Renal outcomes with sodium-glucose cotransporter 2 inhibitors in Japanese people with grade 3 chronic kidney disease and type 2 diabetes: Analysis of medical administrative databases.

AIM: To evaluate whether sodium-glucose cotransporter 2 inhibitor (SGLT2i) therapy is associated with a reduction of renal events compared with other glucose-lowering drugs (oGLDs) among Japanese people with type 2 diabetes (T2D) and grade 3 (G3) chronic kidney disease (CKD) in a real-world clinical practice setting. MATERIALS AND METHODS: People with T2D who were newly prescribed an SGLT2i or an oGLD from April 2014 to November 2021 (without prior use of index drugs for ≥ 1 year prior to index date) and G3 CKD (estimated glomerular filtration rate [eGFR] ≥ 30 to < 60 mL/min/1.73 m2) were selected from the Medical Data Vision database (MDV-DB) and the Real-World Data database (RWD-DB). SGLT2i and oGLD users were matched (1:1) using propensity score on patient background characteristics. The primary endpoint was a composite of the development of end-stage kidney disease or a sustained decline in eGFR of 50% or more. Hazard ratios (HRs) were estimated using the Cox proportional hazards model. RESULTS: Overall, 3190 (1595 per group) patients in the MDV-DB and 2572 (1286 per group) patients in the RWD-DB were included in the analyses. The composite outcome was significantly lower in the SGLT2i group than in the oGLD group in the MDV-DB (HR 0.49, 95% confidence interval [CI] 0.33 to 0.74, P < 0.001) and in the RWD-DB (HR 0.57, 95% CI 0.37 to 0.88, P = 0.011). CONCLUSIONS: Japanese people with T2D and G3 CKD initiating an SGLT2i had a lower risk of renal events than people initiating an oGLD.

Amino acid residues critical for DNA binding and inducer recognition in CbnR, a LysR-type transcriptional regulator from Cupriavidus necator NH9.

CbnR, a LysR-type transcriptional regulator from Cupriavidus necator NH9, activates the transcription of chlorocatechol-degradative enzymes. To activate the transcription, CbnR needs to bind not only to the cbnA promoter but also to the inducer. In this study, the transcriptional activity and DNA-binding activity of twenty-five mutants of CbnR were analyzed. Of the 17 mutants of the DNA-binding domain, 11 mutants lost their ability to activate transcription. While most mutants without transcriptional activation did not show DNA-binding activity, Asn17Ala, Gln29Ala, and Pro30Ala retained DNA-binding activity, suggesting that transcriptional activation by CbnR requires more than its binding to promoter DNA. Of the 8 mutants of the regulatory domain, 6 mutants changed their responses to the inducer, when compared with wild-type CbnR. Interestingly, Arg199Ala and Val246Ala induced constitutive expression of the cbnA promoter without the inducer, suggesting that these mutations brought about a conformational change mimicking that induced by the inducer molecule.

Ortholog-Finder: A Tool for Constructing an Ortholog Data Set.

Orthologs are widely used for phylogenetic analysis of species; however, identifying genuine orthologs among distantly related species is challenging, because genes obtained through horizontal gene transfer (HGT) and out-paralogs derived from gene duplication before speciation are often present among the predicted orthologs. We developed a program, "Ortholog-Finder," to obtain ortholog data sets for performing phylogenetic analysis by using all open-reading frame data of species. The program includes five processes for minimizing the effects of HGT and out-paralogs in phylogeny construction: 1) HGT filtering: Genes derived from HGT could be detected and deleted from the initial sequence data set by examining their base compositions. 2) Out-paralog filtering: Out-paralogs are detected and deleted from the data set based on sequence similarity. 3) Classification of phylogenetic trees: Phylogenetic trees generated for ortholog candidates are classified as monophyletic or polyphyletic trees. 4) Tree splitting: Polyphyletic trees are bisected to obtain monophyletic trees and remove HGT genes and out-paralogs. 5) Threshold changing: Out-paralogs are further excluded from the data set based on the difference in the similarity scores of genuine orthologs and out-paralogs. We examined how out-paralogs and HGTs affected phylogenetic trees constructed for species based on ortholog data sets obtained by Ortholog-Finder with the use of simulation data, and we determined the effects of confounding factors. We then used Ortholog-Finder in phylogeny construction for 12 Gram-positive bacteria from two phyla and validated each node of the constructed tree by comparison with individually constructed ortholog trees.

HGT-Gen: a tool for generating a phylogenetic tree with horizontal gene transfer.

