Biology subjects

Jacobson, K. R.

Publications and source records attributed to Jacobson, K. R..

8 recordsLinked to original sources

bioRxiv2024

Systematic review and meta-analysis of protocols and yield of direct from sputum sequencing of Mycobacterium tuberculosis

Direct sputum whole genome sequencing (dsWGS) can revolutionize Mycobacterium tuberculosis (Mtb) diagnosis by enabling rapid detection of drug resistance and strain diversity without the biohazard of culture. We searched PubMed, Web of Science and Google scholar, and identified 8 studies that met inclusion criteria for testing protocols for dsWGS. Utilising meta-regression we identify several key factors positively associated with dsWGS success, including higher Mtb bacillary load, mechanical disruption, and enzymatic/chemical lysis. Specifically, smear grades of 3+ (OR = 14.7, 95% CI: 3.5, 62.1; p = 0.0005) were strongly associated with improved outcomes, whereas decontamination with sodium hydroxide (NaOH) was negatively associated (OR = 0.005, 95% CI: 0.001, 0.03; p = 7e-06), likely due to its harsh effects on Mtb cells. Furthermore, mechanical lysis (OR = 193.3, 95% CI: 11.7, 3197.8; p = 0.008) and enzymatic/chemical lysis (OR = 18.5, 95% CI: 1.9, 183.1; p = 0.02) were also strongly associated with improved dsWGS. Across the studies, we observed a high degree of variability in approaches to sputum pre-processing prior to dsWGS highlighting the need for standardized best practices. In particular we conclude that optimizing pre-processing steps including decontamination with the exploration of alternatives to NaOH to better preserve Mtb cells and DNA, and best practices for cell lysis during DNA extraction as priorities. Further and considering the strong association between Mtb load and successful dsWGS, protocol improvements for optimal sputum sample collection, handling, and storage could also further enhance the success rate of dsWGS.

microbiology↗

bioRxiv2024

Analysis of the limited M. tuberculosis accessory genome reveals potential pitfalls of pan-genome analysis approaches

Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety of methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. To quantify sources of bias and error related to common pan-genome analysis approaches, we evaluated different approaches applied to curated collection of 151 Mycobacterium tuberculosis (Mtb) isolates. Mtb is characterized by its clonal evolution, absence of horizontal gene transfer, and limited accessory genome, making it an ideal test case for this study. Using a state-of-the-art graph-genome approach, we found that a majority of the structural variation observed in Mtb originates from rearrangement, deletion, and duplication of redundant nucleotide sequences. In contrast, we found that pan-genome analyses that focus on comparison of coding sequences (at the amino acid level) can yield surprisingly variable results, driven by differences in assembly quality and the softwares used. Upon closer inspection, we found that coding sequence annotation discrepancies were a major contributor to inflated Mtb accessory genome estimates. To address this, we developed panqc, a software that detects annotation discrepancies and collapses nucleotide redundancy in pan-genome estimates. When applied to Mtb and E. coli pan-genomes, panqc exposed distinct biases influenced by the genomic diversity of the population studied. Our findings underscore the need for careful methodological selection and quality control to accurately map the evolutionary dynamics of a bacterial species.

bioinformatics↗

bioRxiv2023

Assessment and validation of enrichment and target capture approaches to improve Mycobacterium tuberculosis WGS from direct patient samples

BackgroundWithin-host M. tuberculosis (Mtb) diversity may capture antibiotic resistance or predict tuberculosis treatment failure and is best captured through sequencing directly from sputum. Here we compared three sample preprocessing steps for DNA decontamination and studied the yield of a new target enrichment protocol for optimal whole-genome sequencing (WGS) from direct patient samples. Design/MethodsMtb positive NALC - NaOH treated patient sputum sediments were pooled, and heat inactivated, split in replicates, and treated by either a wash, DNase I or Benzonase digestion. Levels of contaminating host DNA and target Mtb DNA was assessed by Quantitative PCR (qPCR), followed by WGS with and without custom dsDNA target enrichment. ResultsThe pre-treatment sample has a high host-to-target ratio of DNA (6 168 {+/-} 1 638 host copies/ng to 212,3 {+/-} 59,4 Mtb copies/ng) that significantly decreased with all three treatments. Benzonase treatment resulted in the highest enrichment of Mtb DNA at 100-fold compared with control (3 422 {+/-} 2 162 host copies/ng to 11 721 {+/-} 7 096 Mtb copies/ng). The custom dsDNA probe panel successfully enriched libraries from as little as 0.45 pg of Mtb DNA (100 genome copies). Applied to direct sputum after benzonase treatment the dsDNA target enrichment panel increased the percent of sequencing reads mapping to the Mtb to 90,95% from 1,18 % compared with benzonase treatment without enrichment. ConclusionWe demonstrate a low limit of detection (LoD) for a new custom dsDNA Mtb target enrichment panel that has a favourable cost profile. The results also demonstrate that pre-processing to remove contaminating extracellular DNA prior to cell lysis and DNA extraction improves the host-to-Mtb DNA ratio but is not adequate to support average coverage WGS without target capture.

