how nonneuronal cells modulate neuronal activities.

neuroscience↗

bioRxiv2023

Conditional Chemoconnectomics: A Set of Libraries Targeting All Chemical Transmission Corresponding Genes Efficiently

Dissection of neural circuitry underlying behaviors is a central theme in neurobiology. We have previously proposed the concept of chemoconnectome (CCT) to cover the entire chemical transmission between neurons and target cells in an organize and created tools for studying it (CCTomics) by targeting all genes related to the CCT in Drosophila. Here we have created lines targeting the CCT in conditional manners after modifying GFP RNA interference, Flp-out and CRISPR/Cas9 technologies. All three strategies are validated to be highly effective with the best using chromatin-peptide fused Cas9 variants and scaffold optimized sgRNAs. As a proof of principle, we conduct a comprehensive intersection analysis of CCT genes expression profiles in the clock neurons, uncovering 43 CCT genes present in clock neurons. Specific elimination of each from clock neurons revealed that loss of the neuropeptide CNMa in two posterior dorsal clock neurons (DN1ps) or its receptor (CNMaR) caused advanced morning activity, indicating a suppressive role of CNMa-CNMaR on morning anticipation, opposite to the promoting role of PDF-PDFR on morning anticipation. These results demonstrate the effectiveness of conditional CCTomics and its tools created by us here and establish an antagonistic relationship between CNMa-CNMaR and PDF-PDFR signaling in regulating morning anticipation.

neuroscience↗

bioRxiv2023

CD4+ T cell senescence is associated with reduced reactogenicity in severe/critical COVID-19

BackgroundAging is a critical risk factor for unfavorable clinical outcomes among COVID-19 patients and may affect vaccine efficacy. However, whether the senescence of T cells impact the progression to severe COVID-19 in the elderly individuals remains unclear. MethodsBy using flow cytometry, we analyzed the frequency of senescent T cells (Tsens) in the peripheral blood from 100 elderly patients hospitalized for COVID-19 and compared the difference between mild/moderate and severe/critical illness. We also assessed correlations between the percentage of Tsens and the quantity and quality of spike-specific antibodies by ELISA, neutralizing antibody test kit and Elispot assay respectively, cytokine production profile of COVID-19 reactive T cells as well as plasma soluble factors by cytometric bead array (CBA). ResultsWe found a significant elevated level of CD4+ Tsens in severe/critical disease compared to mild/moderate illness and patients with a higher level of CD4+ Tsens (>19.78%) showed a decreased survival rate as compared to those with a lower level (<19.78%), especially in the breakthrough infection. The percentage of CD4+ Tsens was negatively correlated with spike-specific antibody titers, neutralization ability and COVID-19 reactive IL-2+ CD4+ T cells. Additionally, IL-2 producing T cells and plasma levels of IL-2 were positively correlated with antibody levels. ConclusionOur data illustrated that the percentage of CD4+ Tsens in the peripheral blood could act as an efficient biomarker for the capacity of spike-specific antibody production and the prognosis of severe COVID-19, especially in the breakthrough infection. Therefore, restoration of the immune response of CD4+ Tsens is one of the key factors to prevent severe illness and improve vaccine efficacy in older adults.

immunology↗

bioRxiv2023

Biochemical and chemical biological approaches to mammalian sleep: roles of calcineurin in site-specific dephosphorylation and sleep regulation

Understanding of sleep mechanisms traditionally rely on electrophysiology and genetics but here we have initiated biochemical and chemical biological studies. Sleep was increased in mouse mutants with an alanine replacing threonine at residue 469 (T469A) of the salt inducible kinase 3 (SIK3). We searched for T469 phosphatases by classic purification with HEK293 cells and by a new photo-crosslinking method with mouse brains. Both led to PPP3CA, a catalytic subunit of calcium/calmodulin activated phosphatase (calcineurin). It dephosphorylated T469 and serine (S) 551 but not T221 in SIK3 in vitro. PPP3CA knockdown increased phosphorylation of T469 and S551 but not T221 in mouse brains. Knockdown of its regulatory subunit PPP3R1 significantly reduced daily sleep by more than 5 hours, exceeding other known mouse mutants. Our results have uncovered in vitro and in vivo evidence for site-specific SIK3 dephosphorylation by calcineurin, demonstrated a physiological role for calcineurin in sleep, and suggested sleep control by calcium dependent dephosphorylation.

neuroscience↗

bioRxiv2023

Gender-specific impact of trans 10, cis 12-conjugated linoleic acid on pai transgenic female mice

