Biology subjects

Hu, G.

Publications and source records attributed to Hu, G..

At least 109 records · Page 6Linked to original sources

bioRxiv2022

Network-based hub biomarker discovery for glaucoma

Glaucoma is an optic neuropathy, and the leading cause of irreversible blindness worldwide. However, the early detection of glaucoma remains challenging as chronic forms of glaucoma remain largely asymptomatic until considerable irreversible visual field deficits have ensued. Thus, biomarkers that facilitate early diagnosis and treatment for patients with a high risk of progression are critical. Network medicine approaches can be useful in identifying key relationships and important biomolecules for complex diseases. In this paper, we identified several hub biomarkers/drug targets for the diagnosis, treatment and prognosis for glaucoma and explored their associations for glaucoma based on human disease-biomarker and disease-target-drug networks. These results were verified by text-mining and genomic/epidemiology data. We also predicted the new application of BMP1 and MMP9 to diagnose glaucoma and confirm the theory of hub biomarkers with multiple clinical applications. Further, relevant pivotal pathways (regulation of the multicellular organismal process, regulation of localisation, and cytoplasmic vesicle for biomarkers; signal transduction and developmental process for targets) for these hub biomolecules were discovered, which may be foundations for future biomarker and drug target prediction for glaucoma. In conclusion, based on complex networks, hub biomolecules, essential pathways, and close diseases were identified for glaucoma in diagnosis, treatment and prognosis.

bioinformatics↗

bioRxiv2022

RACK1 may participate in placental development via regulating proliferation and migration of trophoblast cell in pigs following intrauterine growth restriction

Intrauterine growth restriction (IUGR) is a severe complication in swine production. Placental insufficiency is responsible for inadequate fetal growth, but the specific etiology of placental dysfunction-induced IUGR in pigs remains poorly understood. In this work, placenta samples supplying the lightest-weight (LW) and mean-weight (MW) pig fetuses in the litter at day 65 (D65) of gestation were collected, and the relationship between fetal growth and placental morphologies and functions was investigated using histomorphological analysis, RNA sequencing, quantitative polymerase chain reaction, and in-vitro experiment in LW and MW placentas. Results showed that the folded structure of the epithelial bilayer of LW placentas followed a poor and incomplete development compared with that of MW placentas. A total of 632 differentially expressed genes (DEGs) were screened out between the LW and MW placentas, and RACK1 was found to be downregulated in LW placentas. The DEGs were mainly enriched in translation, ribosome, protein synthesis, and mTOR signaling pathway according to GO and KEGG enrichment analyses. In-vitro experiments indicated that the decreased RACK1 in LW placentas may be involved in abnormal development of placental folds (PFs) by inhibiting the proliferation and migration of porcine trophoblast cells. Taken together, these results revealed that RACK1 may be a vital regulator in the development of PFs via regulating trophoblast ribosome function, proliferation, and migration in pigs.

genetics↗

bioRxiv2022

Identifying Lymph Node Metastasis-related Factors in Breast Cancer using Differential Modular and Mutational Structural Analysis

Complex diseases are generally caused by disorders of biological networks and/or mutations in multiple genes. Network theory provides useful tools to study the underlying laws governing complex diseases. Within this framework, comparisons of network topologies, including the node, edge, and community, between different disease states can highlight key factors within these dynamic processes. Here, we propose a differential modular analysis approach that integrates protein-protein interactions with gene expression profiles for modular analysis, and introduces inter-modular edges and date hubs to identify the "core network module" that quantifies the significant phenotypic variation. Then, based on this core network module, key factors including functional protein-protein interactions, pathways, and drive mutations are predicted by the topological-functional connection score and structural modeling. We applied the approach to analyze the lymph node metastasis (LNM) process in breast cancer. The functional enrichment analysis showed that both inter-modular edges and date hubs play important roles in cancer metastasis and invasion, and in metastasis hallmarks. The structural mutation analysis suggested that the LNM of breast cancer may be the outcome of the dysfunction of rearranged during transfection (RET) proto-oncogene-related interactions and the non-canonical calcium signaling pathway via an allosteric mutation of RET. We believe that the proposed method can provide new insights into disease progression such as cancer metastasis. Author summaryMetastasis is the hallmark of cancer that is responsible for the greatest number of cancer-related deaths. However, it remains poorly understood. PPI networks not only provide a static picture of cellular function and biological processes, but also have emerged as new paradigms in the study of the dynamic process of disease progression, including cancer metastasis. Herein, a network-based strategy was proposed based on the integration of expression profiles with protein interactions, by filtering with "date hubs" and "inter-modular edges", demonstrating that different network modules may provide robust predictors to represent the dynamic mechanisms involved in metastasis formation. Furthermore, the mapping of protein structure and mutation data on the network module level provides insight into signaling mechanisms; helps understand the mechanism of disease-related mutations; and helps in drug discovery. The application of our method to study the LNM in breast cancer highlights network modules defining protein communities that respond to therapeutics, and the implications of detailed structural and mechanistic insight into oncogenic activation and how it can advance allosteric precision oncology.

