Biology subjects

Zhou, C.

Publications and source records attributed to Zhou, C..

At least 127 records · Page 7Linked to original sources

bioRxiv2024

Intermittent energy restriction inhibits tumor growth and enhances paclitaxel response in a transgenic mouse model of endometrial cancer

ObjectiveOverweight/obesity is the strongest risk factor for endometrial cancer (EC), and weight management can reduce that risk and improve survival. We aimed to establish the differential abilities of intermittent energy restriction (IER) and low-fat diet (LFD), alone and in combination with paclitaxel, to reverse the procancer effects of high-fat diet (HFD)-induced obesity in a mouse model of EC. MethodsLkb1fl/flp53fl/fl mice were fed high-fat diet (HFD) or LFD to generate obese and lean phenotypes, respectively. Obese mice were maintained on HFD or switched to LFD (HFD-LFD) or IER (HFD-IER). Ten weeks after induction of endometrial tumor, mice in each group received paclitaxel or placebo for 4 weeks. Body and tumor weights; tumoral transcriptomic, metabolomic and oxylipin profiles; and serum metabolic hormones and chemocytokines were assessed. ResultsHFD-IER and HFD-LFD, relative to HFD, reduced body weight; reversed obesity-induced alterations in serum insulin, leptin and inflammatory factors; and decreased tumor incidence and mass, often to levels emulating those associated with continuous LFD. Concurrent paclitaxel, versus placebo, enhanced tumor suppression in each group, with greatest benefit in HFD-IER. The diets produced distinct tumoral gene expression and metabolic profiles, with HFD-IER associated with a more favorable (antitumor) metabolic and inflammatory environment. ConclusionIn Lkb1fl/flp53fl/fl mice, IER is generally more effective than LFD in promoting weight loss, inhibiting obesity-related endometrial tumor growth (particularly in combination with paclitaxel), and reversing detrimental obesity-related metabolic effects. These findings lay the foundation for further investigations of IER as a EC prevention strategy in women with overweight/obesity.

cancer biology↗

bioRxiv2024

Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA

Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3'[->]5' exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII (Escherichia coli) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labelled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5'-bond of phosphodiester from 3' to 5' end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3'-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.

biochemistry↗

bioRxiv2024

BAMBI: Integrative biostatistical and artificial-intelligence method discover coding and non-coding RNA genes as biomarkers

Accurate disease diagnosis and prognosis are crucial for effective treatment management and improving patient outcomes. However, accurately detecting early signs of certain diseases or recurrence remains challenging. Existing machine-learning methods for identifying gene expression biomarkers have several limitations, including poor performance on independent test datasets, inability to directly process omics data, and difficulty in identifying noncoding RNA genes as biomarkers. Additionally, these methods may not provide sufficient biological interpretation of their results, and the panel biomarkers they identify may not be suitable for clinical application. To address these limitations, we have developed a new computational method called BAMBI, which integrates multiple machine-learning algorithms and statistical approaches to identify putative coding and noncoding genes as biomarkers for disease diagnosis and prognosis. We evaluated BAMBI ability to identify diagnostic and prognostic biomarkers by analyzing multiple RNA-seq datasets from cancerous and non-cancerous diseases at population levels. The results from BAMBI demonstrate significant biological interpretability and state-of-the-art prediction performance. When the singular gene identified by BAMBI is used as a diagnostic biomarker, it achieves a balance accuracy exceeding 95% in studies of both breast cancer and psoriasis. Additionally, the prognostic biomarkers that BAMBI identifies from RNA-seq data of Acute Myeloid Leukemia (AML) patients significantly correlate with the survival rates in an independent AML patient cohort. Additionally, BAMBI outperforms existing methods by delivering more robust results, identifying biomarkers with fewer genes, and simultaneously achieving superior prediction accuracy. We have implemented BAMBI into user-friendly software for the research community. In summary, BAMBI serves as a more reliable pipeline for identifying both coding and noncoding genes as biosignature markers, enhancing the accuracy of disease diagnosis and prognosis. BAMBI is available via https://github.com/CZhouLab/BAMBI.

bioinformatics↗

bioRxiv2023

An Endogenous Aryl Hydrocarbon Receptor Ligand Induces Preeclampsia-like Phenotypes: Transcriptome, Phosphoproteome, and Cell Functions