molecular biology↗

bioRxiv2022

Mycobacterium tuberculosis disease associates with higher HIV-1-specific antibody responses

Mycobacterium tuberculosis (Mtb) can enhance immune responses against unrelated pathogens. Although Mtb is the most common co-infection in people living with HIV (PWH), there has been no examination of its impact on HIV-1 immune responses. Plasma neutralization and antibody dependent cellular cytotoxicity (ADCC) was compared among PWH and Mtb disease (PWH/Active Mtb) and PWH/No Mtb both prior to and after antiretroviral treatment (ART) and completion of Mtb therapy. We assessed HIV-1 sequences, total antibody quantities and isotypes, and plasma cytokine levels to ascertain mechanisms that affect humoral responses. HIV-1 neutralizing antibodies (nAbs) were broader and more potent in PWH/Active Mtb as compared to PWH/No Mtb, and nAbs increased among PWH who developed Mtb after ART initiation. ADCC was also higher in the PWH who had Mtb disease after starting ART. PWH/Active Mtb as compared to PWH/No Mtb had unique HIV-1 envelope sequence motifs associated with neutralization resistance further implying differences in humoral selection. The Mtb-linked antibody augmentation associated with elevated plasma cytokine levels important for B cells and antibody production, namely interleukin-6, a proliferation-inducing ligand (APRIL), and B-cell activating factor (BAFF). Increased plasma virus levels, greater HIV-1 envelope diversity, higher levels of all antibodies, and cross-reactive responses did not explain the enhanced HIV-1 humoral responses in those with Mtb. Mtb disease enhances HIV-1 humoral responses likely by perturbing pathways important for antibody production in lymphoid tissue that has both pathogens. These findings have implications for using antibody-based therapies and inducing optimal HIV-1 antibody responses. Author SummaryMycobacterium tuberculosis (Mtb) is the most common infection among people with HIV (PWH) in the world. Mtb infection can enhance immune responses against unrelated pathogens. Previous studies have not examined the impact of Mtb disease on HIV antibodies in PWH. This information has importance for future strategies aimed at enhancing HIV antibody responses in naive individuals or PWH. We show that HIV neutralizing antibodies and antibody-dependent cellular cytotoxicity are broader and more potent in PWH in the presence as compared to the absence of Mtb disease. PWH and Mtb disease as compared to those without Mtb also harbor unique HIV envelope sequences, which further indicates that there is differential antibody selection pressure. The Mtb linked HIV antibody enhancement associated with specific mediators important for B cell and antibody development. Importantly, the Mtb mediated HIV antibody augmentation was not due to cross-reactivity, a generalized increase in all antibodies, or a higher level, more diverse, or longer duration of antigen exposure. We speculate that more potent HIV antibodies arise in lymphatic tissue that harbors both Mtb and HIV. Our findings have implications for both future uses of HIV antibodies as prophylaxis or treatment and strategies aimed inducing better HIV antibody responses.

microbiology↗

bioRxiv2022

Mechanical loading is required for initiation of extracellular matrix deposition at the developing murine myotendinous junction

The myotendinous junction (MTJ) contributes to the generation of motion by connecting muscle to tendon. At the adult MTJ, a specialized extracellular matrix (ECM) is thought to contribute to the mechanical integrity of the muscle-tendon interface, but the factors that influence MTJ formation during mammalian development are unclear. Here, we combined 3D imaging and proteomics with murine models in which muscle contractility and patterning are disrupted to resolve morphological and compositional changes in the ECM during MTJ development. We found that MTJ-specific ECM deposition can be initiated via static loading due to growth; however, it required cyclic loading to develop a mature morphology. Furthermore, the MTJ can mature without the tendon terminating into cartilage. Based on these results, we describe a model wherein MTJ development depends on mechanical loading but not insertion into an enthesis.

developmental biology↗

bioRxiv2022

Extracellular matrix deposition precedes muscle-tendon integration during murine forelimb morphogenesis

