CUBN is a gene locus for albuminuria

Identification of genetic risk factors for albuminuria may alter strategies for early prevention of CKD progression, particularly among patients with diabetes. Little is known about the influence of common genetic variants on albuminuria in both general and diabetic populations. We performed a meta-analysis of data from 63,153 individuals of European ancestry with genotype information from genome-wide association studies (CKDGen Consortium) and from a large candidate gene study (CARe Consortium) to identify susceptibility loci for the quantitative trait urinary albumin-to-creatinine ratio (UACR) and the clinical diagnosis microalbuminuria. We identified an association between a missense variant (I2984V) in the CUBN gene, which encodes cubilin, and both UACR (P = 1.1 × 10(-11)) and microalbuminuria (P = 0.001). We observed similar associations among 6981 African Americans in the CARe Consortium. The associations between this variant and both UACR and microalbuminuria were significant in individuals of European ancestry regardless of diabetes status. Finally, this variant associated with a 41% increased risk for the development of persistent microalbuminuria during 20 years of follow-up among 1304 participants with type 1 diabetes in the prospective DCCT/EDIC Study. In summary, we identified a missense CUBN variant that associates with levels of albuminuria in both the general population and in individuals with diabetes.     

The Dental Caries Pandemic and Disparities Problem

Understanding caries etiology and distribution is central to understanding potential opportunities for and likely impact of new biotechnologies and biomaterials to reduce the caries burden worldwide. This review asserts the appropriateness of characterizing caries as a "pandemic" and considers static and temporal trend reports of worldwide caries distribution. Oral health disparities within and between countries are related to sugar consumption, fluoride usage, dental care, and social determinants of health. Findings of international and U.S. studies are considered in promoting World Health Organization's and others' recommendations for science-based preventive and disease management interventions at the individual, clinical, public health, and public policy levels.     

Association of heat shock proteins with all-cause mortality

Experimental mild heat shock is widely known as an intervention that results in extended longevity in various models along the evolutionary lineage. Heat shock proteins (HSPs) are highly upregulated immediately after a heat shock. The elevation in HSP levels was shown to inhibit stress-mediated cell death, and recent experiments indicate a highly versatile role for these proteins as inhibitors of programmed cell death. In this study, we examined common genetic variations in 31 genes encoding all members of the HSP70, small HSP, and heat shock factor (HSF) families for their association with all-cause mortality. Our discovery cohort was the Rotterdam study (RS1) containing 5,974 participants aged 55 years and older (3,174 deaths). We assessed 4,430 single nucleotide polymorphisms (SNPs) using the HumanHap550K Genotyping BeadChip from Illumina. After adjusting for multiple testing by permutation analysis, three SNPs showed evidence for association with all-cause mortality in RS1. These findings were followed in eight independent population-based cohorts, leading to a total of 25,007 participants (8,444 deaths). In the replication phase, only HSF2 (rs1416733) remained significantly associated with all-cause mortality. Rs1416733 is a known cis-eQTL for HSF2. Our findings suggest a role of HSF2 in all-cause mortality.     

Soluble serum Klotho levels in healthy subjects. Comparison of two different immunoassays

Objective

Soluble serum Klotho is a new biomarker linked to chronic kidney disease, cardiovascular disease and diabetes. This study describes the evaluation and comparison of two different immunoassays and establishment of assay specific reference intervals in adults.

Design and methods

Serum Klotho concentrations were determined in 120 healthy adults aged 19–66 years. Blood samples were collected, and stored sera were assayed for Klotho according to age and gender. In addition several other clinical and laboratory characteristics were determined in the cohort and compared to the levels of serum Klotho.

Results

Serum Klotho levels were significantly higher in time-resolved fluorescence immunoassay (TRF) compared to an ELISA (IBL) and no correlation was found between the assays. No signal was obtained in either assay when the standard curve was switched between the two different immunoassays. The median serum Klotho concentration using TRF was 61 ng/mL (2.5–97.5% reference limits; 11–181 ng/mL) for males and 99 ng/mL (2.5–97.5% reference limits; 19–316 ng/mL) for females while the ELISA gave a mean value of 472 pg/mL (2.5–97.5% reference limits; 204–741 pg/mL) with no difference between genders. Concentrations of serum Klotho were independently associated with estimated glomerular filtration rate (eGFR) and body weight using TRF whereas serum Klotho concentrations were associated with age using the ELISA.

Conclusion

Comparison of two different immunoassays for serum Klotho indicate, that the protein exists in human beings in different forms which may function as independent factors and whose role and potential value as biomarkers needs to be evaluated separately. Reference intervals specific for the different forms recognized by the different assays were calculated in this study.     