BackgroundPreeclampsia (PE) is one hypertensive disorder and a leading cause of maternal and fetal mortality and morbidity during human pregnancy. Aryl hydrocarbon receptor (AhR) is a transcription factor, which regulates vascular functions. Exogenous and endogenous AhR ligands can induce hypertension in animals. However, if dysregulation of endogenous AhR ligands contributes to the pathophysiology of PE remains elusive. MethodsWe measured AhR activities in human maternal and umbilical vein sera. We also applied physiological, cellular, and molecular approaches to dissect the role of endogenous AhR ligands in vascular functions during pregnancy using pregnant rats and primary human umbilical vein endothelial cells (HUVECs) as models. ResultsPE elevated AhR activities in human umbilical vein sera. Exposure of pregnant rats to an endogenous AhR ligand, 2-(1H-indole-3-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) increased blood pressure and proteinuria, while decreased uteroplacental blood flow and reduced fetal and placental weights, all of which are hallmarks of PE. ITE dampened vascular growth and fetal sex-specifically altered immune cell infiltration in rat placentas. ITE also decreased cell proliferation and cell monolayer integrity in HUVECs in vitro. RNA sequencing analysis revealed that ITE dysregulated transcriptome in rat placentas and HUVECs in a fetal sex-specific manner. Bottom-up phosphoproteomics showed that ITE disrupted phosphoproteome in HUVECs. These ITE-dysregulated genes and phosphoproteins were enriched in biological functions and pathways which are highly relevant to diseases of heart, liver, and kidney, vascular functions, inflammation responses, cell death, and kinase inhibition. ConclusionsDysregulation of endogenous AhR ligands during pregnancy may lead to the development of PE with underlying impaired vascular functions, fetal sex-specific immune cell infiltration and transcriptome, and phosphoproteome. Thus, this study has provided a novel mechanism for the development of PE and potentially other forms of hypertensive pregnancies. These AhR ligand-activated genes and phosphoproteins might represent promising therapeutic and fetal sex-specific targets for PE-impaired vascular functions.

pathology↗

bioRxiv2023

Site Effects in Multisite Fetal Brain MRI: Morphological Insights into Early Brain Development

BackgroundMultisite MRI studies have become prevalent given their advantage in revealing reliable biological or clinical findings. Adult and adolescent multisite studies have demonstrated inevitable site-related non-biological effects that introduce bias. However, the site effect on fetal brain MRI remains unknown. PurposeTo identify crucial acquisition factors affecting fetal brain structural measurements and developmental patterns, while assessing the effectiveness of existing harmonization methods in mitigating site effects. Materials and MethodsBetween May 2017 and March 2022, T2-weighted fast spin-echo sequences in-utero MRI were performed in healthy fetuses from prospectively recruited pregnant volunteers on four different scanners at four sites. A generalized additive model (GAM) was used to quantitatively assess site effects, including field strength (FS), manufacture (M), in-plane resolution (R), and slice thickness (ST), on subcortical volume and cortical morphological measurements, including cortical thickness, curvature, and sulcal depth. Growth models were selected to elucidate developmental trajectories of these morphological measurements. Welchs test was performed to evaluate the influence of site effects on developmental trajectories. ComBat-GAM harmonization method was applied to mitigate site-related biases. ResultsThe final analytic sample consisted of 340 MRI scans from 218 fetuses (mean gestational age, 30.1 weeks {+/-} 4.4 [range, 21.7-40 weeks]). GAM results showed that low FS and low spatial resolution led to overestimations in selected brain regions of subcortical volumes and cortical morphological measurements, and cortical measurements were more susceptible to site effects than subcortical volumes. Only the peak cortical thickness in developmental trajectories was significantly influenced by the effects of FS and R. Notably, ComBat-GAM harmonization effectively removed site effects while preserving developmental patterns. ConclusionOur findings pinpointed the key acquisition factors in in-utero fetal brain MRI and underscored the necessity of data harmonization when pooling multisite data for fetal brain morphology investigations.