Dietary trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) is a potential candidate in anti-obesity trials. A transgenic mouse was previously successfully established to determine the anti-obesity properties of t10c12-CLA in male mice that could produce endogenous t10c12-CLA. To test whether there is a different impact of t10c12-CLA on lipid metabolism in both sexes, this study investigated the adiposity and metabolic profiles of female Pai mice that exhibited a dose-dependent expression of foreign Pai gene and a shift of t10c12-CLA content in tested tissues. Compared to their gender-match wild-type littermates, Pai mice had no fat reduction but exhibited enhanced lipolysis and thermogenesis by phosphorylated hormone-sensitive lipase and up-regulating uncoupling proteins in brown adipose tissue. Simultaneously, Pai mice showed hepatic steatosis and hypertriglyceridemia by decreasing gene expression involved in lipid and glucose metabolism. Further investigations revealed that t10c10-CLA induced excessive prostaglandin E2, adrenaline, corticosterone, glucagon and inflammatory factors in a dose-dependent manner, resulting in less heat release and oxygen consumption in Pai mice. Moreover, fibroblast growth factor 21 overproduction only in monoallelic Pai/wt mice indicates that it was sensitive to low doses of t10c12-CLA. These results suggest that chronic t10c12-CLA has system-wide effects on female health via synergistic actions of various hormones.

physiology↗

bioRxiv2023

Metabolic impact of trans 10, cis 12-conjugated linoleic acid in pai transgenic mice

Trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) from ruminant-derived foodstuffs can induce body fat loss in mammals after oral administration, while its mechanism on fat reduction has yet to be clarified fully until now. In the current study, a transgenic mouse that produced t10c12-CLA had been generated by inserting the Propionibacterium acnes isomerase (Pai) expression cassette into the Rosa26 locus, and its male offspring were used to decipher an irreversible long-term impact of t10c12-CLA on health and its mechanism of action. Compared to their wild-type C57BL/6J littermates, comprehensive phenotype profiling of biallelic pai/pai mice indicated that white fat was decreased while brown fat was increased reversely; meanwhile, more heat was released and the central activities were reduced. Besides decreased plasma triglycerides in both pai genotypes and increased serum FGF21 in pai/wt mice, RNA and protein analysis revealed that the fatty acid oxidation and thermogenesis capacity of brown adipose tissues were elevated via CPT1B and UCP1/2 over-expression. The results indicate that the t10c12-CLA-induced fat loss might be caused by the excess FGF21 and the increased mass and extra thermogenesis of brown adipose tissue in transgenic mice.

physiology↗

bioRxiv2023

Functional genomic analysis of adult and pediatric brain tumor isolates

BackgroundAdult and pediatric tumors display stark differences in their mutation spectra and chromosome alterations. Here, we attempted to identify common and unique gene dependencies and their associated biomarkers among adult and pediatric tumor isolates using functional genetic lethal screens and computational modeling. MethodsWe performed CRISRP-Cas9 lethality screens in two adult glioblastoma (GBM) tumor isolates and five pediatric brain tumor isolates representing atypical teratoid rhabdoid tumors (ATRT), diffuse intrinsic pontine glioma, GBM, and medulloblastoma. We then integrated the screen results with machine learning-based gene-dependency models generated from data from >900 cancer cell lines. ResultsWe found that >50% of candidate dependencies of 280 identified were shared between adult GBM tumors and individual pediatric tumor isolates. 68% of screen hits were found as nodes in our network models, along with shared and tumor-specific predictors of gene dependencies. We investigated network predictors associated with ADAR, EFR3A, FGFR1 (pediatric-specific), and SMARCC2 (ATRT-specific) gene dependency among our tumor isolates. ConclusionsThe results suggest that, despite harboring disparate genomic signatures, adult and pediatric tumor isolates share a preponderance of genetic dependences. Further, combining data from primary brain tumor lethality screens with large cancer cell line datasets produced valuable insights into biomarkers of gene dependency, even for rare cancers. Importance of the StudyOur results demonstrate that large cancer cell lines data sets can be computationally mined to identify known and novel gene dependency relationships in adult and pediatric human brain tumor isolates. Gene dependency networks and lethality screen results represent a key resource for neuro-oncology and cancer research communities. We also highlight some of the challenges and limitations of this approach.

cancer biology↗

bioRxiv2022

Evidence suggesting creatine as a new central neurotransmitter: presence in synaptic vesicles, release upon stimulation, effects on cortical neurons and uptake into synaptosomes and synaptic vesicles