The development of a functional vertebrate musculoskeletal system requires the combination of contractile muscle and extracellular matrix (ECM)-rich tendons that transmit muscle-generated force to bone. Despite the different embryologic origins, muscle and tendon integrate at the myotendinous junction (MTJ) to seamlessly connect cells and ECM across this interface. While the cell-cell signaling factors that direct development have received considerable attention, how and when the ECM linking these tissues is deposited remains unknown. To address this gap, we analyzed the 3D distribution of different ECM and the influence of skeletal muscle in forelimbs from wildype (WT) and muscle-less Pax3Cre/Cre mice. At E11.5, prior to MTJ integration, an aligned ECM was present at the presumptive insertion of the long triceps into the WT ulna. Mechanically robust tendon-like and muscle compartmentalization structures, positive for type I collagen, type V collagen, and fibrillin-2, still formed when muscle was knocked out. However, MTJ-specific ECM was not observed when muscle was absent. Our results show that an ECM-based template forms independent of muscle, but muscle is needed for the proper assembly of ECM at the MTJ. Summary statementAn aligned ECM template connects tendon and muscle during limb development, independent of muscle progenitor migration into the limb; however, the assembly of MTJ-specific ECM requires the presence of muscle.

developmental biology↗

bioRxiv2020

3D mapping reveals a complex and transient interstitial matrix during murine renal development

BackgroundThe extracellular matrix (ECM) is a network of proteins and glycosaminoglycans that provides structural and biochemical cues to cells. In the kidney, the ECM is critical for nephrogenesis; however, the dynamics of ECM composition and how it relates to 3D structure during development is unknown. MethodsUsing embryonic day (E)14.5, E18.5, postnatal day (P)3, and adult kidneys, we fractionated proteins based on differential solubilities, performed liquid chromatography tandem-mass spectrometry, and identified changes in ECM protein content (matrisome). Decellularized kidneys were stained for ECM proteins and imaged in 3D using confocal microscopy. ResultsWe observed an increase in interstitial ECM that connect the stromal mesenchyme to the basement membrane (TNXB, COL6A1, COL6A2, COL6A3) between the embryo and adult, and a transient elevation of interstitial matrix proteins (COL5A2, COL12A1, COL26A1, ELN, EMID1, FBN1, LTBP4, THSD4) at perinatal timepoints. Basement membrane proteins critical for metanephric induction (FRAS1, FREM2) were highest in the embryo, whereas proteins necessary for glomerular basement membrane integrity (COL4A3, COL4A4, COL4A5, LAMB2) were more abundant in the adult. 3D visualization revealed a complex interstitial matrix that dramatically changed over development, including the perinatal formation of fibrillar structures that appear to support the medullary rays. ConclusionBy correlating 3D ECM spatiotemporal organization with global protein abundance, we identified novel changes in the interstitial matrix during kidney development. This new information regarding the ECM in developing kidneys offers the potential to inform the design of regenerative scaffolds that can guide nephrogenesis in vitro. Significance statementEnd-stage renal disease is increasing and there are a limited number of organs available for transplantation. Therefore, researchers have focused on understanding how cellular signaling influences kidney development to expand strategies to rebuild a kidney. However, the extracellular matrix (ECM), another critical component that biomechanically regulates nephrogenesis, has been largely neglected. This paper combines proteomics and 3D imaging of the murine kidney to resolve previously undescribed dynamics of the interstitial matrix in the cortex and corticomedullary junction during development. Combined with cell and growth factors, scaffolds modeled after the composition and organization of the developmental ECM have the potential to improve tissue engineering models of the kidney, like organoids.

developmental biology↗

bioRxiv2020

Extracellular matrix protein composition dynamically changes during murine forelimb development

The extracellular matrix (ECM) is an integral part of multicellular organisms, connecting different cell layers and tissue types. During morphogenesis and growth, tissues undergo substantial reorganization involving cellular proliferation, migration, and differentiation. While it is intuitive that the ECM remodels in concert, little is known regarding how matrix composition and organization change during development. We utilized tissue fractionation and mass spectrometry to define ECM protein (matrisome) dynamics during murine forelimb development and resolved significant differences in ECM composition as a function of development, disease and tissue type. Additionally, we used bioorthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized ECM within the developing forelimb. We demonstrate the feasibility of using BONCAT to enrich for newly synthesized matrisome components and identified differences in ECM synthesis between morphogenesis and growth. This resource will guide future research investigating the role of the matrisome during complex tissue development.

developmental biology↗