systems biology↗

bioRxiv2022

Altered tau in rTg4510 mice after a single interfaced CHIMERA traumatic brain injury

Traumatic brain injury (TBI) in an established risk factor for neurodegenerative disease. In this report, we used the Closed Head Injury Model of Engineered Rotational Acceleration (CHIMERA) to study the effects of a single moderate-severe TBI in rTg4510 mice, a mouse model of tauopathy. Fifteen male rTg4510 mice were impacted at 4.0J at 4-mo of age using interfaced CHIMERA and compared to sham controls. Immediately after injury, moderate-severe TBI induced significant mortality (7/15; 47%), and a prolonged duration of loss of righting reflex. At 2-mo post-injury, surviving mice displayed significant histological evidence of microgliosis (Iba1) and axonal injury (Neurosilver). Western blotting showed that TBI mice had a reduced p-GSK-3{beta} (S9):GSK-3{beta} ratio, suggesting greater tau kinase activity. However, tauopathy in surviving TBI mice showed a divergent response, with 3/8 mice having a very low level of tau protein in brain lysates (i.e. lower than sham), and were thus analyzed separately from the other 5/8 TBI mice that maintained the expected level of total tau. Compared to sham controls, TBI mice with normal tau levels had increased p-tau (PHF1 and AT8), increased autophagolysosome accumulation (p62 and Cathepsin D) and decreased hippocampal size. These findings were not observed in TBI mice with low total tau levels. These observations suggest that TBI leads to chronic white matter injury and altered GSK-3{beta} activity. However, post-injury tauopathy and autophagolysosome accumulation diverged in surviving mice through mechanisms that remain to be defined.

neuroscience↗

bioRxiv2022

EDoF-Miniscope: pupil engineering for extended depth-of-field imaging in a fluorescence miniscope

Extended depth of field (EDoF) microscopy has emerged as a powerful solution to greatly increase the access into neuronal populations in table-top imaging platforms. Here, we present EDoF-Miniscope, which integrates an optimized thin and lightweight binary diffractive optical element (DOE) onto the gradient refractive index (GRIN) lens of a head-mounted fluorescence miniature microscope, i.e. "miniscope". We achieve an alignment accuracy of 70 m to allow a 2.8X depth-of-field extension between the twin foci. We optimize the phase profile across the whole back aperture through a genetic algorithm that considers the primary GRIN lens aberrations, optical property of the submersion media, and axial intensity loss from tissue scattering in a Fourier optics forward model. Compared to other computational miniscopes, our EDoF-Miniscope produces high-contrast signals that can be recovered by a simple algorithm and can successfully capture volumetrically distributed neuronal signals without significantly compromising the speed, signal-to-noise, signal-to-background, and maintain a comparable 0.9-m lateral spatial resolution and the size and weight of the miniature platform. We demonstrate the robustness of EDoF-Miniscope against scattering by characterizing its performance in 5-m and 10-m beads embedded in scattering phantoms. We demonstrate that EDoF-Miniscope facilitates deeper interrogations of neuronal populations in a 100-m thick mouse brain sample, as well as vessels in a mouse brain. Built from off-the-shelf components augmented by a customizable DOE, we expect that this low-cost EDoF-Miniscope may find utility in a wide range of neural recording applications.

bioengineering↗

bioRxiv2022

The LncRNA Carmn is a Critical Regulator for Gastrointestinal Smooth Muscle Contractile Function and Motility

