Purification and characterization of major gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo

The hyaline layer is an apically located extracellular matrix that surrounds the sea urchin embryo from the time of fertilization until metamorphosis occurs. Gelatin-cleavage activities were absent from freshly prepared hyaline layers but a dynamic pattern of activities appeared when layers were incubated at 15°C or 37°C in UV-irradiated and millipore-filtered sea water. Three major gelatin cleavage activities at 55-, 41-, and 32 kDa were induced. The 55 kDa species was identified as the major activity at 37°C, while the 41- and 32 kDa activities were the minor species. In contrast, both the 41- and 32 kDa activities were the major species at 15°C, the ambient temperature for Strongylocentrotus purpuratus. The 55 kDa and other higher molecular weight species appeared only after 92 hours of incubation at 15°C. Interestingly, the gelatin-cleavage activities were absent when the hyaline layers were incubated at 37°C in millipore-filtered sea water containing 10 mM benzamidine hydrochloride, a reversible serine protease inhibitor. The minimum ionic requirements for the 15°C induction of the three activities were determined: both the 55- and 41 kDa species required 300 mM NaCl, while the 32 kDa activity required both 3 mM CaCl₂ and 200 mM NaCl. However, none of these activities required MgCl₂ for their induction. -- The 55-, 41- and 32 kDa gelatinase activities could be dissociated from the hyaline layers when incubated in 50 mM Tris-HCI, pH 8.0 containing 5 mM EDTA for 24 hours at 37°C. All three species were purified using gel filtration chromatography. In both qualitative and quantitative assays, the 55 kDa gelatinase activity was inhibited by 1,10 phenanthroline (a Zn²⁺ - specific chelator), ethylenediamine tetraacetic acid or ethylenebis (oxyethylenenitrilo) acetic acid. In the presence of 2 mM 1, 1 0-phenanthroline, the 55 kDa species was inhibited 93.1 %. Variations in the percent inhibition of the 55 kDa activity, in the range of 33-1 00% were observed in the presence of 5 mM ethylenebis (oxyethylenenitrilo) acetic acid. The quantitative gelatinase assay demonstrated that 10 mM CaCl2 stimulated 58.1% of the EGTA-inhibited (33%) 55 kDa species, which resulted in complete recovery of the activity. On the other hand, only 28.2% activation of the EGTA-inhibited (33%) 55 kDa was noticed in the presence of 50 mM, which recovered up to 85% of the total activity. The calcium reactivation of EGTA-inhibited (100%) 55 kDa occurred with an apparent dissociation constant of 1.2 mM. Since the hyaline layer is in direct contact with sea water, which contains 10 mM CaCl₂, 50 mM MgCl₂ and 500 mM NaCl, the effects of MgCl₂ and NaCl were also studied. Our results show that 85% of the total activity of the 33% EGTA-inhibited 55 kDa was reconstituted in the presence of 50 mM MgCl₂. The 55 kDa gelatin-cleavage activity was inhibited 57.6% in the presence of 500 mM NaCl. The NaCl-dependent inhibition suggests a possible mechanism for regulating the 55 kDa gelatinase activity. -- Developmental substrate gel analysis was performed using embryos of various developmental stages. The 55 kDa species comigrated with a gelatin cleavage activity present in 45 hours-old embryos and persisted until 93 hours after fertilization. This result confirms the presence of the 55 kDa species in the sea urchin embryo. We conclude that the sea urchin ECM, the hyaline layer has several gelatin-cleavage activities including a 55 kDa species, which is a Zn²⁺- and Ca²⁺ dependent, gelatin-specific endopeptidases that belongs to the matrix metalloproteinase class of extracellular matrix remodeling enzymes. Our results suggest that the 55 kDa is secreted as an inactive precursor, which is activated by one or more serine proteinase activity following incubation in MFSW at 15°C or 37°C. Our data indicate that the 55 kDa species might be the precursor of the 41 kDa gelatinase activity

Plasma chemokines CXCL10 and CXCL9 as potential diagnostic markers of drug-sensitive and drug-resistant tuberculosis

Tuberculosis (TB) diagnosis still remains to be a challenge with the currently used immune based diagnostic methods particularly Interferon Gamma Release Assay due to the sensitivity issues and their inability in differentiating stages of TB infection. Immune markers are valuable sources for understanding disease biology and are easily accessible. Chemokines, the stimulant, and the shaper of host immune responses are the vital hub for disease mediated dysregulation and their varied levels in TB disease are considered as an important marker to define the disease status. Hence, we wanted to examine the levels of chemokines among the individuals with drug-resistant, drug-sensitive, and latent TB compared to healthy individuals. Our results demonstrated that the differential levels of chemokines between the study groups and revealed that CXCL10 and CXCL9 as potential markers of drug-resistant and drug-sensitive TB with better stage discriminating abilities

Differential Frequencies of Intermediate Monocyte Subsets Among Individuals Infected With Drug-Sensitive or Drug-Resistant Mycobacterium tuberculosis