bioengineering↗

bioRxiv2023

Xanthomonas euvesicatoria effector XeAvrRxo1 triggers a Rxo1-mediated defense response in Nicotiana benthamiana and its chaperone Xe4429 functions as an antitoxin, an expression repressor, and an enhancer of XeAvrRxo1 secretion

Xanthomonas euvesicatoria (X. euvesicatoria) is the causal agent of bacterial spot disease that threatens pepper and tomato production around the globe. X. euvesicatoria gene Xe4428 encodes a type III effector (T3E) that shares 89.67% amino acid identity with Xanthomonas oryzae pv. oryzicola (Xoc) T3E AvrRxo1. Deletion of Xe4428 in the genome of X. euvesicatoria (strain Xcv85-10) compromised its virulence to infect pepper and Nicotiana benthamiana plants. Transient co-expression of Xe4428 and Rxo1 on pepper and N. benthamiana plant leaves results in a robust hypersensitive reaction. Thus, Xe4428, renamed as XeAvrRxo1, is a bona fide orthologue of XocAvrRxo1 that possesses both virulence and avirulence functions. Expression of XeAvrRxo1 in E. coli and X. euvesicatoria is toxic to both bacterial cells. Another X. euvesicatoria gene Xe4429, encodes a putative chaperone of XeAvrRxo1, which can interact with XeAvrRxo1 to suppress its toxicity in X. euvesicatoria and E. coli bacterial cells. Xe4429 also binds to the promoter region of XeavrRxo1 and represses its transcription/translation in X. euvesicatoria bacterial cells. In addition, expression of Xe4429 can enhance the secretion and translocation of XeAvrRxo1 into plant cells. Therefore, Xe4429 functions as an antitoxin, a transcription repressor, and a type III chaperone that is capable of enhancing the secretion and translocation of XeAvrRxo1 during pathogenesis.

pathology↗

bioRxiv2023

DNA Methylation (DM) data format and DMtools for efficient DNA methylation data storage and analysis

DNA methylation is a critical epigenetic mechanism that plays a pivotal role in various biological processes. Currently, larger datasets from whole-genome bisulfite sequencing for DNA methylation pose challenges throughout the computational analysis pipeline, including storage and memory constraints. Unfortunately, storage formats and analysis tools have not kept pace with these increased resource demands. In this study, we present a new and efficient design for storing DNA methylation (DM) data after mapping in compressed binary indexed DM format. Our format significantly reduces storage space by 80%-95% compared to commonly used file formats for DNA methylation data after mapping. To enhance the processing of DNA methylation data in DM format, we have developed DMtools, a comprehensive toolkit that offers utilities such as rapid and random access, computation of DNA methylation profiles across genes, and analysis of differential DNA methylation. The analysis speed is improved by over 100 times compared to existing methods. Furthermore, we have created pyDMtools, a Python package that efficiently processes DM format files for Python users. The integration of the DM format and its associated tools represents significant progress in handling and exploring DNA methylation data, offering the potential to significantly reduce storage needs and improve downstream analysis capabilities.

bioinformatics↗

bioRxiv2023

Preoptic area controls sleep-related seizure onset in a genetic epilepsy mouse model

In genetic and refractory epileptic patients, seizure activity exhibits sleep-related modulation/regulation and sleep and seizure are intermingled. In this study, by using one het Gabrg2Q390X KI mice as a genetic epilepsy model and optogenetic method in vivo, we found that subcortical POA neurons were active within epileptic network from the het Gabrg2Q390XKI mice and the POA activity preceded epileptic (poly)spike-wave discharges(SWD/PSDs) in the het Gabrg2Q390X KI mice. Meanwhile, as expected, the manipulating of the POA activity relatively altered NREM sleep and wake periods in both wt and the het Gabrg2Q390X KI mice. Most importantly, the short activation of epileptic cortical neurons alone did not effectively trigger seizure activity in the het Gabrg2Q390X KI mice. In contrast, compared to the wt mice, combined the POA nucleus activation and short activation of the epileptic cortical neurons effectively triggered or suppressed epileptic activity in the het Gabrg2Q390X KI mice, indicating that the POA activity can control the brain state to trigger seizure incidence in the het Gabrg2Q390X KI mice in vivo. In addition, the suppression of POA nucleus activity decreased myoclonic jerks in the Gabrg2Q390XKI mice. Overall, this study discloses an operational mechanism for sleep-dependent seizure incidence in the genetic epilepsy model with the implications for refractory epilepsy. This operational mechanism also underlies myoclonic jerk generation, further with translational implications in seizure treatment for genetic/refractory epileptic patients and with contribution to memory/cognitive deficits in epileptic patients.