Background & aimsVisceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract and are critical for regulating motility. SMC contraction is regulated by changes in post-translational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality but the mechanisms regulating the expression levels of SMC-specific contractile proteins, including the role of long non-coding RNAs (lncRNAs), remains largely unexplored. Herein, we have uncovered an important role of Carmn (Cardiac mesoderm enhancer-associated noncoding RNA), a SMC-specific lncRNA, in regulating the phenotype of visceral SMCs of the GI tract. MethodsAnalysis of GTEx and publicly available single-cell RNA sequencing (scRNA-seq) datasets from embryonic, adult human and mouse GI tissues were used to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using a novel GFP knock-in (KI) reporter/knockout (KO) mouse model. Bulk RNA sequencing (RNA-seq) and single nuclei RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms. ResultsUnbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is specifically expressed in SMCs in human and mouse GI tract. Premature lethality was observed in global Carmn KO (gKO) and inducible SMC-specific KO (iKO) mice due to colonic pseudo-obstruction, severe distension of the GI tract with blockages in cecum and colon segments. Histology, whole-gut GI transit time and muscle myography analysis revealed severe dilation, significantly delayed GI transit and impaired GI contractility in Carmn KO mice versus control mice. Bulk RNA-seq of colonic muscularis revealed that Carmn deficiency promotes SMC de-differentiation as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes including Mylk, a key regulator of SMC contraction. SnRNA-seq further revealed SMC Carmn deficiency not only compromised myogenic motility by reducing expression of contractile genes but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance as silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression including MYLK and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program. ConclusionOur data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice, and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.

cell biology↗

bioRxiv2022

A New Classification Method Based on Dynamic Ensemble Selection and its Application to Predict Variance Patterns in HIV-1 Env

Therapeutics that target the envelope glycoproteins (Envs) of human immunodeficiency virus type 1 (HIV-1) effectively reduce virus levels in patients. However, due to mutations, new Env variants are frequently generated, which may be resistant to the treatments. The appearance of such sequence variance at any Env position is seemingly random. A better understanding of the spatiotemporal patterns of variance across Env may lead to the development of new therapeutic strategies. We hypothesized that, at any time point in a patient, positions with sequence variance are clustered on the three-dimensional structure of Env. To test this hypothesis, we examined whether variance at any Env position can be predicted by the variance measured at adjacent positions. Sequences from 300 HIV-infected patients were applied to a new algorithm we developed. The k-best classifiers (KBC) method is a dynamic ensemble selection technique that identifies the best classifier(s) within the neighborhood of a new observation. It applies bootstrap resampling to generate out-of-bag samples that are used with the resampled set to evaluate each classifier. For many positions of Env, primarily in the CD4-binding site, KBC accurately predicted variance based on the variance at their adjacent positions. KBC improved performance compared to the initial learners, static ensemble, and other baseline models. KBC also outperformed other algorithms for predicting variance at multi-position footprints of therapeutics on Env. These understandings can be applied to refine models that predict future changes in HIV-1 Env. More generally, we propose KBC as a new high-performance dynamic ensemble selection technique.

bioinformatics↗

bioRxiv2022

Patterns of Volatility Across the Spike Protein Accurately Predict the Emergence of Mutations within SARS-CoV-2 Lineages

Mutations in the spike glycoprotein of SARS-CoV-2 allow the virus to probe the sequence space in search of higher-fitness states. New sublineages of SARS-CoV-2 variants-of-concern (VOCs) continuously emerge with such mutations. Interestingly, the sites of mutation in these sublineages vary between the VOCs. Whether such differences reflect the random nature of mutation appearance or distinct evolutionary spaces of spike in the VOCs is unclear. Here we show that each position of spike has a lineage-specific likelihood for mutations to appear and dominate descendent sublineages. This likelihood can be accurately estimated from the lineage-specific mutational profile of spike at a protein-wide level. The mutability environment of each position, including adjacent sites on the protein structure and neighboring sites on the network of comutability, accurately forecast changes in descendent sublineages. Mapping of imminent changes within the VOCs can contribute to the design of immunogens and therapeutics that address future forms of SARS-CoV-2.

bioinformatics↗

bioRxiv2022

Dissecting Mutational Allosteric Effects in Alkaline Phosphatases Associated with Different Hypophosphatasia Phenotypes: An Integrative Computational Investigation