The discovery of a new neurotransmitter, especially one in the central nervous system (CNS), is both important and difficult. We have been searching for new neurotransmitters for 12 years. We detected creatine (Cr) in synaptic vesicles (SVs), at a level lower than glutamate (Glu) and gamma-aminobutyric acid (GABA) but higher than acetylcholine (ACh) and 5-hydroxytryptamine (5-HT). SV Cr was reduced in mice lacking either arginine:glycine amidinotransferase (AGAT, a Cr synthetase) or SLC6A8, a Cr transporter with mutations among the most common causes of intellectual disability (ID) in men. Calcium-dependent release of Cr was detected after stimulation in brain slices. Cr release was reduced in SLC6A8 and AGAT mutants. Cr inhibited neocortical pyramidal neurons. SLC6A8 was necessary for Cr uptake into synaptosomes. Cr was found by us to be taken up into SVs in an ATP dependent manner. Our biochemical, chemical, genetic and electrophysiological results are consistent with the possibility of Cr as a neurotransmitter, though not yet reaching the level of proof for the now classic transmitters. Our novel approach to discover neurotransmitters is to begin with analysis of contents in SVs before defining their function and physiology.

neuroscience↗

bioRxiv2022

ppGpp is Present in and Functions to Regulate Sleep in Drosophila

Sleep is essential for animals, and receives inputs from circadian, homeostasis, and environment, yet the mechanisms of sleep regulation remain elusive. Discovery of molecules in living systems and demonstration of their functional roles are pivotal in furthering our understanding of the molecular basis of biology. Here we report that ppGpp (guanosine-5-diphosphate, 3-diphosphate), a molecule that has been detected in prokaryotes for more than five decades, is present in Drosophila, and plays an important role in regulation of sleep and SISL (starvation induced sleep loss). ppGpp is detected in germ-free Drosophila and hydrolyzed by an enzyme encoded by the mesh1 gene in Drosophila. Nighttime sleep and SISL were defected in mesh1 mutant flies, and rescued by expression of wildtype Mesh1, but not the enzymatically defective mutant Mesh1E66A. Ectopic expression of RelA, the E. coli synthetase for ppGpp, phenocopied mesh1 knockout mutants, whereas overexpression of Mesh1 resulted in the opposite phenotypes, supporting that ppGpp is both necessary and sufficient in sleep regulation. A chemoconnectomic screen followed by genetic intersection experiments implicate the Dilp2 neurons in the pars intercerebralis (PI) brain region as the site of ppGpp function. Our results have thus supported that ppGpp is present in animals after long lag since its discovery in bacteria, and revealed a physiological role of ppGpp in sleep regulation for the first time.

genetics↗

bioRxiv2022

Importance of Glutamine in Synaptic Vesicles Revealed by Functional Studies of SLC6A17 and Its Mutations Pathogenic for Intellectual Disability

Human mutations in the gene encoding the solute carrier (SLC) 6A17 caused intellectual disability (ID). The physiological role of SLC6A17 and pathogenesis of Slc6a17-based-ID were both unclear. Here we report learning deficits in SLC6A17 knockout and point mutants. Biochemistry, proteomics and electron microscopy (EM) support SLC6A17 protein localization in synaptic vesicles (SVs). Chemical analysis of SVs by liquid chromatography coupled to mass spectrometry (LC-MS) revealed glutamine (Gln) in SVs containing SLC6A17. Virally mediated overexpression of SLC6A17 increased Gln in SVs. Either genetic or virally mediated targeting of SLC6A17 reduced Gln in SVs. One ID mutation caused SLC6A17 mislocalization while the other caused defective Gln transport. Multidisciplinary approaches with 7 types of genetically modified mice have shown Gln as an endogenous substrate of SLC 6A17, uncovered Gln as a new molecule in SVs, established the necessary and sufficient roles of SLC6A17 in Gln transport into SVs, and suggested SV Gln decrease as the key pathogenetic mechanism in human ID.

neuroscience↗

bioRxiv2022

Proliferative Exhausted CD8 T Cells Exacerbate Long-lasting Anti-tumor Effects in Human Papillomavirus Positive Head and Neck Squamous Cell Carcinoma