neuroscience↗

bioRxiv2023

A high-performance brain-sentence communication designed for logosyllabic language

Many severe neurological diseases, such as stroke and amyotrophic lateral sclerosis, can impair or destroy the ability of verbal communication. Recent advances in brain-computer interfaces (BCIs) have shown promise in restoring communication by decoding neural signals related to speech or motor activities into text. Existing research on speech neuroprosthesis has predominantly focused on alphabetic languages, leaving a significant gap of logosyllabic languages such as Mandarin Chinese which are spoken by more than 15% of the world population. Logosyllabic languages pose unique challenges to brain-to-text decoding due to extended character sets (e.g., 50,000+ for Mandarin Chinese) and complex mapping between characters and pronunciation. To address these challenges, we established a speech BCI designed for Mandarin, decoding speech-related stereoelectroencephalography (sEEG) signals into coherent sentences. We leverage the unique acoustic features of Mandarin Chinese syllables, constructing prediction models for syllable components (initials, tones, and finals), and employ a language model to resolve pronunciation to character ambiguities according to the semantic context. This method leads to a high-performance decoder with a median character accuracy of 71.00% over the full character set, demonstrating huge potentials for clinical application. To our knowledge, we are the first to report brain-to-sentence decoding for logosyllabic languages over full character set with a large intracranial electroencephalography dataset.

neuroscience↗

bioRxiv2023

Vascular abnormalities in heart and brain are associated with cardiovascular and neurological symptoms in a novel mouse model for Williams syndrome

Williams syndrome is a developmental disorder caused by a microdeletion entailing the loss of a single copy of 25-27 genes on chromosome 7q11.23. Patients suffer from cardiovascular and neuropsychological symptoms. Structural abnormalities of the cardiovascular system in Williams syndrome have been attributed to the hemizygous loss of the elastin (ELN) gene. In contrast, the neuropsychological consequences of Williams syndrome, including sensorimotor deficits, hypersociability and cognitive impairments, have been mainly attributed to altered expression of transcription factors like LIMK1, GTF2I and GTF2IRD1, while the potential impact of altered cerebrovascular function has been largely overlooked. To study the relationship between Williams syndrome mutations and vascularization of both the heart and brain, we generated a mouse model carrying a relatively long microdeletion (LD) that includes the Ncf1 gene, thereby minimizing the confounding impact of hypertension. LD mice had elongated and tortuous aortas but, unlike Eln haploinsufficient mice, showed no signs of structural cardiac hypertrophy. Remarkably, LD mice also displayed structural abnormalities in coronary and brain vessels, including disorganized extracellular matrices. Importantly, LD mice faithfully replicated both cardiovascular and neuropsychological symptoms observed in patients. The phenotype was even more comprehensive than former models, with structure-function correlations evident in aberrant auditory and motor behaviors resembling those in patients with Williams syndrome. Together, our findings suggest that not only cardiovascular but also neuropsychological symptoms in Williams syndrome may be driven in part by vascular abnormalities affecting both heart and brain. Significance StatementWilliams syndrome is caused by microdeletion of 25-27 genes on chromosome 7q11.23, resulting in cardiovascular and neuropsychological symptoms. It remains unclear how the affected genes interact and whether cardiovascular deficits influence brain function. We developed and characterized a mouse model with the longest Williams syndrome microdeletion to date. This model reveals interactions between genes that can be compensatory or additive: haploinsufficiency of Ncf1 may counteract the cardiac hypertrophy caused by Eln deletion, while vascular defects that are potentially due to Eln haploinsufficiency extend to the brain and may worsen neuropsychological symptoms. Our findings support the hypothesis that structural vascular deficits putatively contribute to both cardiac and cognitive phenotypes in Williams syndrome, opening new avenues for understanding and treating this syndrome.

systems biology↗

bioRxiv2023

Enhancing Mental Well-being: Efficacy and Neural Mechanisms of Leyi Training in Emotional and Cognitive Intervention