Hypophosphatasia (HPP) is a rare inherited disorder characterized by defective bone mineralization, is highly variable in its clinical phenotype. The disease occurs due to various loss-of-function mutations in ALPL, the gene encoding tissue-nonspecific alkaline phosphatase (TNSALP). In this work, a data-driven and biophysics-based approach for large-scale analysis of ALPL mutations - from nonpathogenic to severe HPPs is proposed. Allosteric molecular signatures of ALPL mutations were determined using an integrated pipeline of synergistic approaches including sequence and energetic-based analysis, structural topology network modeling and elastic network models. Statistical analysis of molecular features computed for the ALPL mutations showed a significant difference between the control and mild and severe HPP phenotypes, and the developed machine learning model suggested that the topological network parameters could serve as is a robust indicator for severe mutations. Molecular dynamics simulations coupled with protein structure network was employed to analyze the effect of single-residue variation on conformational dynamics of TNSALP dimers, and we found that severe disease-associated mutations have a significantly greater effect on allosteric communications. The results of this study suggested that ALPL mutations associated with mild and severe HPPs tend to have different effects on protein stability on local scale and long-range communications caused by network rewiring. By linking disease phenotypes with allosteric molecular signatures, the proposed integrative computational approach elucidates the complex sequence-structure-allostery relationships of ALPL mutations and dissects the role of allosteric effects in the pathogenesis of HPPs. Author SummaryThe understanding of mutational genotype- disease phenotype relationship is a fundamental step for enabling precision medicine. It remains a challenging task to assess the molecular principle of the genotype- phenotype relationship. By focusing on Hypophosphatasia, a rare inherited disorder, as an example, we performed comprehensive analysis of single-amino-acid mutations in the encoded protein of Tissue Nonspecific Alkaline Phosphatase associated in terms of their sequence, structure, and dynamics properties. We further developed a machine learning -based method to classify different disease phenotypes, and the interpretability of the classification model was addressed by the structural-functional analysis of network topological important mutations. Our results highlighted the allosteric propensity of severe mutations, and show the potential allosteric principle of genotype- phenotype relationship.

biophysics↗

bioRxiv2022

Adeno-associated Virus Receptor-binding: Flexible Domains and Alternative Conformations through cryo-Electron Tomography of AAV2 and AAV5 complexes

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAVs molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near universal entry receptor, AAVR, and structures by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now {beta}-strand A is the minor conformer and, for the major conformer, partially ordered N-termini near the 2-fold axis join the canonical capsid jellyroll fold at the {beta}A-{beta}B turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCEWith 150 clinical trials for 30 diseases underway, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction, has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.

microbiology↗

bioRxiv2021

Uterine-specific SIRT1 deficiency confers premature uterine aging and impairs invasion and spacing of blastocyst and stromal cell decidualization in mice.

A distinct age-related alteration in the uterine environment has recently been identified as a prevalent cause of the reproductive decline in older female mice. However, the molecular mechanisms that underlie age-associated uterine adaptability to pregnancy are not known. Sirtuin 1 (SIRT1), a multifunctional NAD+-dependent deacetylase that regulates cell viability, senescence and inflammation during aging, is reduced in aged decidua. Thus, we hypothesize that SIRT1 plays a critical role in uterine adaptability to pregnancy and that uterine-specific ablation of Sirt1 gene accelerates premature uterine aging. Female mice with uterine ablation of Sirt1 gene using progesterone receptor Cre (PgrCre) exhibit subfertility and signs of premature uterine aging. These Sirt1-deficient mothers showed decreases in litter size from their 1st pregnancy and became sterile (25.1{+/-}2.5 weeks of age) after giving birth to the 3rd litter. We report that uterine-specific Sirt1 deficiency impairs invasion and spacing of blastocysts, and stromal cell decidualization, leading to abnormal placentation. We found that these problems traced back to the very early stages of hormonal priming of the uterus. During the window of receptivity, Sirt1 deficiency compromises uterine epithelial-stromal crosstalk, whereby estrogen (E2), progesterone (P4) and Indian hedgehog (IHH) signaling pathways are dysregulated, hampering stromal cell priming for decidualization. Uterine transcriptomic analyses also link these causes to perturbations of histone proteins and epigenetic modifiers, as well as adrenomedullin signaling, hyaluronic acid metabolism, and cell senescence. Strikingly, our results also identified genes with significant overlaps with the transcriptome of uteri from aged mice and transcriptomes related to master regulators of decidualization (e.g., Foxo1, Wnt4, Sox17, Bmp2, Egfr and Nr2f2). Our results also implicate accelerated deposition of aging-related fibrillar type I and III collagens in Sirt1-deficient uteri. Collectively, SIRT1 is an indispensable age-related regulator of invasion and spacing of blastocysts, as well as decidualization of stromal cells. Author SummaryAdvanced maternal age (i.e., [≥]35 years old) is considered a significant risk factor for birth defects, including fetal growth restriction, stillbirth, preterm birth, and preeclampsia. Lifestyle factors such as tobacco smoke, alcohol usage and other environmental toxins are facilitators of premature reproductive aging. Thus, understanding the precise mechanisms by which female reproductive organs age is a prerequisite for ultimately developing counteracting therapies. Much attention has been focused on ovarian function and oocyte quality, but we provide evidence that severe placentation defects are a major cause of age-related reproductive decline, which results from an impaired decidual response by uterine stromal cells. This problem is due to a blunted progesterone (P4) responsiveness of the aging uterus, via its cognate receptor, PGR. In the present study, the uterine SIRT1 gene was deleted to advance understanding of the genetics of premature uterine aging in a mouse model of blunted PGR actions that is similar to physiological aging. Our results have informed as to molecular changes in response to blunting PGR actions in aging uteri that are unrelated to oocyte quality, and provide new insights into strategies for developing counteracting measures for pregnancy in females at an advanced reproductive age.

