Biology subjects

Posey, J. E.

Publications and source records attributed to Posey, J. E..

8 recordsLinked to original sources

bioRxiv2025

Identification and characterization of host-directed therapeutics for tuberculosis using a versatile human 3D tuberculoma bioplatform

Host-directed therapies (HDT) represent a pivotal strategy in combating both drug-susceptible and drug-resistant tuberculosis (TB). Current evaluations, however, show limited success in animal models and clinical settings, emphasizing the need for more effective HDT candidates. With few druggable targets validated within granulomas, it is essential to verify the effectiveness of HDT candidates identified through traditional macrophage cultures in the context of the tuberculous granuloma milieus. Bioengineering a scalable, high-throughput screening (HTS) platform that replicates the physiological microenvironments in the hallmark tubercular lesions could significantly improve the identification of relevant HDT candidates and new treatment strategies. Here, we developed a facile, HTS-compatible bioplatform that generates tuberculoma-emulating structures, following three-dimensional (3D) co-cultures of human cells and pathogenic mycobacteria. Employing high-content imaging alongside immunological and transcriptomic approaches, we demonstrated that these 3D structures exhibit classic tuberculoma attributes and develop crucial transformations. Utilizing this system, we screened antibody biosimilars and potential HDT compounds. Our findings demonstrate the systems versatility in discovering antimicrobials and HDT candidates that effectively reduce mycobacterial burdens and granuloma lesions, while elucidating their immune mechanisms within 3D milieus. Many compounds effective in two-dimensional (2D) cultures were ineffective once granulomas formed in our 3D model. Notably, several promising compounds were found to induce rapid autophagy flux, and we validated the effectiveness of one such compound, the multi-kinase inhibitor AT9283, in a mouse model. Our findings highlight several HDT candidates for potential repurposing in TB treatment, offering a robust tool for accelerating therapeutic discoveries and advancing translational research for TB and other granulomatous diseases. SummarySafer and shorter treatment regimens for tuberculosis (TB) are urgently needed. Host-directed therapies (HDT) are being explored to enhance antibiotic regimens and address antimicrobial resistance. To expedite the discovery of HDT candidates and the development of new treatment strategies, scalable in vitro systems are needed that can replicate the critical features and microenvironments of TB lesions in a format compatible with high-throughput screening (HTS). We developed an HTS-compatible bioplatform using 3D co-cultures of human cells and fluorescent mycobacteria, creating tuberculoma-like structures with classic solid, necrotic, and cavitary transformations that exhibit crucial microenvironments. This facile system enabled us to screen antibody biosimilars and HDT compounds in 3D human in vitro tuberculomas, identifying candidates that inhibited mycobacterial growth and granuloma lesions while revealing potential innate immune mechanisms. The study revealed several promising HDT candidates that could be repurposed for TB treatment, introducing a versatile tool for screening therapeutic libraries. Additionally, it presents a framework for enhancing human in-vitro granuloma models, as advances in tissue-like systems emerge to recapitulate the architecture and multilineage differentiation in the lungs.

immunology↗

bioRxiv2025

Methodology for a freshly engineered or cryo-preserved 3D tuberculoma bioplatform for studying tuberculosis biology and high-content screening of therapeutics

Tuberculomas are the conglomeration of tuberculous granulomas into structurally organized three-dimensional (3D) masses that result from Mycobacterium tuberculosis infection and represent one of the more severe morphological forms of tuberculosis (TB). Several in vitro models that mimic human TB granulomas have been reported to decipher complex host-pathogen interactions and discover new prophylactic and therapeutic interventions. They serve as ethical bridge approaches to human studies. However, these models need improvements in generating well-organized granuloma lesions, classic tuberculoma structures, and relevant microenvironments. They are impractical for screening extensive chemical and genetic libraries owing to their low throughput, limited scalability, batch-to-batch variability, and high costs. Here, we describe a mycobacteria-in-spheroid co-culture workflow in a standard 96-well plate format that generates a robust 3D cell culture model. This model reproduces key attributes and microenvironments in human tuberculomas and can be scaled up as a high-throughput screening (HTS)-compatible bioplatform. The tuberculoma-like structures generated encompass organized, florid granulomatous lesions and exhibit solid, necrotic, and cavitary morphologies. This model can be developed using freshly isolated human primary cells or a monocytic cell line with virulent mycobacteria. The platform combines the entire workflow from generation to imaging of tuberculoma-like structures in situ. It permits the serial quantitation of drug efficacy and monitoring of lesion resolution over several days to weeks following a single treatment. Additionally, we outline a methodology for adopting this workflow for cryo-preservation, enhancing its potential for commercial application. The ease of generation, pliability, cryo-shelf stability, and reproducibility of the bioplatform make it ideal for HTS applications and implementation in the discovery programs of TB and other granulomatous diseases.