The rampant increase in drug-resistant tuberculosis (TB) remains a major challenge not only for treatment management but also for diagnosis, as well as drug design and development. Drug-resistant mycobacteria affect the quality of life owing to the delayed diagnosis and require prolonged treatment with multiple and toxic drugs. The phenotypic modulations defining the immune status of an individual during tuberculosis are well established. The present study aims to explore the phenotypic changes of monocytes & dendritic cells (DC) as well as their subsets across the TB disease spectrum, from latency to drug-sensitive TB (DS-TB) and drug-resistant TB (DR-TB) using traditional immunophenotypic analysis and by uniform manifold approximation and projection (UMAP) analysis. Our results demonstrate changes in frequencies of monocytes (classical, CD14(++)CD16(-), intermediate, CD14(++)CD16(+) and non-classical, CD14(+/-)CD16(++)) and dendritic cells (DC) (HLA-DR(+)CD11c(+) myeloid DCs, cross-presenting HLA-DR(+)CD14(-)CD141(+) myeloid DCs and HLA-DR(+)CD14(-)CD16(-)CD11c(-)CD123(+) plasmacytoid DCs) together with elevated Monocyte to Lymphocyte ratios (MLR)/Neutrophil to Lymphocyte ratios (NLR) and alteration of cytokine levels between DS-TB and DR-TB groups. UMAP analysis revealed significant differential expression of CD14(+), CD16(+), CD86(+) and CD64(+) on monocytes and CD123(+) on DCs by the DR-TB group. Thus, our study reveals differential monocyte and DC subset frequencies among the various TB disease groups towards modulating the immune responses and will be helpful to understand the pathogenicity driven by Mycobacterium tuberculosis

Whole-Genome Sequencing to Identify Missed Rifampicin and Isoniazid Resistance Among Tuberculosis Isolates—Chennai, India, 2013–2016

India has a high burden of drug-resistant tuberculosis (DR TB) and many cases go undetected by current drug susceptibility tests (DSTs). This study was conducted to identify rifampicin (RIF) and isoniazid (INH) resistance associated genetic mutations undetected by current clinical diagnostics amongst persons with DR TB in Chennai, India. Retrospectively stored 166 DR TB isolates during 2013–2016 were retrieved and cultured in Löwenstein-Jensen medium. Whole genome sequencing (WGS) and MGIT DST for RIF and INH were performed. Discordant genotypic and phenotypic sensitivity results were repeated for confirmation and the discrepant results considered final. Further, drug resistance-conferring mutations identified through WGS were analyzed for their presence as targets in current WHO-recommended molecular diagnostics. WGS detected additional mutations for rifampicin and isoniazid resistance than WHO-endorsed line probe assays. For RIF, WGS was able to identify an additional 10% (15/146) of rpoB mutant isolates associated with borderline rifampicin resistance compared to MGIT DST. WGS could detect additional DR TB cases than commercially available and WHO-endorsed molecular DST tests. WGS results reiterate the importance of the recent WHO revised critical concentrations of current MGIT DST to detect low-level resistance to rifampicin. WGS may help inform effective treatment selection for persons at risk of, or diagnosed with, DR TB

A Compendium of Potential Biomarkers of Pancreatic Cancer

Akhilesh Pandey and colleagues describe a compendium of potential biomarkers that can be systematically validated by the pancreatic cancer community

Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

Telomere length is a risk factor in disease and the dynamics of telomere length are crucial to our understanding of cell replication and vitality. The proliferation of whole genome sequencing represents an unprecedented opportunity to glean new insights into telomere biology on a previously unimaginable scale. To this end, a number of approaches for estimating telomere length from whole-genome sequencing data have been proposed. Here we present Telomerecat, a novel approach to the estimation of telomere length. Previous methods have been dependent on the number of telomeres present in a cell being known, which may be problematic when analysing aneuploid cancer data and non-human samples. Telomerecat is designed to be agnostic to the number of telomeres present, making it suited for the purpose of estimating telomere length in cancer studies. Telomerecat also accounts for interstitial telomeric reads and presents a novel approach to dealing with sequencing errors. We show that Telomerecat performs well at telomere length estimation when compared to leading experimental and computational methods. Furthermore, we show that it detects expected patterns in longitudinal data, repeated measurements, and cross-species comparisons. We also apply the method to a cancer cell data, uncovering an interesting relationship with the underlying telomerase genotype

Biological heterogeneity in idiopathic pulmonary arterial hypertension identified through unsupervised transcriptomic profiling of whole blood

Idiopathic pulmonary arterial hypertension (IPAH) is a rare but fatal disease diagnosed by right heart catheterisation and the exclusion of other forms of pulmonary arterial hypertension, producing a heterogeneous population with varied treatment response. Here we show unsupervised machine learning identification of three major patient subgroups that account for 92% of the cohort, each with unique whole blood transcriptomic and clinical feature signatures. These subgroups are associated with poor, moderate, and good prognosis. The poor prognosis subgroup is associated with upregulation of the ALAS2 and downregulation of several immunoglobulin genes, while the good prognosis subgroup is defined by upregulation of the bone morphogenetic protein signalling regulator NOG, and the C/C variant of HLA-DPA1/DPB1 (independently associated with survival). These findings independently validated provide evidence for the existence of 3 major subgroups (endophenotypes) within the IPAH classification, could improve risk stratification and provide molecular insights into the pathogenesis of IPAH

Publisher Correction: Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

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