genetics↗

bioRxiv2021

Dual domestication, diversity, and differential introgression in Old World cotton diploids

Domestication in the cotton genus is remarkable in that it has occurred independently four different times at two different ploidy levels. Relatively little is known about genome evolution and domestication in the cultivated diploid species Gossypium herbaceum and G. arboreum, because of the absence of wild representatives for the latter species, their ancient domestication, and their joint history of human-mediated dispersal and interspecific gene flow. Using in-depth resequencing of a broad sampling from both species, we confirm their independent domestication, as opposed to a progenitor-derivative relationship, showing that diversity (mean {pi} = 2.3x10-3) within species is similar, and that divergence between species is modest (weighted FST=0.4430). Individual accessions were homozygous for ancestral SNPs at over half of variable sites, while fixed, derived sites were at modest frequencies. Notably, two chromosomes with a paucity of fixed, derived sites (i.e., chromosomes 7 and 10) were also strongly implicated in introgression analyses. Collectively, these data demonstrate variable permeability to introgression among chromosomes, which we propose is due to divergent selection under domestication and/or the phenomenon of F2 breakdown in interspecific crosses. Our analyses provide insight into the evolutionary forces influencing diversity and divergence in the diploid cultivated species, and establish a foundation for understanding the contribution of introgression and/or strong parallel selection to the extensive morphological similarities shared between species. Significance statementThe cotton genus (Gossypium) contains four different species that were independently domesticated at least 4,000 years ago. Relatively little is understood about diversity and evolution in the two diploid African-Asian sister-species G. herbaceum and G. arboreum, despite their historical importance in the region and contemporary cultivation, largely in the Indian subcontinent. Here we address questions regarding the relationship between the two species, their contemporary levels of diversity, and their patterns of interspecific gene flow accompanying their several millennia history of human-mediated dispersal and contact. We validate independent domestication of the two species and document the genomic distribution of interspecific genetic exchange.

evolutionary biology↗

bioRxiv2021

Limited variation between SARS-CoV-2-infected individuals in domain specificity and relative potency of the antibody response against the spike glycoprotein

The spike protein of SARS-CoV-2 is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The variation between infected individuals in domain specificity of the antibodies and in their relative neutralization efficacy is still poorly characterized. To this end, we tested serum and plasma samples from 85 COVID-19 convalescent subjects using 7 immunoassays that employ different domains, subunits and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. We observed strong correlations between the levels of NTD- and RBD-specific antibodies, with a fixed ratio of each type to all anti-spike antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects, and was not associated with the overall amount of anti-spike antibodies produced. Accordingly, the ability of immunoassays that use RBD, NTD and different forms of S1 or S1/S2 as capture antigens to estimate the neutralizing efficacy of convalescent samples was largely similar. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency. IMPORTANCEInfection by SARS-CoV-2 elicits antibodies against various domains of the spike protein, including the RBD, NTD and S2. Different infected individuals generate vastly different amounts of anti-spike antibodies. By contrast, as we show here, there is a remarkable similarity in the properties of the antibodies produced. Different individuals generate the same proportions of antibodies against each domain of the spike protein. Furthermore, the relationship between the amount of anti-spike antibodies produced and their neutralization efficacy of SARS-CoV-2 is highly conserved. Therefore, the observed variation in the neutralizing activity of the antibody response in COVID-19 convalescent subjects is caused by differences in the amounts of antibodies rather than their recognition properties or relative antiviral activity. These findings suggest that COVID-19 vaccine strategies that focus on enhancing the overall level of the antibodies will likely elicit a more uniformly efficacious protective response.