The survival prognosis of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) is largely different, and little is known about the anti-tumor mechanism of tumor-infiltrated CD8+ exhausted T cells (Tex) in HNSCC. We performed cell level multi-omics sequencing on human HNSCC samples to decipher the multi-dimensional characteristics of Tex cells. A proliferative exhausted CD8+ T cell cluster (P-Tex) which was beneficial to survival outcomes of patients with HPV-positive HNSCC was identified. Interestingly, P-Tex cells expressed CDK4 genes as high as cancer cells, which could be simultaneously inhibited by CDK4 inhibitors and might be a potential reason for the ineffectiveness of CDK4 inhibitors in treating HPV-positive HNSCC. P-Tex cells could aggregate in the antigen presenting cell niches and activate certain signaling pathways. Together, our findings suggest a promising role for P-Tex cells in the prognosis of patients with HPV-positive HNSCC by providing modest but persistent anti-tumor effects. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/513993v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1f8810forg.highwire.dtl.DTLVardef@1e11d84org.highwire.dtl.DTLVardef@58fb6aorg.highwire.dtl.DTLVardef@1e20a13_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

3D-Printing in Radiation Oncology: Development and Validation of Custom 3D-Printed Brachytherapy Alignment Device and Phantom

Brachytherapy seeks to treat cancer through insertion of radioactive sources aligned by needles within standardized blocking templates. However, many patients have anatomy that does not conform to standard tools. We created a 3D printed device to provide customized needle alignment for a patient. Device was validated through CT scan and treatment plan creation on a custom anatomical phantom based on patient data. CT scan of a cervix tumor from anonymized patient data was used to develop a 3D printed brachytherapy alignment tool and phantom anatomical mold. Multiple materials were evaluated to match patient anatomy in density and Hounsfield Units present on CT scan, with additional considerations for toxicity, compliance, and practicality. Alignment device and molds were developed in PLA. Silicone of T20 hardness was used to create relevant anatomical organs (Uterus, Rectum, Bladder). Tumor tissue was mimicked by addition of 1CC of Iodine contrast agent to silicone. Device and needles were arranged, inserted into anatomical phantom, and scanned by CT to mimic brachytherapy procedure. 3D printed Silicone uterus of 1.08 g/cm^3 and 40 HU mimicked human uterus on CT scan. Constructed uterus dimensions of 6.5 cm x 5.5 cm x 3.3 cm were verified on imaging to be within + 1 mm of original patient scan. The 1 CC of contrast agent provided sufficient differentiation of "tumor" ring from "tissue." CT scan and treatment plan creation verified that the alignment device provided correction insertion of needles into the phantom tumor tissue and uterus. This pilot study provides a potential methodology to develop future anatomical phantoms and alignment devices from CT scans of patient data. Additional modifications could make this a viable training tool for future residents and medical students to learn brachytherapy. Key PointsO_LI3D printing can be leveraged to create a Brachytherapy alignment device from patients anatomy. C_LIO_LIAnatomical phantom can be generated from 3D printing for use testing device or for training. C_LIO_LIRelevant materials to create phantom that mimic patients anatomy on CT scan. C_LIO_LIGeneration of a treatment plan based on CT scan was able to validate device and phantom C_LI

bioengineering↗

bioRxiv2022

MutSigCVsyn: Identification of Thirty Synonymous Cancer Drivers

Synonymous mutations, which change only the DNA sequence but not the encoded protein sequence, can affect protein structure and function, mRNA maturation, and mRNA half-lives. The possibility that synonymous mutations can act as cancer drivers has been explored in several recent studies. However, none of these studies control for all three levels (patient, histology, and gene) of mutational heterogeneity that are known to affect the accurate identification of non-synonymous cancer drivers. Here, we create an algorithm, MutSigCVsyn, an adaptation of MutSigCV, to identify synonymous cancer drivers based on a novel non-coding background model that takes into account the mutational heterogeneity across these levels. Examining 2,572 PCAWG cancer whole-genome sequences, MutSigCVsyn identifies 30 novel synonymous drivers that include mutations in promising candidates like BCL-2. By bringing the best practices in non-synonymous driver identification to the analysis of synonymous drivers, these are promising candidates for future experimental study.

bioinformatics↗

bioRxiv2021

APOBEC-mediated Editing of SARS-CoV-2 Genomic RNA Impacts Viral Replication and Fitness

During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure. One-sentence summaryEfficient Editing of SARS-CoV-2 genomic RNA by Host APOBEC deaminases and Its Potential Impacts on the Viral Replication and Emergence of New Strains in COVID-19 Pandemic

microbiology↗

bioRxiv2021

LKB1 Is Physiologically Required for Sleep from Drosophila to the Mouse

LKB1 is known as a master kinase for 14 kinases related to the adenosine monophosphate (AMP)-activated protein kinase (AMPK). Two of them (SIK3 and AMPK) have previously been implicated in sleep regulation. We generated loss-of-function (LOF) mutants for Lkb1 in both Drosophila and mice. Sleep was reduced in Lkb1-mutant flies and in flies with neuronal deletion of Lkb1. Sleep was reduced in mice after virally mediated reduction of Lkb1 in the brain. Electroencephalography (EEG) analysis showed that non-rapid eye movement (NREM) sleep and sleep need were both reduced in Lkb1-mutant mice. These results indicate that LKB1 plays a physiological role in sleep regulation conserved from flies to mice.