UNLABELLED: Horizontal gene transfer (HGT) is a common event in prokaryotic evolution. Therefore, it is very important to consider HGT in the study of molecular evolution of prokaryotes. This is true also for conducting computer simulations of their molecular phylogeny because HGT is known to be a serious disturbing factor for estimating their correct phylogeny. To the best of our knowledge, no existing computer program has generated a phylogenetic tree with HGT from an original phylogenetic tree. We developed a program called HGT-Gen that generates a phylogenetic tree with HGT on the basis of an original phylogenetic tree of a protein or gene. HGT-Gen converts an operational taxonomic unit or a clade from one place to another in a given phylogenetic tree. We have also devised an algorithm to compute the average length between any pair of branches in the tree. It defines and computes the relative evolutionary time to normalize evolutionary time for each lineage. The algorithm can generate an HGT between a pair of donor and acceptor lineages at the same evolutionary time. HGT-Gen is used with a sequence-generating program to evaluate the influence of HGT on the molecular phylogeny of prokaryotes in a computer simulation study. AVAILABILITY: The database is available for free at http://www.grl.shizuoka.ac.jp/˜thoriike/HGT-Gen.html.

Amyloid oligomer conformation in a group of natively folded proteins.

Recent in vitro and in vivo studies suggest that destabilized proteins with defective folding induce aggregation and toxicity in protein-misfolding diseases. One such unstable protein state is called amyloid oligomer, a precursor of fully aggregated forms of amyloid. Detection of various amyloid oligomers with A11, an anti-amyloid oligomer conformation-specific antibody, revealed that the amyloid oligomer represents a generic conformation and suggested that toxic beta-aggregation processes possess a common mechanism. By using A11 antibody as a probe in combination with mass spectrometric analysis, we identified GroEL in bacterial lysates as a protein that may potentially have an amyloid oligomer conformation. Surprisingly, A11 reacted not only with purified GroEL but also with several purified heat shock proteins, including human Hsp27, 40, 70, 90; yeast Hsp104; and bovine Hsc70. The native folds of A11-reactive proteins in purified samples were characterized by their anti-beta-aggregation activity in terms of both functionality and in contrast to the beta-aggregation promoting activity of misfolded pathogenic amyloid oligomers. The conformation-dependent binding of A11 with natively folded Hsp27 was supported by the concurrent loss of A11 reactivity and anti-beta-aggregation activity of heat-treated Hsp27 samples. Moreover, we observed consistent anti-beta-aggregation activity not only by chaperones containing an amyloid oligomer conformation but also by several A11-immunoreactive non-chaperone proteins. From these results, we suggest that the amyloid oligomer conformation is present in a group of natively folded proteins. The inhibitory effects of A11 antibody on both GroEL/ES-assisted luciferase refolding and Hsp70-mediated decelerated nucleation of Abeta aggregation suggested that the A11-binding sites on these chaperones might be functionally important. Finally, we employed a computational approach to uncover possible A11-binding sites on these targets. Since the beta-sheet edge was a common structural motif having the most similar physicochemical properties in the A11-reactive proteins we analyzed, we propose that the beta-sheet edge in some natively folded amyloid oligomers is designed positively to prevent beta aggregation.

Method for comparing the structures of protein ligand-binding sites and application for predicting protein-drug interactions.

Many drugs, even ones that are designed to act selectively on a target protein, bind unintended proteins. These unintended bindings can explain side effects or indicate additional mechanisms for a drug's medicinal properties. Structural similarity between binding sites is one of the reasons for binding to multiple targets. We developed a method for the structural alignment of atoms in the solvent-accessible surface of proteins that uses similarities in the local atomic environment, and carried out all-against-all structural comparisons for 48,347 potential ligand-binding regions from a nonredundant protein structure subset (nrPDB, provided by NCBI). The relationships between the similarity of ligand-binding regions and the similarity of the global structures of the proteins containing the binding regions were examined. We found 10,403 known ligand-binding region pairs whose structures were similar despite having different global folds. Of these, we detected 281 region pairs that had similar ligands with similar binding modes. These proteins are good examples of convergent evolution. In addition, we found a significant correlation between Z-score of structural similarity and true positive rate of "active" entries in the PubChem BioAssay database. Moreover, we confirmed the interaction between ibuprofen and a new target, porcine pancreatic elastase, by NMR experiment. Finally, we used this method to predict new drug-target protein interactions. We obtained 540 predictions for 105 drugs (e.g., captopril, lovastatin, flurbiprofen, metyrapone, and salicylic acid), and calculated the binding affinities using AutoDock simulation. The results of these structural comparisons are available at http://www.tsurumi.yokohama-cu.ac.jp/fold/database.html.