Stendomycin selectively inhibits TIM23-dependent mitochondrial protein import.

Tim17 and Tim23 are the main subunits of the TIM23 complex, one of the two major essential mitochondrial inner-membrane protein translocon machineries (TIMs). No chemical probes that specifically inhibit TIM23-dependent protein import were known to exist. Here we show that the natural product stendomycin, produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of the TIM23 complex in yeast and mammalian cells. Furthermore, stendomycin-mediated blockage of the TIM23 complex does not alter normal processing of the major regulatory mitophagy kinase PINK1, but TIM23 is required to stabilize PINK1 on the outside of mitochondria to initiate mitophagy upon membrane depolarization.

Diabetic foot syndrome in patients with diabetes. A multicenter German/Austrian DPV analysis on 33 870 patients.

AIMS: The diabetic foot syndrome (DFS) is a serious complication in patients with diabetes increasing the risk for minor/major amputations. This analysis aimed to examine differences in diabetes patients with or without DFS stratified by type 1 (T1D) or type 2 diabetes (T2D). MATERIAL AND METHODS: Adult patients (≥20y of age) with diabetes from the German/Austrian diabetes patients follow-up registry (DPV) were included. The cross-sectional study comprised 45 722 subjects with T1D (nDFS  = 2966) and 313 264 with T2D (nDFS  = 30 904). In DFS, minor/major amputations were analysed. To compare HbA1C , neuropathy, nephropathy, cardiovascular disease risk factors, and macrovascular complications between patients with or without DFS, regression models were conducted. Confounders: age, sex, diabetes duration. RESULTS: In patients with DFS, a minor amputation was documented in 27.2% (T1D) and 25.9% (T2D), a major amputation in 10.2% (T1D) and 11.3% (T2D). Regression models revealed that neuropathy was more frequent in subjects with DFS compared with patients without DFS (T1D: 70.7 vs 29.8%; T2D: 59.4% vs 36.9%; both P < 0.0001). Hypertension, nephropathy, peripheral vascular disease, stroke, or myocardial infarction was more common compared with patients without DFS (all P < 0.0001). In T1D with DFS, a slightly higher HbA1C (8.11% vs 7.95%; P < 0.0001) and in T2D with DFS a lower HbA1C (7.49% vs 7.69%; P < 0.0001) was observed. CONCLUSIONS: One third of the patients with DFS had an amputation of the lower extremity. Especially neuropathy or peripheral vascular disease was more prevalent in patients with DFS. New concepts to prevent DFS-induced amputations and to reduce cardiovascular risk factors before the occurrence of DFS are necessary.

Somatic copy number alterations by whole-exome sequencing implicates YWHAZ and PTK2 in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole-exome sequencing on five CRPC/normal paired formalin-fixed paraffin-embedded (FFPE) samples, using the SOLiD4 next-generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock-down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock-down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole-exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next-generation sequencing technologies.

AspSnFR: A genetically encoded biosensor for real-time monitoring of aspartate in live cells.

Aspartate is crucial for nucleotide synthesis, ammonia detoxification, and maintaining redox balance via the malate-aspartate-shuttle (MAS). To disentangle these multiple roles of aspartate metabolism, tools are required that measure aspartate concentrations in real time and in live cells. We introduce AspSnFR, a genetically encoded green fluorescent biosensor for intracellular aspartate, engineered through displaying and screening biosensor libraries on mammalian cells. In live cells, AspSnFR is able to precisely and quantitatively measure cytosolic aspartate concentrations and dissect its production from glutamine. Combining high-content imaging of AspSnFR with pharmacological perturbations exposes differences in metabolic vulnerabilities of aspartate levels based on nutrient availability. Further, AspSnFR facilitates tracking of aspartate export from mitochondria through SLC25A12, the MAS' key transporter. We show that SLC25A12 is a rapidly responding and direct route to couple Ca2+ signaling with mitochondrial aspartate export. This establishes SLC25A12 as a crucial link between cellular signaling, mitochondrial respiration, and metabolism.

An integrated approach for identification and target validation of antifungal compounds active against Erg11p.

Systemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.

Idiopathic Frozen Shoulder in Individuals with Diabetes: Association with Metabolic Control, Obesity, Antidiabetic Treatment and Demographic Characteristics in Adults with Type 1 or 2 Diabetes from the DPV Registry.