neuroscience↗

bioRxiv2021

Sleep Need, the Key Regulator of Sleep Homeostasis, Is Indicated and Controlled by Phosphorylation of Threonine 221 in Salt Inducible Kinase 3

Sleep need drives sleep and plays a key role in homeostatic regulation of sleep. So far sleep need can only be inferred by animal behaviors and indicated by electroencephalography (EEG). Here we report that threonine 221 (T221) of the salt inducible kinase 3 (SIK3) was important for the catalytic activity and stability of SIK3. T221 phosphorylation in the mouse brain indicates sleep need: more sleep resulting in less phosphorylation and less sleep more phosphorylation during daily sleep/wake cycle and after sleep deprivation (SD). Sleep need was reduced in SIK3 loss of function (LOF) mutants and by T221 mutation to alanine (T221A). Sleep rebound after SD was also decreased in SIK3 LOF and T221A mutant mice. Other kinases such as SIK1 and SIK2 or other sites in SIK3 do not fulfil criteria to be both an indicator and a controller of sleep need. Our results reveal SIK3 T221 phosphorylation as the first and only chemical modification which indicates and controls sleep need.

neuroscience↗

bioRxiv2021

A Potential Novel COVID-19 Vaccine With RBD-HR1/HR2 Hexamer Structure

The COVID-19 pandemic and the continued spreading of the SARS-CoV-2 variants have brought a grave public health consequence and severely devastated the global economy with recessions. Vaccination is considered as one of the most promising and efficient methods to end the COVID-19 pandemic and mitigate the disease conditions if infected. Although a few vaccines have been developed with an unprecedented speed, scientists around the world are continuing pursuing the best possible vaccines with innovations. Comparing to the expensive mRNA vaccines and attenuated/inactivated SARS-CoV-2 vaccines, recombinant protein vaccines have certain advantages, including their safety (non-virus components), potential stronger immunogenicity, broader protection, ease of scaling-up production, reduced cost, etc. In this study, we reported a novel COVID-19 vaccine generated with RBD-HR1/HR2 hexamer that was creatively fused with the RBD domain and heptad repeat 1 (HR1) or heptad repeat 2 (HR2) to form a dumbbell-shaped hexamer to target the spike S1 subunit. The novel hexamer COVID-19 vaccine induced high titers of neutralizing antibody in mouse studies (>100,000), and further experiments also showed that the vaccine also induced an alternative antibody to the HR1 region, which probably alleviated the drop of immunogenicity from the frequent mutations of SARS-CoV-2.

biochemistry↗

bioRxiv2021

ppGpp Is Present in, and Regulates Sleep of, Drosophila

Discovery of molecules in living systems and demonstration of their functional roles are pivotal in furthering our understanding of the molecular basis of biology. ppGpp (guanosine-5-diphosphate, 3-diphosphate) has been detected in prokaryotes for more than five decades. Here we report that a genetic screen followed by chemical analysis revealed the presence of ppGpp in Drosophila. It can be detected in germ-free Drosophila and its level is controlled by an enzyme encoded by the mesh1 gene in Drosophila. Loss of function mutations in mesh1, which encoded the ppGpp degrading enzyme led to longer sleep latency and less total sleep. These phenotypes could be rescued by wild type mesh1, but not by the enzymatically defective mutant Mesh1E66A, functionally implicating ppGpp. Ectopic expression of RelA, the E. coli synthetase for ppGpp, phenocopied mesh1 knockout mutants, whereas overexpression of mesh1 resulted in the opposite phenotypes, supporting that ppGpp is both necessary and sufficient in sleep regulation. mesh1 is expressed in a specific population of neurons, and a chemoconnectomic screen followed by genetic intersection experiments implicate the pars intercerebralis (PI) as the site of ppGpp function. Our results have thus revealed that ppGpp is present in animals after long lag since its discovery in bacteria. Furthermore, we have demonstrated that ppGpp in a specific subset of neurons plays a physiological role in regulating sleep. We speculate that ppGpp may play function in mammals.

neuroscience↗