bioengineering↗

bioRxiv2024

VizCNV: An integrated platform for concurrent phased BAF and CNV analysis with trio genome sequencing data

BackgroundCopy number variation (CNV) is a class of genomic Structural Variation (SV) that underlie genomic disorders and can have profound implications for health. Short-read genome sequencing (sr-GS) enables CNV calling for genomic intervals of variable size and across multiple phenotypes. However, unresolved challenges include an overwhelming number of false-positive calls due to systematic biases from non-uniform read coverage and collapsed calls resulting from the abundance of paralogous segments and repetitive elements in the human genome. MethodsTo address these interpretative challenges, we developed VizCNV. The VizCNV computational tool for inspecting CNV calls uses various data signal sources from sr-GS data, including read depth, phased B-allele frequency, as well as benchmarking signals from other SV calling methods. The interactive features and view modes are adept for analyzing both chromosomal abnormalities [e.g., aneuploidy, segmental aneusomy, and chromosome translocations], gene exonic CNV and non-coding gene regulatory regions. In addition, VizCNV includes a built-in filter schema for trio genomes, prioritizing the detection of impactful germline CNVs, such as de novo CNVs. Upon computational optimization by fine-tuning parameters to maximize sensitivity and specificity, VizCNV demonstrated approximately 83.8% recall and 77.2% precision on the 1000 Genome Project data with an average coverage read depth of 30x. ResultsWe applied VizCNV to 39 families with primary immunodeficiency disease without a molecular diagnosis. With implemented build-in filter, we identified two de novo CNVs and 90 inherited CNVs >10 kb per trio. Genotype-phenotype analyses revealed that a compound heterozygous combination of a paternal 12.8 kb deletion of exon 5 and a maternal missense variant allele of DOCK8 are likely the molecular cause of one proband. ConclusionsVizCNV provides a robust platform for genome-wide relevant CNV discovery and visualization of such CNV using sr-GS data.

genomics↗

bioRxiv2024

Impaired central pattern generators due to abnormal EPHA4 signaling leads to idiopathic scoliosis

Idiopathic scoliosis (IS) is the most common form of spinal deformity with unclear pathogenesis. In this study, we firstly reanalyzed the loci associated with IS, drawing upon previous studies. Subsequently, we mapped these loci to candidate genes using either location-based or function-based strategies. To further substantiate our findings, we verified the enrichment of variants within these candidate genes across several large IS cohorts encompassing Chinese, East Asian, and European populations. Consequently, we identified variants in the EPHA4 gene as compelling candidates for IS. To confirm their pathogenicity, we generated zebrafish mutants of epha4a. Remarkably, the zebrafish epha4a mutants exhibited pronounced scoliosis during later stages of development, effectively recapitulating the IS phenotype. We observed that the epha4a mutants displayed defects in left-right coordination during locomotion, which arose from disorganized neural activation in these mutants. Our subsequent experiments indicated that the disruption of the central pattern generator (CPG) network, characterized by abnormal axon guidance of spinal cord interneurons, contributed to the disorganization observed in the mutants. Moreover, when knocked down efnb3b, the ligand for Epha4a, we observed similar CPG defects and disrupted left-right locomotion. These findings strongly suggested that ephrin B3-Epha4 signaling is vital for the proper functioning of CPGs, and defects in this pathway could lead to scoliosis in zebrafish. Furthermore, we identified two cases of IS in NGEF, a downstream molecule in the EPHA4 pathway. Collectively, our data provide compelling evidence that neural patterning impairments and disruptions in CPGs may underlie the pathogenesis of IS.

genetics↗

bioRxiv2019

Integrated Sequencing & Array Comparative Genomic Hybridization in Familial Parkinson’s Disease