AIMS: To examine the association of frozen shoulder (FS) with demographic and diabetes-related outcomes in individuals with type 1 (T1D) or type 2 (T2D) diabetes aged ≥30 years. MATERIALS AND METHODS: Multivariable logistic regression models, adjusted for demographics were used to calculate the proportion of FS in association with age, gender, diabetes duration, body mass index (BMI), haemoglobin A1C (HbA1c) and diabetes treatment. RESULTS: The unadjusted percentage of FS was higher in T1D compared to T2D (0.22% vs. 0.06%). In T1D, adjusted regression models revealed higher prevalence of FS in women than men (0.26 [0.20-0.34] % vs. 0.15 [0.11-0.21] %, p=0.010). No significant relationship of age and BMI with FS was found in both diabetes types. Longer diabetes duration was associated with a higher proportion of FS in T1D (p<0.001) and T2D (p=0.004). In T1D, HbA1c >7% was related to a higher proportion of FS compared to HbA1c ≤7% (0.25 [0.19-0.32] vs. 0.12 [0.08-0.20] %, p=0.007), while an inverse relationship was found in T2D (HbA1c ≤7%: 0.08 [0.07-0.10] vs. HbA1c >7%: 0.05 [0.04-0.06] %, p=0.001). CONCLUSIONS: Different associations of FS with gender and HbA1c were observed for T1D and T2D; however, longer diabetes duration increases the risk for FS independent of diabetes type. Musculoskeletal diseases are still underreported in individuals with diabetes and awareness should be raised for FS as a specific diabetes complication.

Differences in insulin dosing in women with type 1 diabetes before and after the menopause.

The menstrual cycle increases insulin requirements in a subset of women with type 1 diabetes as a result of reduced insulin action through sexual hormones. If exposure to sexual hormones declines during the menopause, adaptions of insulin dosing may be required. However, there are no validated recommendations available on how to adapt insulin treatment in postmenopausal women with type 1 diabetes. The present study compared insulin dosing profiles of 630 premenopausal and 548 postmenopausal women, who had long-term type 1 diabetes and used continuous subcutaneous insulin infusion. Data were extracted from the German "Diabetes-Patienten- Verlaufsdokumentation". It was found that total daily insulin (p <0.0001), daily insulin per kilogram bodyweight (p <0.0001), total daily basal insulin (p <0.0001), daily basal insulin per kilogram bodyweight (p <0.0001) and estimated glomerular filtration rate (eGFR) (p <0.0001) were lower in postmenopausal women. Total daily bolus insulin, daily bolus insulin per kilogram, glycated haemoglobin A1, body mass index and the incidence of severe hypoglycaemic events were similar in both cohorts.Postmenopausal women with type 1 diabetes used lower insulin doses as compared with premenopausal women, whereas glycaemic control and body mass index were comparable. This observation might be explained by lower exposure to sexual hormones and lower eGFR, even though the contribution of other factors such as body composition and eating habits requires further investigation.

Long-term storage of compound solutions for high-throughput screening by using a novel 1536-well microplate.

This report describes the features and the performance of a new and significantly improved 1536-well microplate design. The design allows for simple, automation-friendly, and cost-effective storage of compound solutions for high-throughput screening. The plate design is based on Society for Biomolecular Sciences standards for microplates and can be molded from polystyrene or cycloolefin copolymer, thus making the plate suitable for use with acoustic dispensing as well as other conventional liquid dispensing in the nanoliter range. For a 9:1 DMSO/water mix as solvent, the novel plate design has shown to perform over 4 months with only minor losses in solvent. Thus, this novel plate design creates the basis for further reductions in compound storage volumes and allows for an increase in the storage times for microliter volumes for up to a year or more. The high protection against solvent evaporation is also visible for aqueous solutions, thus allowing for reduced edge effects during screening campaigns.

Diabetes mellitus in Friedreich Ataxia: A case series of 19 patients from the German-Austrian diabetes mellitus registry.

Friedreich ataxia (FRDA) is a multisystem autosomal recessive disease with progressive clinical course involving the neuromuscular and endocrine system. Diabetes mellitus (DM) is one typical non-neurological manifestation, caused by beta cell failure and insulin resistance. Because of its rarity, knowledge on DM in FRDA is limited. Based on data from 200,301 patients with DM of the German-Austrian diabetes registry (DPV) and two exemplary patient reports, characteristics of patients with DM and FRDA are compared with classical type 1 or type 2 diabetes. Diabetes phenotype in FRDA is intermediate between type 1 and type 2 diabetes with ketoacidosis being frequent at presentation and blood glucose levels similar to T1Dm but higher than in T2Dm (356 ±â€¯165 and 384 ±â€¯203 mg/dl). 63.2% of FRDA patients received insulin monotherapy, 21% insulin plus oral antidiabetics and 15.8% lifestyle change only, applying similar doses of insulin in all three groups. FRDA patients did not show overweight and HbA1c levels were even lower than in T1Dm or T2Dm patients, respectively, indicating good overall diabetes control. FRDADm can be controlled by individualized treatment regimen with insulin or oral antidiabetics. Patients with DM in FRDA may show a relevant risk to ketoacidotic complications, which should be avoided.