microbiology↗

bioRxiv2021

Binding Free energy Decomposition and Multiple Unbinding Paths of Buried Ligands in a PreQ1 Riboswitch

Riboswitches are naturally occurring RNA elements that control bacterial gene expression by binding to specific small molecules. They serve as important models for RNA-small molecule recognition and have also become a novel class of targets for developing antibiotics. Here, we carried out conventional and enhanced-sampling molecular dynamics (MD) simulations, totaling 141.5 s, to characterize the determinants of binding free energies and unbinding paths for the cognate and synthetic ligands of a PreQ1 riboswitch. Binding free energy analysis showed that two triplets of nucleotides U6-C15-A29 and G5-G11-C16, contribute the most to the binding of the cognate ligands, by hydrogen bonding and by base stacking, respectively. Mg2+ ions are essential in stabilizing the binding pocket. For the synthetic ligands, the hydrogen-bonding contributions of the U6-C15-A29 triplet are significantly compromised, and the bound state resembles the apo state in several respects, including the disengagement of the C15-A14-A13 and A32-G33 base stacks. The bulkier synthetic ligands lead to significantly loosening of the binding pocket, including extrusion of the C15 nucleobase and a widening of the C15-C30 groove. Enhanced-sampling simulations further revealed that the cognate and synthetic ligands unbind in almost opposite directions. Our work offers new insight for designing riboswitch ligands.

biophysics↗

bioRxiv2021

The Gossypium anomalum genome as a resource for cotton improvement and evolutionary analysis of hybrid incompatibility

Cotton is an important crop that has been the beneficiary of multiple genome sequencing efforts, including diverse representatives of wild species for germplasm development. Gossypium anomalum is a wild African diploid species that harbors stress-resistance and fiber-related traits with potential application to modern breeding efforts. In addition, this species is a natural source of cytoplasmic male sterility and a resource for understanding hybrid lethality in the genus. Here we report a high-quality de novo genome assembly for G. anomalum and characterize this genome relative to existing genome sequences in cotton. In addition, we use the synthetic allopolyploids 2(A2D1) and 2(A2D3) to discover regions in the G. anomalum genome potentially involved in hybrid lethality, a possibility enabled by introgression of regions homologous to the D3 (G. davidsonii) lethality loci into the synthetic 2(A2D3) allopolyploid.

genomics↗

bioRxiv2021

Prenatal cadmium exposure alters proliferation in mouse CD4+ T cells via LncRNA Snhg7

ObjectivePrenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA hostgene 7 (lncSnhg7) in T cell proliferation. MethodsRNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. ResultsWe identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. ConclusionPrenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

immunology↗

bioRxiv2021

Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone (IMC) expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but both reduced the virus burden, and V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 abolished these effector activities and abrogated virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

immunology↗

bioRxiv2021

The Gossypium stocksii genome as a novel resource for cotton improvement

Cotton is an important textile crop whose gains in production over the last century have been challenged by various diseases. Because many modern cultivars are susceptible to several pests and pathogens, breeding efforts have included attempts to introgress wild, naturally resistant germplasm into elite lines. Gossypium stocksii is a wild cotton species native to Africa, which is part of a clade of vastly understudied species. Most of what is known about this species comes from pest resistance surveys and/or breeding efforts, which suggests that G. stocksii could be a valuable reservoir of natural pest resistance. Here we present a high-quality de novo genome sequence for G. stocksii. We compare the G. stocksii genome with resequencing data from a closely related, understudied species (G. somalense) to generate insight into the relatedness of these cotton species. Finally, we discuss the utility of the G. stocksii genome for understanding pest resistance in cotton, particularly resistance to cotton leaf curl virus.

genomics↗