A genome catalogue of lake bacterial diversity and its drivers at continental scale.

Lakes are heterogeneous ecosystems inhabited by a rich microbiome whose genomic diversity is poorly defined. We present a continental-scale study of metagenomes representing 6.5 million km2 of the most lake-rich landscape on Earth. Analysis of 308 Canadian lakes resulted in a metagenome-assembled genome (MAG) catalogue of 1,008 mostly novel bacterial genomospecies. Lake trophic state was a leading driver of taxonomic and functional diversity among MAG assemblages, reflecting the responses of communities profiled by 16S rRNA amplicons and gene-centric metagenomics. Coupling the MAG catalogue with watershed geomatics revealed terrestrial influences of soils and land use on assemblages. Agriculture and human population density were drivers of turnover, indicating detectable anthropogenic imprints on lake bacteria at the continental scale. The sensitivity of bacterial assemblages to human impact reinforces lakes as sentinels of environmental change. Overall, the LakePulse MAG catalogue greatly expands the freshwater genomic landscape, advancing an integrative view of diversity across Earth's microbiomes.

Discovery of GLPG1972/S201086, a Potent, Selective, and Orally Bioavailable ADAMTS-5 Inhibitor for the Treatment of Osteoarthritis.

There are currently no approved disease-modifying osteoarthritis (OA) drugs (DMOADs). The aggrecanase ADAMTS-5 is key in the degradation of human aggrecan (AGC), a component of cartilage. Therefore, ADAMTS-5 is a promising target for the identification of DMOADs. We describe the discovery of GLPG1972/S201086, a potent and selective ADAMTS-5 inhibitor obtained by optimization of a promising hydantoin series following an HTS. Biochemical activity against rat and human ADAMTS-5 was assessed via a fluorescence-based assay. ADAMTS-5 inhibitory activity was confirmed with human aggrecan using an AGC ELISA. The most promising compounds were selected based on reduction of glycosaminoglycan release after interleukin-1 stimulation in mouse cartilage explants and led to the discovery of GLPG1972/S201086. The anticatabolic activity was confirmed in mouse cartilage explants (IC50 < 1.5 µM). The cocrystal structure of GLPG1972/S201086 with human recombinant ADAMTS-5 is discussed. GLPG1972/S201086 has been investigated in a phase 2 clinical study in patients with knee OA (NCT03595618).

Microbiology in the Field: Construction and Validation of a Portable Incubator for Real-Time Quantification of Coliforms and Other Bacteria.

Performing microbiological assays on environmental samples in field settings poses logistical challenges with respect to the availability of suitable equipment or the ability to get samples to the laboratory in a timely fashion. For example, the viability of some bacteria can decrease greatly between sampling and arrival to the laboratory for processing. We developed and constructed rugged, reliable, and cost-effective portable incubators that were used by 10 independent field teams to perform microbiological assays on surface water samples from lakes across Canada. Rigorous testing and validation of our incubators ensured that incubation conditions were consistent within and across all 10 field teams and 2 sampling years. Samples from all sites were processed in duplicate and bacterial counts were highly repeatable within and across sampling teams. Bacterial counts were also found to be statistically equivalent to counts obtained with standard laboratory techniques using a conventional incubator. Using this method, thermotolerant coliforms (TTCs) and Escherichia coli were quantified from 432 lakes, allowing comparison to both historical datasets that relied on TTCs and those following current guidelines that use E. coli counts. We found higher loads at the shoreline than the middle of lakes and different patterns between ecozones. E. coli was not frequently detected, but many lakes exceeded Canadian guideline values for activities such as swimming and some even exceeded the guideline value for secondary recreational activities such as boating. To the best of our knowledge, this is the largest bacteriological water quality assessment of freshwater lakes to date in terms of both spatial scale and the number of lakes sampled. Our incubator design can be easily adapted for a wide variety of researcher goals and represents a robust platform for field studies and other applications, including those in remote or low-resources settings.

Specific forms of BAFF favor BAFF receptor-mediated epithelial cell survival.