Comparison of MDRD, CKD-EPI, and Cockcroft-Gault equation in relation to measured glomerular filtration rate among a large cohort with diabetes.

AIMS: To analyze the performance of Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), Cockcroft-Gault (CG), and CG calculated with ideal bodyweight (CG-IBW) equations to estimate glomerular filtration rate (eGFR) based on serum creatinine in a large diabetic population. METHODS: 24,516 adults with type-1-diabetes or type-2-diabetes from the multicenter diabetes prospective follow-up registry DPV were analyzed. We compared eGFR and measured GFR (mGFR) based on 24-h urine collection by calculating mean bias (difference), precision (SD of this difference), accuracy (proportion of eGFR within ±10% of mGFR), Bland-Altman-plots. RESULTS: CG overestimates, whereas MDRD, CKD-EPI, and CG-IBW underestimate. Smallest mean bias and highest accuracy (75.3%) were observed for MDRD compared to the other equations (p<0.0001). MDRD and CKD-EPI estimated most accurately in stages 1 (MDRD:57.7%, CKD-EPI:57.3%) and 2 (MDRD:80.2%, CKD-EPI:80.7%). In stages 3 to 5, highest accuracy was observed for the MDRD (stage 3:82.3%, stage 4:77.8%, stage 5:71.0%). Among younger subjects, accuracy was higher using the CKD-EPI (18-<40years:63.7%, 40-<60years:72.8%). Above age 60years, MDRD estimated most accurately (60-<70years:77.3%, ≥70years:78.8%). In males and females, MDRD estimated most accurately (males:75.3%, females:75.3%). CONCLUSION: In this large diabetic cohort, smallest bias and highest accuracy were observed for the MDRD.

Two low complexity ultra-high throughput methods to identify diverse chemically bioactive molecules using Saccharomyces cerevisiae.

The budding yeast S. cerevisiae is widely used as a eukaryotic model organism to elucidate the mechanism of action of low molecular weight compounds. This report describes the development of two high throughput screening methods based on cell viability either by monitoring the reduction of alamarBlue® (resazurin) or by direct optical measurement of cell growth. Both methods can be miniaturized to allow screening of large numbers of samples, and can be performed using S. cerevisiae in 384 and 1536-well format. The alamarBlue® approach achieves Z' values of >0.7 with signal to basal ratios of >6.5, and around 1.1 million low molecular weight compounds were screened, identifying approximately 25,000 primary hits. Dose response curves generated for a subset (1930) using both alamarBlue® and optical density methods showed significant overlap. In genome-wide haploinsufficiency profiling (HIP), 572 of these hits demonstrated a diverse mechanism of action, affecting >25% of all yeast strains.

Variability of Basal Rate Profiles in Insulin Pump Therapy and Association with Complications in Type 1 Diabetes Mellitus.

BACKGROUND: Traditionally, basal rate profiles in continuous subcutaneous insulin infusion therapy are individually adapted to cover expected insulin requirements. However, whether this approach is indeed superior to a more constant BR profile has not been assessed so far. This study analysed the associations between variability of BR profiles and acute and chronic complications in adult type 1 diabetes mellitus. MATERIALS AND METHODS: BR profiles of 3118 female and 2427 male patients from the "Diabetes-Patienten-Verlaufsdokumentation" registry from Germany and Austria were analysed. Acute and chronic complications were recorded 6 months prior and after the most recently documented basal rate. The "variability index" was calculated as variation of basal rate intervals in percent and describes the excursions of the basal rate intervals from the median basal rate. RESULTS: The variability Index correlated positively with severe hypoglycemia (r = .06; p<0.001), hypoglycemic coma (r = .05; p = 0.002), and microalbuminuria (r = 0.05; p = 0.006). In addition, a higher variability index was associated with higher frequency of diabetic ketoacidosis (r = .04; p = 0.029) in male adult patients. Logistic regression analysis adjusted for age, gender, duration of disease and total basal insulin confirmed significant correlations of the variability index with severe hypoglycemia (ß = 0.013; p<0.001) and diabetic ketoacidosis (ß = 0.012; p = 0.017). CONCLUSIONS: Basal rate profiles with higher variability are associated with an increased frequency of acute complications in adults with type 1 diabetes.

Linking phenotypes and modes of action through high-content screen fingerprints.

