Biology subjects

Bitar, M.

Publications and source records attributed to Bitar, M..

6 recordsLinked to original sources

bioRxiv2026

A Murine Model of Mycobacterium abscessus Encapsulated in Alginate-Beads: Advancing Toward a Chronic Infection Model

BackgroundThe increasing incidence of Mycobacterium abscessus (M. abscessus) lung infections, together with its intrinsic multidrug resistance, highlights the need for new therapeutic regimens. However, the lack of a reliable chronic infection model in immunocompetent mice limits preclinical evaluation. MethodsTo mimic the bronchial environment of infected patients, we evaluated the effect of encapsulating M. abscessus in alginate beads on infection progression in BALB/cJRJ mice following intratracheal inoculation, compared with intranasal infection using non-encapsulated bacteria. The impact of dexamethasone treatment (DEX) was also assessed. Bacterial loads in lungs, spleen, liver, and kidneys were quantified over time in untreated and antibiotic-treated mice. Lung inflammation was evaluated by measuring IFN-{gamma} and TNF- levels. In vitro, the activity of imipenem and bedaquiline was assessed against free or alginate-encapsulated M. abscessus. ResultsCompared with intranasal infection, intratracheal infection with alginate-encapsulated bacteria resulted in slower pulmonary clearance and greater extrapulmonary dissemination. DEX further enhanced these features, reducing lung clearance, increasing dissemination, and amplifying lung inflammation. Bedaquiline showed no effect, whereas imipenem efficacy depended on treatment timing. For both drugs, alginate encapsulation reduced in vitro antibacterial activity. ConclusionThis model represents a step toward a chronic M. abscessus infection model characterized by moderate lungs clearance, extrapulmonary dissemination, and pronounced inflammatory responses. Reduced antibiotic activity against alginate-encapsulated bacteria may more accurately predict treatment efficacy in humans than activity measured against free bacteria.

microbiology↗

bioRxiv2024

HyDRA: a pipeline for integrating long- and short-read RNAseq data for custom transcriptome assembly

BackgroundShort-read RNA sequencing (RNAseq) has widely been used to sequence RNA from a wide range of different tissues, developmental stages and species. However, the technology is limited by inherent biases and its inability to capture full-length transcripts. Long-read RNAseq overcomes these issues by providing reads that can span multiple exons, resolve complex repetitive regions and the capability to cover entire transcripts. Unfortunately, this technology is still prone to higher error rates. Noncoding RNA transcripts are highly specific to different cell types and tissues and remain underrepresented in current reference annotations. This problem is exacerbated by the dismissal of sequenced reads that align to genomic regions that do not contain annotated transcripts, resulting in approximately half of the expressed transcripts being overlooked in transcriptional studies. ResultsWe have developed a pipeline, named HyDRA (Hybrid de novo RNA assembly), which combines the precision of short reads with the structural resolution of long reads, enhancing the accuracy and reliability of custom transcriptome assemblies. Deep, short- and long-read RNAseq data derived from ovarian and fallopian tube samples were used to develop, validate and assess the efficacy of HyDRA. We identified more than 50,000 high-confidence long noncoding RNAs, most of which have not been previously detected using traditional methods. ConclusionsHyDRAs assembly performed more than 40% better than a similar assembly obtained with the top-ranked stand-alone de novo transcriptome short-read-only assembly tool and over 30% better than one obtained with the best-in-class multistep short-read-only approach. Although long-read sequencing is rapidly advancing, the vast availability of short-read RNAseq data will ensure that hybrid approaches like the one implemented in HyDRA continue to be relevant, allowing the discovery of high-confidence transcripts within specific cell types and tissues. As the practice of performing hybrid de novo transcriptome assemblies becomes commonplace, HyDRA will advance the annotation of coding and noncoding transcripts and expand our knowledge of the noncoding genome.

bioinformatics↗

bioRxiv2023

CRISPR-Cas13d screens identify KILR a breast cancer risk-associated lncRNA that regulates DNA replication and repair.