BackgroundParkinsons disease (PD) is a genetically heterogeneous condition; both single nucleotide variants (SNVs) and copy number variants (CNVs) are important genetic risk factors. We examined the utility of combining exome sequencing and genome-wide array-based comparative genomic hybridization (aCGH) for identification of PD genetic risk factors. MethodsWe performed exome sequencing on 110 subjects with PD and a positive family history; 99 subjects were also evaluated using genome-wide aCGH. We interrogated exome sequencing and array comparative genomic hybridization data for pathogenic SNVs and CNVs at Mendelian PD gene loci. SNVs were confirmed via Sanger sequencing. CNVs were confirmed with custom-designed high-density aCGH, droplet digital PCR, and breakpoint sequencing. ResultsUsing exome sequencing, we discovered individuals with known pathogenic single nucleotide variants in GBA (p.E365K, p.T408M, p.N409S, p.L483P) and LRRK2 (p.R1441G and p.G2019S). Two subjects were each double heterozygotes for variants in GBA and LRRK2. Based on aCGH, we additionally discovered cases with an SNCA duplication and heterozygous intragenic GBA deletion. Five additional subjects harbored both SNVs (p.N52fs, p.T240M, p.P437L, p.W453*) and likely disrupting CNVs at the PARK2 locus, consistent with compound heterozygosity. In nearly all cases, breakpoint sequencing revealed microhomology, a mutational signature consistent with CNV formation due to DNA replication errors. ConclusionsIntegrated exome sequencing and aCGH yielded a genetic diagnosis in 19.3% of our familial PD cohort. Our analyses highlight potential mechanisms for SNCA and PARK2 CNV formation, uncover multilocus pathogenic variation, and identify novel SNVs and CNVs for further investigation as potential PD risk alleles.

genetics↗

bioRxiv2019

Genetic and molecular mechanism for distinct clinical phenotypes conveyed by allelic truncating mutations implicated in FBN1

The molecular and genetic mechanisms by which different single nucleotide variant (SNV) alleles in specific genes, or at the same genetic locus, bring about distinct disease phenotypes often remain unclear. Allelic truncating mutations of fibrillin-1(FBN1) cause either classical Marfan syndrome (MFS) or a more severe phenotype associated with Marfanoid-progeroid-lipodystrophy syndrome (MPLS). A total of three Marfan syndrome/Marfanoid patients (2 singletons and 1 parent-offspring trio) were recruited. Targeted next-generation sequencing was performed on all the participants. We analyzed the molecular diagnosis, patient clinical features, and the potential molecular mechanism involved in the MPLS subject in our cohort. We investigated a small cohort, consisting of two classical MFS and one MPLS patient from China, whose clinical presentation included scoliosis potentially requiring surgical intervention. We provide evidence that most nonsense and frameshift mutations lead to FBN1 null alleles due to mutant mRNA transcript degradation. In contrast, the more severe disease phenotype, MPLS, is caused by mutant mRNAs that are predicted to escape the nonsense mediated decay (NMD) surveillance pathway, making a mutant protein that exerts a dominant negative interference effect to FBN1 thus generating a gain-of-function (GoF) rather than a loss-of-function (LoF) allele as in MFS. Overall, we provide direct evidence that a dominant negative interaction of FBN1 potentially explains the distinct clinical phenotype in MPLS patients through genetic and functional analysis of the first Chinese patient with MPLS. Moreover, our study expands the mutation spectrum of FBN1 and highlights the potential molecular mechanism for MPLS patients.

genetics↗

bioRxiv2018

Validation of novel Mycobacterium tuberculosis isoniazid resistance mutations not detectable by common molecular tests

Resistance to the first-line anti-tuberculosis (TB) drug, isoniazid (INH), is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a minimum inhibitory concentration of [≥]0.25 g/mL and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a superior method for detection of INH-R MTBC compared to current targeted molecular testing methods and could provide earlier diagnosis of drug-resistant TB.

microbiology↗

bioRxiv2018

Phenotypic expansion in DDX3X -- a common cause of intellectual disability in females

De novo variants in DDX3X account for 1-3% of unexplained intellectual disability (ID), one of the most common causes of ID, in females. Forty-seven patients (44 females, 3 males) have been described. We identified 29 additional individuals carrying 27 unique DDX3X variants in the setting of complex clinical presentations including developmental delay or ID. In addition to previously reported manifestations, rare or novel phenotypes were identified including respiratory problems, congenital heart disease, skeletal muscle mitochondrial DNA depletion, and late-onset neurologic decline. Our findings expand the spectrum of DNA variants and phenotypes associated with DDX3X disorders.

genetics↗