Antagonism of protease-activated receptor 2 protects against experimental colitis

Many trypsin-like serine proteases such as β-tryptase are involved in the pathogenesis of colitis and inflammatory bowel diseases. Inhibitors of individual proteases show limited efficacy in treating such conditions, but also probably disrupt digestive and defensive functions of proteases. Here, we investigate whether masking their common target, protease-activated receptor 2 (PAR2), is an effective therapeutic strategy for treating acute and chronic experimental colitis in rats. A novel PAR2 antagonist (5-isoxazoyl-Cha-Ile-spiro[indene-1,4'-piperidine]; GB88) was evaluated for the blockade of intracellular calcium release in colonocytes and anti-inflammatory activity in acute (PAR2 agonist-induced) versus chronic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] models of colitis in Wistar rats. Disease progression (disease activity index, weight loss, and mortality) and postmortem colonic histopathology (inflammation, bowel wall thickness, and myeloperoxidase) were measured. PAR2 and tryptase colocalization were investigated by using immunohistochemistry. GB88 was a more potent antagonist of PAR2 activation in colonocytes than another reported compound, N1-3-methylbutyryl-N?-6-aminohexanoyl-piperazine (ENMD-1068) (IC?? 8 μM versus 5 mM). Acute colonic inflammation induced in rats by the PAR2 agonist SLIGRL-NH? was inhibited by oral administration of GB88 (10 mg/kg) with markedly reduced edema, mucin depletion, PAR2 receptor internalization, and mastocytosis. Chronic TNBS-induced colitis in rats was ameliorated by GB88 (10 mg/kg/day p.o.), which reduced mortality and pathology (including colon obstruction, ulceration, wall thickness, and myeloperoxidase release) more effectively than the clinically used drug sulfasalazine (100 mg/kg/day p.o.). These disease-modifying properties for the PAR2 antagonist in both acute and chronic experimental colitis strongly support a pathogenic role for PAR2 and PAR2-activating proteases and therapeutic potential for PAR2 antagonism in inflammatory diseases of the colon.     

From the Cover: Leinamycin E1 acting as an anticancer prodrug activated by reactive oxygen species

