The identification of tuberculosis biomarkers in human urine samples.

We aimed to determine whether shotgun proteomic approaches could be used to identify tuberculosis (TB)-specific biomarkers in the urine of well-characterised patients with active TB versus no TB. Patients with suspected TB (n=63) were classified as: definite TB (Mycobacterium tuberculosis positive culture, n=21); presumed latent-TB infection (LTBI) (M. tuberculosis negative culture, no radiological features of active TB, a positive QuantiFERON-TB Gold In-Tube (QFT-IT) test and a positive T-SPOT.TB test, n=24); and presumed non-TB/non-LTBI (M. tuberculosis negative culture, no radiological features of active TB, a negative QFT-IT test and a negative T-SPOT.TB test, n=18). Urine proteins, in the range of 3-50 kDa, were collected, separated by a one-dimensional SDS-PAGE gel and digested using trypsin, after which high-performance liquid chromatography-tandem mass spectrometry was used to identify the urinary proteome. 10 mycobacterial proteins were observed exclusively in the urine of definite TB patients, while six mycobacterial proteins were found exclusively in the urine of presumed LTBI patients. In addition, a gene ontology enrichment analysis identified a panel of 20 human proteins that were significant discriminators (p<0.05) for TB disease compared to no TB disease. Furthermore, seven common human proteins were differentially over- or under-expressed in the TB versus the non-TB group. These biomarkers hold promise for the development of new point-of-care diagnostics for TB.

Dual-Approach Electrochemical Surface-Enhanced Raman Scattering Detection of Mycobacterium tuberculosis in Patient-Derived Biological Specimens: Proof of Concept for a Generalizable Method to Detect and Identify Bacterial Pathogens.

The recent surge in infectious disease-causing pathogens, resulting in global catastrophe, has merited a pivotal quest toward point-of-care (POC) diagnostics. Mycobacterium tuberculosis (MTB) is still the top bacterium-based infectious disease-causing pathogen worldwide. In a concerted effort toward simplifying and decentralizing the discriminatory screening of MTB causing pathogens, electrochemical surface-enhanced Raman scattering (EC-SERS) was adopted to create a customized screening tool. The development strategy combined five key factors, including (i) a simplified Tollens'-based chemical synthesis method for bulk supply of silver nanoparticles, (ii) the deliberate surface modification of nanoparticles with carefully selected polyelectrolytes to resemble the conditioning layer usually found on a natural substratum, (iii) uniform SERS-active films formed through simple unprogrammed assembly, (iv) the controlled manipulation of the local electric field through applied voltage using a technique that does not conform to the limitations of classical EC-SERS, and (v) the inherent specificity of the target-specific SERS vibrational signature. The EC-SERS platform was able to discriminatively detect and identify TB-derived mycobacteria, including three clinically relevant MTB strains, TB-H37Rv, TB-HN878, and TB-CDC1551. Moreover, a customized voltage stepping protocol, compatible with either the inclusion of a short preincubation step or with in situ EC-SERS is illustrated. From the obtained SERS vibrational signatures, a band indicating a mode unique to TB-derived/TB-affiliated mycobacteria and thus not observed for other bacterial types used in this study was illustrated. Furthermore, provisional investigation, done as prelude for assessing the potential for translational adaptability of the EC-SERS technique toward POC clinical settings for sputum and urine specimens, was carried out.

Acute lymphoblastic leukemia-derived extracellular vesicles affect quiescence of hematopoietic stem and progenitor cells.

Patient-derived xenografted (PDX) models were generated through the transplantation of primary acute lymphoblastic leukemia (ALL) cells into immunodeficient NSG mice. We observed that ALL cells from mouse bone marrow (BM) produced extracellular vesicles (EVs) with specific expression of inducible heat shock protein HSP70, which is commonly activated in cancer cells. Taking advantage of this specific expression, we designed a strategy to generate fluorescent HSP70-labeled ALL EVs and monitor the impact of these EVs on endogenous murine BM cells ex vivo and in vivo. We discovered that hematopoietic stem and progenitor cells (HSPC) were mainly targeted by ALL EVs, affecting their quiescence and maintenance in the murine BM environment. Investigations revealed that ALL EVs were enriched in cholesterol and other metabolites that contribute to promote the mitochondrial function in targeted HSPC. Furthermore, using CD34+ cells isolated from cord blood, we confirmed that ALL EVs can modify quiescence of human HSPC. In conclusion, we have discovered a new oncogenic mechanism illustrating how EVs produced by proliferative ALL cells can target and compromise a healthy hematopoiesis system during leukemia development.

HSP90 inhibitor NVP-BEP800 affects stability of SRC kinases and growth of T-cell and B-cell acute lymphoblastic leukemias.

T-cell and B-cell acute lymphoblastic leukemias (T-ALL, B-ALL) are aggressive hematological malignancies characterized by an accumulation of immature T- or B-cells. Although patient outcomes have improved, novel targeted therapies are needed to reduce the intensity of chemotherapy and improve the prognosis of high-risk patients. Using cell lines, primary cells and patient-derived xenograft (PDX) models, we demonstrate that ALL cells viability is sensitive to NVP-BEP800, an ATP-competitive inhibitor of Heat shock protein 90 (HSP90). Furthermore, we reveal that lymphocyte-specific SRC family kinases (SFK) are important clients of the HSP90 chaperone in ALL. When PDX mice are treated with NVP-BEP800, we found that there is a decrease in ALL progression. Together, these results demonstrate that the chaperoning of SFK by HSP90 is involved in the growth of ALL. These novel findings provide an alternative approach to target SRC kinases and could be used for the development of new treatment strategies for ALL.