Long noncoding RNAs (lncRNAs) have surpassed the number of protein-coding genes, yet the majority have no known function. We previously discovered >800 lncRNAs at regions identified by breast cancer genome-wide association studies (GWAS). Here, we performed a pooled CRISPR-Cas13d RNA knockdown screen to identify which of these lncRNAs altered cell proliferation. We found that KILR, a lncRNA that functions as a tumor suppressor, safeguards breast cells against uncontrolled proliferation. The half-life of KILR is significantly reduced by the risk haplotype, revealing an alternative mechanism by which variants alter cancer risk. We showed that KILR sequesters RPA1, a subunit of the RPA complex, required for DNA replication and repair. Reduced KILR expression promotes cell proliferation by increasing the available pool of RPA1 and the speed of DNA replication. Our findings confirm lncRNAs as mediators of breast cancer risk, emphasize the need to annotate noncoding transcripts in relevant cell types when investigating GWAS variants and provide a scalable platform for mapping phenotypes associated with lncRNAs.

cancer biology↗

bioRxiv2022

Redefining normal breast cell populations using long noncoding RNAs

Single-cell RNAseq has allowed unprecedented insight into gene expression across different cell populations in normal tissue and disease states. However, almost all studies rely on annotated gene sets to capture gene expression levels and sequencing reads that do not align to known genes are discarded. Here, we discover thousands of long noncoding RNAs (lncRNAs) expressed in human mammary epithelial cells and analyze their expression in individual cells of the normal breast. We show that lncRNA expression alone can discriminate between luminal and basal cell types and define subpopulations of both compartments. Clustering cells based on lncRNA expression identified additional basal subpopulations, compared to clustering based on annotated gene expression, suggesting that lncRNAs can provide an additional layer of information to better distinguish breast cell subpopulations. In contrast, these breast-specific lncRNAs poorly distinguish brain cell populations, highlighting the need to annotate tissue-specific lncRNAs prior to expression analyses. We also identified a panel of 100 breast lncRNAs that could discern breast cancer subtypes better than protein-coding markers. Overall, our results suggest that lncRNAs are an unexplored resource for new biomarker and therapeutic target discovery in the normal breast and breast cancer subtypes.

cell biology↗

bioRxiv2022

An Alzheimer's disease patient-derived olfactory cell model identifies gene expression changes associated with cognition

An early symptom of Alzheimers disease (AD) is an impaired sense of smell, for which the molecular basis remains elusive. Here, we generated human olfactory neurosphere-derived (ONS) cells from people with AD and mild cognitive impairment (MCI), and performed global RNA sequencing to determine gene expression changes. ONS cells expressed markers of neuroglial differentiation, providing a unique cellular model to explore early AD-associated disease pathways. Our transcriptomics data from ONS cells revealed differentially expressed genes (DEGs) associated with cognitive processes in AD cells compared to MCI, or matched healthy controls (HC). A-Kinase Anchoring Protein 6 (AKAP6) was the most significantly altered gene in AD compared to both MCI and HC, and has been linked to cognitive function. The greatest change in gene expression of all DEGs occurred between AD and MCI. Gene pathway analysis revealed defects in multiple cellular processes with aging, intellectual deficiency and alternative splicing being the most significantly dysregulated in AD ONS cells. Our results demonstrate that ONS cells can provide a cellular model for AD that recapitulates disease-associated differences. We have revealed potential novel genes, including AKAP6 that may have a role in AD, particularly MCI to AD transition, and should be further examined.

neuroscience↗

bioRxiv2019

C/EBPδ demonstrates dichotomous tumor initiation and promotion in high grade serous ovarian carcinogenesis

C/EBP{delta} (CEBPD), a gene part of the highly conserved basic-leucine zipper (b-ZIP) domain of transcriptional factors, is downregulated in 65% of high grade serous carcinoma of the ovary (HGSC). Overexpression of C/EBP{delta} in different tumors as glioblastoma and breast cancer either promotes tumor progression or inhibits growth. Despite these contradictory roles in different cancer types, we show that C/EBP{delta} overexpression has a consistent function of downregulating proliferation and promoting migration in fallopian tube epithelial cells (FTE). We show that the FTE have both mesenchymal and epithelial cell characteristics. Further, our data supports a role for C/EBP{delta} as an early regulatory transcriptional factor that promotes a mesenchymal to epithelial (MET) phenotype by upregulating E-cadherin and downregulating vimentin and N-cadherin in FTE cells. We demonstrate that overexpression of C/EBP{delta} in ovarian and breast cancer cell lines have consistent effects and phenotype as the FTE cells. Our findings suggest a role for C/EBP{delta} in the early events of ovarian serous carcinogenesis which may be used to help further understand how the disease develops from a premalignant cells.

cancer biology↗