Biology subjects

Limberis, J. D.

Publications and source records attributed to Limberis, J. D..

4 recordsLinked to original sources

bioRxiv2025

Impaired IFNγ responsiveness of lung monocyte-derived cells limits immunity to Mycobacterium tuberculosis

Lung mononuclear phagocyte (MNP) subsets differ in their ability to restrict Mycobacterium tuberculosis (Mtb) during chronic infection, yet the mechanisms underlying this difference are not well defined. Here, we show that CD11clo monocyte-derived cells (MNC1), the subset of lung cells that is most permissive for Mtb viability during chronic infection, express lower levels of interferon-gamma (IFN{gamma}) signaling proteins, resulting in reduced responses to IFN{gamma} compared to alveolar macrophages (AM) and CD11chi MNC2. Moreover, type I IFN signaling suppresses IFN{gamma}-mediated MHC-II expression, impairing antigen-specific CD4 T cell activation by MNC1 cells. Importantly, prior immunity conferred by contained Mtb infection enhances IFN{gamma} responsiveness of monocyte-derived cells, reducing bacterial burdens in lungs and within MNC subsets. Our findings indicate that heterogeneous IFN{gamma} responsiveness is exploited by Mtb for persistence in vivo. Overcoming or bypassing impaired IFN{gamma} responsiveness may guide the development of more effective TB vaccines and host-directed therapies.

immunology↗

bioRxiv2025

Beating the gold standard: A review of Mycobacterium tuberculosis lysis using bead beating and the need for standardization.

Bead beating is widely used for mechanical lysis of Mycobacterium tuberculosis, a bacterium with a highly resistant, lipid-rich cell wall. Despite its status as a de facto gold standard for mycobacterial lysis, there is no standardized protocol for bead beating, resulting in significant variability across studies. We conducted a literature review of 73 studies, identifying 38 with explicit mycobacterial bead beating protocols. Our analysis revealed heterogeneity in bead types, sizes, device models, and operational parameters, with 37% of studies failing to report critical details such as lysis speed. We experimentally assessed the impact of key variables--tube type, bead quantity, and device settings--on lysis efficiency using qPCR of M. tuberculosis DNA. Results showed that even minor changes, such as tube shape or bead volume, can significantly affect DNA yield. These findings underscore the need for standardized bead-beating protocols to improve reproducibility and comparability. Future efforts should prioritize developing consensus methods tailored to sample type and analytical application.

molecular biology↗

bioRxiv2025

URAdime - a tool for analyzing primer sequences in sequencing data to identify dimers and super-amplicons.

Targeted sequencing of multiplex PCR amplicons is commonplace in research laboratories and clinical diagnostics. There are numerous tools for the a priori optimization of primers and reactions, but no tools to detect specific problematic primers post hoc. We developed URAdime, a tool for analyzing primer sequences in sequencing data to identify dimers and super-amplicons. We show that it successfully detects these unwanted amplicons and accurately attributes their generation to specific primers.

bioinformatics↗

bioRxiv2024

spotPCR: A Rapid and Efficient Approach for Indexing Individual Template Molecules using Unique Molecular Identifiers

Low-frequency mutations provide valuable insights in various fields, including drug resistance identification, cancer and infectious disease research. One promising strategy to enhance the sensitivity and specificity of mutation detection is the incorporation of unique molecular identifiers (UMIs) during polymerase chain reaction (PCR) amplification and before deep sequencing. However, conventional methods for UMI incorporation oRen necessitate multiple labor-intensive steps. spotPCR (Specific Primer Limited Unique Molecular Identifier Tagging PCR) overcomes these challenges, streamlining the UMI tagging process.

molecular biology↗