Activity-based probes for functional interrogation of retaining β-glucuronidases

Humans express at least two distinct β-glucuronidase enzymes that are involved in disease: exo-acting β-glucuronidase (GUSB), whose deficiency gives rise to mucopolysaccharidosis type VII, and endo-acting heparanase (HPSE), whose overexpression is implicated in inflammation and cancers. The medical importance of these enzymes necessitates reliable methods to assay their activities in tissues. Herein, we present a set of β-glucuronidase-specific activity-based probes (ABPs) that allow rapid and quantitative visualization of GUSB and HPSE in biological samples, providing a powerful tool for dissecting their activities in normal and disease states. Unexpectedly, we find that the supposedly inactive HPSE proenzyme proHPSE is also labeled by our ABPs, leading to surprising insights regarding structural relationships between proHPSE, mature HPSE, and their bacterial homologs. Our results demonstrate the application of β-glucuronidase ABPs in tracking pathologically relevant enzymes and provide a case study of how ABP-driven approaches can lead to discovery of unanticipated structural and biochemical functionality

Functionally heterogeneous human satellite cells identified by single cell RNA sequencing.

Although heterogeneity is recognized within the murine satellite cell pool, a comprehensive understanding of distinct subpopulations and their functional relevance in human satellite cells is lacking. We used a combination of single cell RNA sequencing and flow cytometry to identify, distinguish, and physically separate novel subpopulations of human PAX7+ satellite cells (Hu-MuSCs) from normal muscles. We found that, although relatively homogeneous compared to activated satellite cells and committed progenitors, the Hu-MuSC pool contains clusters of transcriptionally distinct cells with consistency across human individuals. New surface marker combinations were enriched in transcriptional subclusters, including a subpopulation of Hu-MuSCs marked by CXCR4/CD29/CD56/CAV1 (CAV1+). In vitro, CAV1+ Hu-MuSCs are morphologically distinct, and characterized by resistance to activation compared to CAV1- Hu-MuSCs. In vivo, CAV1+ Hu-MuSCs demonstrated increased engraftment after transplantation. Our findings provide a comprehensive transcriptional view of normal Hu-MuSCs and describe new heterogeneity, enabling separation of functionally distinct human satellite cell subpopulations

Metabolomics for bioactivity assessment of natural products.

Natural products historically have been a rich source of lead molecules in drug discovery, based on their capability to create unique and diverse chemical structures. However, it is also true that the vast number of metabolites typically present in natural products and their huge dynamic range results in the loss of many possibly bioactive natural compounds, becoming an inextricable obstacle for drug development. Recently, new strategies which favour a holistic approach as opposed to the traditional reductionist methods used previously, have been introduced with the purpose of overcoming the bottlenecks in natural product research. This approach is based on the application of new technologies, including metabolomics, for example. Metabolomics allows a systematic study of a complex mixture such as a phytochemical preparation, which can be linked to observations obtained through biological testing systems without the need for isolating active principles. This may put drug discovery from natural products back in the limelight again. In this review paper, the description of some examples of successful metabolomics applications in several important fields related to drug discovery from natural sources aims at raising the potential of metabolomics in reducing the gap between natural products (NP) and modern drug discovery demand

SLC2A10 genetic polymorphism predicts development of peripheral arterial disease in patients with type 2 diabetes. SLC2A10 and PAD in type 2 diabetes

<p>Abstract</p> <p>Background</p> <p>Recent data indicate that loss-of-function mutation in the gene encoding the facilitative glucose transporter GLUT10 (<it>SLC2A10</it>) causes arterial tortuosity syndrome via upregulation of the TGF-β pathway in the arterial wall, a mechanism possibly causing vascular changes in diabetes.</p> <p>Methods</p> <p>We genotyped 10 single nucleotide polymorphisms and one microsatellite spanning 34 kb across the <it>SLC2A10 </it>gene in a prospective cohort of 372 diabetic patients. Their association with the development of peripheral arterial disease (PAD) in type 2 diabetic patients was analyzed.</p> <p>Results</p> <p>At baseline, several common SNPs of <it>SLC2A10 </it>gene were associated with PAD in type 2 diabetic patients. A common haplotype was associated with higher risk of PAD in type 2 diabetic patients (haplotype frequency: 6.3%, <it>P </it>= 0.03; odds ratio [OR]: 14.5; 95% confidence interval [CI]: 1.3- 160.7) at baseline. Over an average follow-up period of 5.7 years, carriers with the risk-conferring haplotype were more likely to develop PAD (<it>P </it>= 0.007; hazard ratio: 6.78; 95% CI: 1.66- 27.6) than were non-carriers. These associations remained significant after adjustment for other risk factors of PAD.</p> <p>Conclusion</p> <p>Our data demonstrate that genetic polymorphism of the <it>SLC2A10 </it>gene is an independent risk factor for PAD in type 2 diabetes.</p

The Sec-independent function of Escherichia coli YidC is evolutionary conserved and essential

YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Sec-associated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc

A minor merger scenario for the ultraluminous X-ray source ESO 243-49 HLX-1-II. Constraints from photometry

The point-like X-ray source HLX-1, close to the S0 galaxy ESO 243-49, is the brightest knownultraluminous X-ray source and one the strongest intermediate-mass black hole candidates.Weargue that the counterpart of HLX-1 may be the nucleus of a satellite galaxy, undergoing minormerger with the S0 galaxy. We investigate this scenario by running a set of N-body/smoothedparticle hydrodynamics simulations of the minor merger between an S0 galaxy and a gas-richbulgy satellite galaxy, and by comparing the results with the available photometric HubbleSpace Telescope (HST) data of ESO 243-49 and of the HLX-1 counterpart. In particular, wederive synthetic surface brightness profiles for the simulated counterpart of HLX-1 in six HSTfilters, ranging from far-ultraviolet (FUV) to infrared wavelengths. Such synthetic profilesinclude a contribution from the stellar population associated with the simulated disruptedsatellite and a contribution from an irradiated disc model. These are in reasonable agreementwith the observed surface brightness profiles of the HLX-1 counterpart, provided that themerger is at sufficiently late stage (2.5 Gyr since the first pericentre passage). The maindifference between models and observations is in the FUV band, where the HST image showsa fuzzy and extended emission.We showthat the spectral energy distribution of the bulge of ESO 243-49 cannot be explainedwith a single old stellar population, but requires the existence of a younger stellar component.This is in good agreement with the star formation history derived from our N-body simulations,and is a further hint for the minor merger scenario