Leyi mental training comprises physical and mental cultivation methods, such as relaxation, breathing, meditation, koan, and cognitive regulation, with the ultimate goal of achieving enlightenment. Over a decade of practical experience has demonstrated its potential to enhance individuals mental well-being. In our study, we employed EEG and psychological scales to investigate the efficacy of Leyi training in emotional and cognitive intervention, as well as its underlying neural mechanisms. The results revealed significant improvements in both emotion and cognition following the intervention. We propose that the effectiveness of Leyi training in emotional regulation may stem from the heightened sensitivity to present experiences and the robust cognitive control ability cultivated through the practice, which enables practitioners to allocate equal, non-reactive attention to all stimuli, regardless of their emotional valence. Moreover, this training facilitates more effective allocation of cognitive resources to deal with current tasks, thus improving cognitive abilities.

neuroscience↗

bioRxiv2023

Prothoracicotropic hormone controls female sexual receptivity through the function of ecdysone in pC1 neurons of Drosophila

Female sexual receptivity is essential for reproduction of a species. Neuropeptides play the main role in regulating female receptivity. However, whether neuropeptides regulate female sexual receptivity during the neurodevelopment is unknown. Here we found the peptide hormone prothoracicotropic hormone (PTTH), which belongs to the insect PG axis, negatively regulated virgin female receptivity through ecdysone during neurodevelopment in Drosophila melanogaster. We identified PTTH neurons as doublesex-positive neurons, they regulated virgin female receptivity before the metamorphosis during the 3rd-instar larval stage. PTTH deletion resulted in the increased EcR-A expression in the whole newly formed prepupae. Furthermore, the ecdysone receptor EcR-A in pC1 neurons positively regulated virgin female receptivity during metamorphosis. The decreased EcR-A in pC1 neurons induced abnormal morphological development of pC1 neurons without changing neural activity. Among all subtypes of pC1 neurons, the function of EcR-A in pC1b neurons was necessary for virgin female copulation rate. These suggested that the changes of synaptic connections between pC1b and other neurons decreased female copulation rate. Moreover, female receptivity significantly decreased when the expression of PTTH receptor Torso was reduced in pC1 neurons. This suggested that PTTH not only regulates female receptivity through ecdysone but also through affecting female receptivity associated neurons directly. The PG axis has similar functional strategy as the HPG axis in mammals to trigger the juvenile-adult transition. Our work suggests a general mechanism underlying which the neurodevelopment during maturation regulates female sexual receptivity.

neuroscience↗

bioRxiv2023

Spatial transcriptomics and single-nucleus RNA sequencing reveal a transcriptomic atlas of adult human spinal cord

Despite the recognized importance of the spinal cord in sensory processing, motor behaviors, and neural diseases, the underlying organization of neuronal clusters and their spatial location remain elusive. Recently, several studies have attempted to define the neuronal types and functional heterogeneity in the spinal cord using single-cell or single-nucleus RNA sequencing in animal models or developing humans. However, molecular evidence of cellular heterogeneity in the adult human spinal cord is limited. Here, we classified spinal cord neurons into 21 subclusters and determined their distribution from nine human donors using single-nucleus RNA sequencing and spatial transcriptomics. Moreover, we compared the human findings with previously published single-nucleus data of the mouse adult spinal cord, which revealed an overall similarity in the neuronal composition of the spinal cord between the two species while simultaneously highlighting some degree of heterogeneity. Additionally, we examined the sex differences in the spinal neuronal subclusters. Several genes, such as SCN10A and HCN1, showed sex differences in motor neurons. Finally, we classified human dorsal root ganglia (DRG) neurons using spatial transcriptomics and explored the putative interactions between DRG and spinal cord neuronal subclusters. In summary, these results illustrate the complexity and diversity of spinal neurons in humans and provide an important resource for future research to explore the molecular mechanisms underlying spinal cord physiology and diseases.