High-content screening (HCS) is a powerful technique for monitoring phenotypic responses to treatments on a cellular and subcellular level. Cellular phenotypes can be characterized by multivariate image readouts such as shape, intensity, or texture. The corresponding feature vectors can thus be defined as HCS fingerprints that serve as a powerful biological compound descriptor. Therefore, clustering or classification of HCS fingerprints across compound treatments allows for the identification of similarities in protein targets or pathways. We developed an HCS-based profiling panel that serves as basis for characterizing the mode of action of compounds. This panel measures phenotypic effects in six different compartments of U-2OS cells, namely the nucleus, the cytoplasm, the endoplasmic reticulum, the Golgi apparatus, and the cytoskeleton. We profiled a set of 2,725 well-annotated compounds and clustered their corresponding HCS fingerprints to establish links between predominant cellular phenotypes and cellular processes and protein targets. We found various different clusters enriched for individual targets (e.g., HDAC, HSP90, TOP1, HMGCR, TUB), signaling pathways (e.g., PIK3/AKT/mTOR), or gene sets associated with diseases (e.g., psoriasis, leukemia). Based on this clustering we were able to identify novel compound-target associations for selected compounds such as a submicromolar inhibitory activity of Silmitasertib (a casein kinase inhibitor) on PI3K and mTOR.

Impact of Physical Activity on Glycemic Control and Prevalence of Cardiovascular Risk Factors in Adults With Type 1 Diabetes: A Cross-sectional Multicenter Study of 18,028 Patients.

OBJECTIVE: Physical activity (PA) can improve cardiovascular risk in the general population and in patients with type 2 diabetes. Studies also indicate an HbA(1c)-lowering effect in patients with type 2 diabetes. Since reports in patients with type 1 diabetes are scarce, this analysis aimed to investigate whether there is an association between PA and glycemic control or cardiovascular risk in subjects with type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 18,028 adults (≥18 to <80 years of age) from Germany and Austria with type 1 diabetes from the Diabetes-Patienten-Verlaufsdokumentation (DPV) database were included. Patients were stratified according to their self-reported frequency of PA (PA0, inactive; PA1, one to two times per week; PA2, more than two times per week). Multivariable regression models were applied for glycemic control, diabetes-related comorbidities, and cardiovascular risk factors. Data were adjusted for sex, age, and diabetes duration. P values for trend were given. SAS 9.4 was used for statistical analysis. RESULTS: An inverse association between PA and HbA(1c), diabetic ketoacidosis, BMI, dyslipidemia (all P < 0.0001), and hypertension (P = 0.0150), as well as between PA and retinopathy or microalbuminuria (both P < 0.0001), was present. Severe hypoglycemia (assistance required) did not differ in PA groups (P = 0.8989), whereas severe hypoglycemia with coma was inversely associated with PA (P < 0.0001). CONCLUSIONS: PA seemed to be beneficial with respect to glycemic control, diabetes-related comorbidities, and cardiovascular risk factors without an increase of adverse events. Hence, our data underscore the recommendation for subjects with type 1 diabetes to perform regular PA.

High throughput screening technologies for direct cyclic AMP measurement.

A study comparing five different cAMP detection technologies in terms of sensitivity, robustness, and feasibility for HTS is presented. In this report, the following methods are described: a nonhomogeneous DELFIA, and the homogeneous methods based on time-resolved fluorescence (HTRF), luminescent singlet oxygen channeling or ALPHAScreen, FP, and high-affinity enzyme complementation. DELFIA had the highest sensitivity, whereas ALPHAScreen and HTRF shared several advantages, including high sensitivity, broad dynamic range, and minimal reagent addition steps. For G(s)-coupled antagonist screens, we found HTRF and ALPHAScreen the more sensitive and HTS-compatible techniques.

Multiplexed reporter gene assays: monitoring the cell viability and the compound kinetics on luciferase activity.

High-throughput screening assays with multiple readouts enable one to monitor multiple assay parameters. By capturing as much information about the underlying biology as possible, the detection of true actives can be improved. This report describes an extension to standard luciferase reporter gene assays that enables multiple parameters to be monitored from each sample. The report describes multiplexing luciferase assays with an orthogonal readout monitoring cell viability using reduction of resazurin. In addition, this technical note shows that by using the luciferin substrate in live cells, an assay time course can be recorded. This enables the identification of nonactive or unspecific compounds that act by inhibiting luciferase, as well as compounds altering gene expression or cell growth.

Hypothesis-driven screening.

Phenotypic chemogenomics studies require screening strategies that account for the complex nature of the experimental system. Unknown mechanism of action and high frequency of false positives and false negatives necessitate iterative experiments based on hypotheses formed on the basis of results from the previous step. Process-driven High Throughput Screening (HTS), aiming to "industrialize" lead finding and developed to maximize throughput, is rarely affording sufficient flexibility to design hypothesis-based experiments.In this contribution, we describe a High Throughput Cherry Picking (HTCP) system based on acoustic dispensing technology that was developed to support a new screening paradigm. We demonstrate the power of hypothesis-based screening in three chemogenomics studies that were recently conducted.