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140, featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. Upon reductive activation in the presence of cellular thiols, LNM exerts its antitumor activity by an episulfonium ion-mediated DNA alkylation. Previously, we have cloned the lnm gene cluster from S. atroolivaceus S-140 and characterized the biosynthetic machinery responsible for the 18-membered lactam backbone and the alkyl branch at C3 of LNM. We now report the isolation and characterization of leinamycin E1 (LNM E1) from S. atroolivacues SB3033, a ΔlnmE mutant strain of S. atroolivaceus S-140. Complementary to the reductive activation of LNM by cellular thiols, LNM E1 can be oxidatively activated by cellular reactive oxygen species (ROS) to generate a similar episulfonium ion intermediate, thereby alkylating DNA and leading to eventual cell death. The feasibility of exploiting LNM E1 as an anticancer prodrug activated by ROS was demonstrated in two prostate cancer cell lines, LNCaP and DU-145. Because many cancer cells are under higher cellular oxidative stress with increased levels of ROS than normal cells, these findings support the idea of exploiting ROS as a means to target cancer cells and highlight LNM E1 as a novel lead for the development of anticancer prodrugs activated by ROS. The structure of LNM E1 also reveals critical new insights into LNM biosynthesis, setting the stage to investigate sulfur incorporation, as well as the tailoring steps that convert the nascent hybrid peptide–polyketide biosynthetic intermediate into LNM.Leinamycin (LNM) (Fig. 1A, 1) is an antitumor antibiotic produced by Streptomyces atroolivaceus S-140 (1). It features an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring, a molecular architecture not found to date in any other natural product (Fig. 1A). LNM shows potent antitumor activity in vitro and in vivo and is active against tumors that are resistant to clinically important anticancer drugs, such as cisplatin, doxorubicin, mitomycin, and cyclophosphamide; therefore 1 has been pursued as a promising anticancer drug lead (2–4).Open in a separate windowFig. 1.LNM (1) and LNM E1 (2) as novel anticancer drug leads via episulfonium ion-mediated DNA alkylation upon complementary activation: (A) reductive activation of 1 by cellular thiols and (B) oxidative activation of 2 by cellular ROS, affording a pair of episulfonium ions that efficiently alkylate the N7 position of deoxyguanosine bases of DNA, thereby causing DNA cleavage and eventual cell death.The mode of action of 1 has been extensively investigated. The 1,3-dioxo-1,2-dithiolane moiety of 1 is essential for its antitumor activity. Upon reductive activation in the presence of thiol agents, 1 exerts its antitumor activity by an episulfonium ion-mediated DNA alkylation, a mode of action that is unprecedented among all DNA-damaging natural products (Fig. 1A). Thus, under a reductive cellular environment, the 1,3-dioxo-1,2-dithiolane moiety of 1 is first attacked by a thiol to produce a sulfenic acid intermediate (1a) that can cyclize, by ejecting a persulfide, to form a 1,2-oxathiolan-5-one intermediate (1b). Subsequent rearrangement of 1b, through an intramolecular attack of the 1,2-oxathiolan-5-one moiety by the C-6/C-7 alkene, affords an episulfonium ion intermediate (1c), which can efficiently alkylate the N7 position of deoxyguanosine bases of DNA (1d), ultimately causing DNA cleavage and cell death. The episulfonium ion 1c exists in equilibrium with an epoxide 1e through the intramolecular backside attack by the C-8-hydroxyl group (Fig. 1A) (4–11). Although 1c formation does not require DNA, because the hydrolysis product (1f) forms rapidly in the absence of DNA (5), noncovalent DNA binding by the Z,E-5-(thiazol-4-yl)-penta-2,4-dienone moiety of 1 significantly enhances its DNA alkylation activity (9). In addition, the persulfides generated in the formation of 1b also contribute to the observed DNA damaging activity of 1, but via an independent mechanism mediated by reactive oxygen species (ROS) (4, 12–14).We have previously cloned and sequenced the lnm biosynthetic gene cluster from S. atroolivaceus S-140, which consists of 27 ORFs (orfs) (15–17). In vivo and in vitro characterizations of the 1 biosynthetic machinery have since established that (i) the thiazole-containing 18-membered lactam backbone of 1 is synthesized by a hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)–less type I polyketide synthase (PKS), consisting of LnmQ (adenylation protein), LnmP [peptidyl carrier protein (PCP)], LnmI (a hybrid NRPS-AT–less type I PKS), LnmJ (AT-less type I PKS), and LnmG (AT) (17–19) and (ii) the alkyl branch at C-3 of 1 is installed by a novel pathway for β-alkylation in polyketide biosynthesis, featuring LnmK (acyltransferase/decarboxylase), LnmL [acyl carrier protein (ACP)], LnmM [hydroxymethylglutaryl-CoA synthase (HCS)], and LnmF [enoyl-CoA hydratase (ECH)] (Fig. 2) (20–22). However, the mechanism of sulfur incorporation to form the 1,3-dioxo-1,2-dithiolane moiety, which is essential for the DNA damage activity of 1, as well as the tailoring steps that convert the nascent hybrid peptide–polyketide intermediate into 1, remains elusive.Open in a separate windowFig. 2.Proposed biosynthetic pathway for LNM (1) featuring (i) the LnmQPIJ hybrid NRPS-AT–less type I PKS with the discrete LnmG AT loading the malonyl CoA extender units to all six PKS modules, (ii) the LnmKLMF enzymes catalyzing the β-alkyl branch at C3, and (iii) LNM E1 (2) as a key intermediate for 1 biosynthesis, setting the stage to investigate the mechanism of sulfur incorporation and the tailoring steps that convert 2 to 1, requiring minimally hydroxylations at C-8 and C-4′, oxidation at S-1′, S-insertion at 2′, and 1,3-dioxo-1,2-dithiolane ring formation. Color coding indicates the moieties installed by NRPS (blue), PKS (red), β-alkyl branch (green), and other tailoring enzymes (black). SAM, S-adenosylmethionine. The green oval denotes an AT docking domain. Domain abbreviations are: A, adenylation; AT, acyltransferase; Cy, cyclization; DH, dehydratase; KR, ketoreductase; KS, ketosynthase; MT, methyltransferase, Ox, oxidation; PCP, peptidyl carrier protein; TE, thioesterase; ?, domain of unknown function.To shed light into these steps, we systematically inactivated all genes within the lnm cluster whose functions or roles in 1 biosynthesis cannot be readily predicted on the basis of bioinformatics analysis alone (16, 17). Each of the mutant strains was then fermented, with the S. atroolivaceus S-140 WT as a control, to isolate new metabolites accumulated to account for their roles in 1 biosynthesis. Here, we report the isolation and characterization of leinamycin E1 (LNM E1, 2) from SB3033, a ΔlnmE mutant strain of S. atroolivaceus S-140. Significantly, the structure of 2 reveals critical new insights into 1 biosynthesis, setting the stage to investigate sulfur incorporation, as well as the tailoring steps that convert the nascent hybrid peptide–polyketide biosynthetic intermediate into 1 (Fig. 2). Most strikingly, 2 can be readily oxidized into a sulfenic acid intermediate (2a), which, in a mechanistic analogy to 1a and via a similar 1,2-oxathiolan-5-one intermediate (2b) to 1b, can undergo further rearrangement to form an episulfonium ion intermediate (2c). Thus, complementary to the reductive activation of 1 by cellular thiols to generate an episulfonium ion intermediate, 2 can be oxidatively activated by cellular ROS to generate a similar episulfonium ion intermediate, thereby alkylating DNA and leading to eventual cell death (Fig. 1B). The feasibility of exploiting 2 as an anticancer prodrug activated by ROS was demonstrated in two prostate cancer cell lines, LNCaP and DU-145, that are known to exist under higher oxidative stress than normal tissues. Because many cancer cells are under higher cellular oxidative stress with increased levels of ROS than normal cells (23, 24), our results suggest a means of exploiting ROS to target cancer cells and highlight 2 as a novel lead for the development of anticancer prodrugs activated by ROS.