Although B cell activating factor (BAFF) and its receptor BR3 are produced and expressed by many cells, their role has been restricted to the lymphocyte lineage. Using various techniques (RT-PCR, indirect immunofluorescence, flow cytometry analysis), we observed the expression of BR3 and the production of BAFF by the human salivary gland cell line, by epithelial cells from biopsies of Sjögren's syndrome patients and their controls, but also by salivary gland epithelial cells in culture. To decipher the role of BAFF and BR3 on epithelial cells, BAFF and BR3 were neutralized by blocking antibodies or RNA specific inhibitor (siBR3) and epithelial cell survival was analyzed. Blocking BR3 promotes epithelial cell apoptosis in vitro. This apoptosis resulted in the nuclear translocation of PKCδ. BAFF neutralization by various anti-BAFF antibodies leads to different effects depending on the antibody used suggesting that only some forms of BAFF are required for epithelial cell survival. Our study demonstrates that BR3 is involved in the survival of cultured epithelial cells due to an autocrine effect of BAFF. It also suggests that epithelial cells produce different forms of BAFF and that only some of them are responsible for this effect.

Epigenetics and Sjögren's syndrome.

There is growing evidence that epigenetics, the study of heritable changes in gene expression that do not involve mutations in the DNA itself, may play an essential role in autoimmune diseases (AID). In Sjögren's syndrome (SS), a chronic AID characterized by an epithelis of the exocrine glands, epigenetic studies have focused on three mechanisms: DNA methylation and its consequences including human endogenous retrovirus (HERV) expression; microRNA expression; and protein post-translational modifications associated with autoantibody production. Although in its infancy, comprehension of the epigenetic (dys)regulation in SS may help us to understand: why SS affects predominantly middle-aged women; why genetically predisposed individuals develop SS but not others; why flare-ups occur; why treatment responses differ between patients; and why some patients develop lymphoma. From these studies will arise a better comprehension of the pathophysiology of SS as well as development of new diagnostic and prognostic biomarkers, and novel therapeutics for prevention and perhaps early intervention.

In Sjögren's syndrome, B lymphocytes induce epithelial cells of salivary glands into apoptosis through protein kinase C delta activation.

Sjögren's syndrome (SS) is a chronic autoimmune epithelitis associated with diffuse lymphocytic infiltration that varies in composition and differs according to lesion severity. T lymphocytes have been viewed as competent in their own right in the destruction of epithelial cells, whereas B lymphocytes that predominate in severe lesions have never been implicated in direct tissue damage. Using co-culture experiments with human salivary gland (HSG) cell line cells and tonsilar B lymphocytes, we observed that direct HSG cell-B lymphocyte contacts were able to induce apoptosis in epithelial cells. This B lymphocyte-mediated cell death could not be ascribed to Fas-Fas ligand interactions but required translocation of protein kinase C delta (PKC δ) into the nucleus of epithelial cells. Ultimately, activation of PKCδ resulted in histone H2B phosphorylation on serine 14 and poly (ADP-ribose) polymerase cleavage. Our results suggest that B lymphocytes infiltrating the SGs of patients with SS could evoke epithelial cell apoptosis.

B-cell tolerance breakdown in Sjögren's syndrome: focus on BAFF.

Sjögren's syndrome (SS) is an autoimmune epithelitis hallmarked by a destruction of epithelial cells, the subsequent lymphocytic infiltration of exocrine glands and their ensuing dryness. Given the prominent role currently assigned to B cells in SS, it is not surprising that the B cell activating factor belonging to the Tumor Necrosis Factor family (BAFF) is involved in its pathogenesis. When overexpressed, this cytokine leads to self-reactive B cells emergence at the transitional B-cell stage. BAFF overexpression that has been observed in SS, is associated with B-cell tolerance breakdown and autoantibody production. Furthermore, BAFF is responsible for the abnormal distribution of B cells subsets and B-cell hyperactivity described in the blood. In the salivary glands, a minority of B-cell clusters represent ectopic germinal center cells, while the majority manifest features consistent with transitional type 2 (T2) and marginal-zone (MZ)-like B cells. Interestingly, both types of B-cell cluster include autoreactive B cells and BAFF is associated with expansion of T2 B cells and MZ-like B cells in the salivary glands. Finally, BAFF plays a major role in B-cell repopulation after their depletion by rituximab in SS.

A new access to dihydrotropones through ring expansion of spirocyclohexadienones: synthesis and mechanism.

In this paper we report the rearrangement of spirocyclohexadienones into dihydrotropones in basic conditions as a new method for the preparation of seven-membered ring ketones, which are key building blocks for the synthesis of tropoloalkaloids. DFT calculations and deuterium labeling studies support the mechanism we propose for this rearrangement, involving the ring opening of a spirocyclopropane intermediate followed by successive base-catalyzed 1,3-hydrogen shifts. The X-ray structure of the resulting dihydrotropone shows near-perfect planarity and the conjugation gain is likely to be the driving force of the reaction.