neuroscience↗

bioRxiv2023

Exploring Degradation of Intrinsically Disordered Protein YAP induced by PROTACs

Yes-associated protein (YAP), a potent oncogene and a key player in the Hippo tumor suppression pathway, has long been considered challenging to target due to its partially intrinsically disordered nature. However, recent advances in High-throughput Screening (HTS) have led to the discovery of a few YAP binders. Building upon this progress, a novel approach utilizing Proteolysis-Targeting Chimera (PROTAC) technology was employed to design and synthesize a series of YAP degraders. Here, our degraders were created by linking NSC682769, a previously reported YAP binder, with either VHL ligand 2 or pomalidomide using various linkers of different lengths and types. The most promising degrader YZ-6 recruits the E3 ligase VHL, inducing rapid and sustained YAP degradation leading to suppression of YAP/TEAD-led transcription in both YAP-dependent NCI-H226 and Huh7 cancer cell lines. In addition to its degradation capabilities, YZ-6 also exhibited potent antiproliferative activity in both cell lines. Importantly, YZ-6 efficiently suppresses tumor development in the Huh7 xenograft mouse model without adverse effects on the mice. These findings highlight the potential of PROTAC-mediated degradation as a viable strategy for reducing oncogenic YAP levels and attenuating downstream signaling in cancer cells. Moreover, the development of PROTACs based on NSC672869 holds promise for treating YAP-driven malignancies.

cancer biology↗

bioRxiv2023

Post-Mating Inhibition of Female Sexual Drive via Heterogeneous Neuronal Ensembles in the Medial Preoptic Area

Male ejaculation acutely suppresses sexual motivation in male mice. In contrast, relatively little is known about how male ejaculation affects sexual motivation and sexual behavior in female mice. How the brain responds to completion of mating is also unclear. Here, by using self-paced mating assay, we first demonstrate that female mice show decreased sexual motivation acutely after experiencing male ejaculation. By using brain-wide analysis of activity-dependent labeling, we next pinpointed the medial preoptic area as a brain region strongly activated during the post-ejaculatory period. Furthermore, using freely moving in vivo calcium imaging to compare neural activity of inhibitory and excitatory neurons in the medial preoptic area, we revealed that a subset of the neurons in this region respond significantly and specifically to male ejaculation but not to female-to-male sniffing or to male mounting. While there were excitatory and inhibitory neurons that showed increased response to male ejaculation, the response magnitude as well as the proportion of neurons responding to the event was significantly larger in the inhibitory neuron population. Next, by unbiased classification of their responses, we also found a subpopulation of neurons that increase their activity late after the onset of male ejaculation. These neurons were all inhibitory indicating that male ejaculation induces a prolonged inhibitory activity in the medial preoptic area. Lastly, we found that chemogenetic activation of medial preoptic area neurons that were active during post-ejaculatory period, but not during appetitive or consummatory periods, were sufficient to suppress female sexual motivation. Together, our data illuminate the importance of medial preoptic area as a brain node which encodes a negative signal that sustains low sexual motivation state after the female mice experience ejaculation. HighlightsO_LIFemale mice show decreased sexual motivation in the post-ejaculatory period. C_LIO_LIA subset of MPOA neurons in female respond specifically to male ejaculation. C_LIO_LIMale-ejaculation evokes persistent activity in MPOA inhibitory neurons in females. C_LIO_LIActivation of a subset of MPOA neurons is sufficient to suppress female sexual motivation. C_LI

neuroscience↗

bioRxiv2023

High-efficiency gene editing in Anopheles sinensis using ReMOT control

CRISPR/Cas9-mediated gene editing provides an effective method for deciphering the molecular mechanisms underlying mosquito development and mosquito-borne disease transmission, as well as for exploring genetic control strategies. However, delivering the Cas9 ribonucleoprotein complex by embryo injection to produce genetic modifications is challenging, is mostly confined to model mosquitoes and specialized laboratories, and has low editing efficiency. Here, we established an effective Receptor-Mediated Ovary Transduction of Cargo (ReMOT) control method, enabling the introduction of heritable mutations into Anopheles sinensis, the major malaria vector in China and Southeast Asia, via the injection of female adult mosquitoes. Injection of a mixture of P2C-DsRed and saponin resulted in red fluorescence in the ovaries, with a 100% success rate. Using this system, we knocked-out the pigment synthesis genes, Aswhite and Asyellow, using injected wild-type (WT) females mated with WT males, resulting in the highest efficiency of gene editing among mosquitoes under the same mating conditions. Furthermore, the gene-editing efficiency was increased by at least 2.1-fold using injected WT females mated with mutant males. This improved ReMOT control method exhibits high editing efficiency, with important benefits in terms of functional genomics research and genetic control strategies in An. sinensis. Moreover, this represents a convenient method for gene manipulation in laboratories that are unable to perform embryo injection or that lack embryo-injection equipment.

genetics↗

bioRxiv2023

Prevotella copri-linked effects of a therapeutic food for malnutrition

Preclinical and clinical studies are providing evidence that the healthy growth of infants and children reflects, in part, healthy development of their gut microbiomes1-5. This process of microbial community assembly and functional maturation is perturbed in children with acute malnutrition. Gnotobiotic animals, colonized with microbial communities from children with severe and moderate acute malnutrition, have been used to develop microbiome-directed complementary food (MDCF) formulations for repairing the microbiomes of these children during the weaning period5. Bangladeshi children with moderate acute malnutrition (MAM) participating in a previously reported 3-month-long randomized controlled clinical study of one such formulation, MDCF-2, exhibited significantly improved weight gain compared to a commonly used nutritional intervention despite the lower caloric density of the MDCF6. Characterizing the metagenome assembled genomes (MAGs) of bacterial strains present in the microbiomes of study participants revealed a significant correlation between accelerated ponderal growth and the expression by two Prevotella copri MAGs of metabolic pathways involved in processing of MDCF-2 glycans1. To provide a direct test of these relationships, we have now performed reverse translation experiments using a gnotobiotic mouse model of mother-to-offspring microbiome transmission. Mice were colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains cultured from Bangladeshi infants/children in the study population, with or without P. copri isolates resembling the MAGs. By combining analyses of microbial community assembly, gene expression and processing of glycan constituents of MDCF-2 with single nucleus RNA-Seq and mass spectrometric analyses of the intestine, we establish a principal role for P. copri in mediating metabolism of MDCF-2 glycans, characterize its interactions with other consortium members including Bifidobacterium longum subsp. infantis, and demonstrate the effects of P. copri-containing consortia in mediating weight gain and modulating the activities of metabolic pathways involved in lipid, amino acid, carbohydrate plus other facets of energy metabolism within epithelial cells positioned at different locations in intestinal crypts and villi. Together, the results provide insights into structure/function relationships between MDCF-2 and members of the gut communities of malnourished children; they also have implications for developing future prebiotic, probiotic and/or synbiotic therapeutics for microbiome restoration in children with already manifest malnutrition, or who are at risk for this pervasive health challenge.

microbiology↗

bioRxiv2023

Conserved long noncoding RNA TILAM promotes liver fibrosis through interaction with PML in hepatic stellate cells

Background & AimsFibrosis is the common endpoint for all forms of chronic liver injury, and progression of fibrosis leads to the development of end-stage liver disease. Activation of hepatic stellate cells (HSCs) and their transdifferentiation to myofibroblasts results in the accumulation of extracellular matrix (ECM) proteins that form the fibrotic scar. Long noncoding (lnc) RNAs regulate the activity of HSCs and may provide targets for fibrotic therapies. MethodsWe identified lncRNA TILAM as expressed near COL1A1 in human HSCs and performed loss-of-function studies in human HSCs and liver organoids. Transcriptomic analyses of HSCs isolated from mice defined the murine ortholog of TILAM. We then generated Tilam-deficient GFP reporter mice and quantified fibrotic responses to carbon tetrachloride (CCl4) and choline-deficient L-amino acid defined high fat diet (CDA-HFD). Co-precipitation studies, mass spectrometry, and gene expression analyses identified protein partners of TILAM. ResultsTILAM is conserved between human and mouse HSCs and regulates expression of ECM proteins, including collagen. Tilam is selectively induced in HSCs during the development of fibrosis in vivo. In both male and female mice, loss of Tilam results in reduced fibrosis in the setting of CCl4 and CDA-HFD injury models. TILAM interacts with promyelocytic leukemia protein (PML) to stabilize PML protein levels and promote the fibrotic activity of HSCs. ConclusionTILAM is activated in HSCs and interacts with PML to drive the development of liver fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end stage liver disease.

cell biology↗