High overall copy number variation burden by genome-wide methylation profiling holds negative prognostic value in surgically treated pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of cancer with an overall 5-year survival of around 10 %. New prognostic tools to stratify patients are needed. Our main aim was to evaluate the prognostic value of overall copy number variation (CNV) burden in surgically treated PDAC. DNA extracted from 108 surgical PDAC specimens was examined to collect data on the genome-wide DNA methylation status of &gt;850,000 CpG sites in promoter, gene body, and enhancer regions (Illumina Infinium Methylation EPIC BeadChip Kit). CNV profiles were obtained and all PDACs were stratified into one of three groups: Low, moderate, or high overall CNV burden. Tumors histologically showing a dominant conventional and/or tubulopapillary pattern in 60 %–100 % and 0–59 % were categorized as Group A and Group B as per Kalimuthu. We also performed targeted next-generation sequencing (NGS) and immunohistochemistry. High overall CNV burden held independent negative prognostic value with poor survival (HR 4.01 (95%CI 1.96–8.19), p = 0.00014) and was more frequent in Group B (p = 0.0003). Most frequent chromosomal arm-level aberrations were gains of 8q (29 %) and 1q (19 %) and losses of 17p (55 %), 18q (43 %), 6q (37 %), 9p (36 %), 6p (26 %), 19p (26 %), and 8p (25 %). Most frequent mutations found were in KRAS (95 %), TP53 (62 %), CDKN2A (24 %), SMAD4 (23 %), ATM (9 %), ARID1A (7 %), RNF43 (7 %), GNAS (6 %), and KDM6A (6 %). Group A PDACs showed more frequently KRAS variants other than Gly12Val and Gly12Asp (p = 0.012). Our data indicate that overall CNV burden using genome-wide methylation profiling may be a useful prognostic tool in surgically treated PDAC. Importantly, our approach, using data from genome-wide methylation profiling for analysis of overall CNV burden, can be performed on formalin-fixed and paraffin embedded PDAC tissues. Future studies should examine the prognostic value of overall CNV burden in unresectable PDAC.</p

K(2P)18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K(2P)18.1 is a relevant regulator. Here, we identify K(2P)18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-kappa B-mediated K(2P)18.1 upregulation in tTreg progenitors. K(2P)18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-kappa B- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K(2P)18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K(2P)18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K(2P)18.1 variant that is associated with poor clinical outcomes indicate that K(2P)18.1 also plays a role in human Treg development. Pharmacological modulation of K(2P)18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K(2P)18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K(2P)18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.Peer reviewe

Association of Serum Afamin Concentrations With Kidney Failure in Patients With CKD: Findings From the German CKD Cohort Study

Rationale & objective: Afamin is a vitamin E-binding glycoprotein primarily expressed in the liver and kidney. This study investigated whether serum afamin concentrations are associated with kidney function and incident kidney failure. Study design: Prospective cohort study with 6.5 years follow-up. Setting & participants: 5,041 White patients enrolled in the German Chronic Kidney Disease (GCKD) study with measured afamin concentrations and either an estimated glomerular filtration rate (eGFR) of 30-60mL/min/1.73m2 or an eGFR>60mL/min/1.73m2 with a urinary albumin-creatinine ratio (UACR) of≥300mg/g at study entry. Exposure: Serum afamin concentrations (mg/L). Outcome: Incident kidney failure (initiation of kidney replacement therapy or kidney-related death). Analytical approach: Generalized linear regression and quantile regression models fit to investigate the association of afamin concentrations with eGFR and UACR. Adjusted Cox regression analysis to examine the association of afamin concentrations with incident kidney failure. Results: The mean±SD afamin concentration at study entry was 73.2±17.6mg/L. Higher afamin concentrations were associated with better kidney function with a 2.60mL/min/1.73m2 higher eGFR (95% CI, 2.30-2.89) and a 5.97mg/g lower UACR (95% CI, 3.04-8.90) for each 10mg/L higher level of afamin concentration in adjusted analysis. During the follow-up period, each 10mg/L higher level of afamin concentration was associated with a 14% lower risk of kidney failure (HR, 0.86 [95%CI, 0.81-0.92], P<0.001). Limitations: Residual confounding, and potential limited generalizability to non-White populations and people with mild stages of chronic kidney disease (CKD) or no CKD. Conclusions: Higher serum afamin concentrations appear to be associated with a higher eGFR, less albuminuria, and a lower risk for future kidney failure in patients with CKD

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Interactive exploration of adverse events and multimorbidity in CKD

Background: Persons with chronic kidney disease (CKD) are at increased risk of adverse events, early mortality and multimorbidity. A detailed overview of adverse event types and rates from a large CKD cohort under regular nephrological care is missing. We generated an interactive tool to enable exploration of adverse events and their combinations in the prospective, observational German CKD (GCKD) study. Methods: The GCKD study enrolled 5217 participants under regular nephrological care with an estimated glomerular filtration rate of 30-60 or >60 mL/min/1.73 m2 and an overt proteinuria. Cardiovascular, cerebrovascular and peripheral vascular, kidney, infection, and cancer events, as well as deaths were adjudicated following a standard operation procedure. We summarized these time-to-event data points for exploration in interactive graphs within an R shiny app. Multivariable adjusted Cox models for time to first event were fitted. Cumulative incidence functions, Kaplan-Meier curves and intersection plots were used to display main adverse events and their combinations by sex and CKD etiology. Results: Over a median of 6.5 years, 10 271 events occurred in 2947 participants (56.5%), of which 680 participants (13.0%) died. The new publicly available interactive platform enables readers to scrutinize adverse events and their combinations as well as mortality trends as a gateway to better understand multimorbidity in CKD: incident rates per 1000 patient-years varied by event type, CKD etiology and baseline characteristics. Incidence rates for the most frequent events and their recurrence were 113.6 (cardiovascular), 75.0 (kidney) and 66.0 (infection). Participants with presumed diabetic kidney disease and men were more prone to experiencing events. Conclusion: This comprehensive explorative tool to visualize adverse events (https://www.gckd.org/studienhintergrund/previous-study-results/event-analysis/), their combination, mortality and multimorbidity among persons with CKD may serve as a valuable resourec for patient care, identification of high-risk groups, health services and public health policy planning

Expression tuning in E.coli: discussion of state-of-the-art technologies and multivariate investigation of the pBAD mixed feed system

Background: Tuning of transcription is a powerful process technological tool for efficient recombinant protein production in E. coli. Many challenges such as product toxicity, formation of inclusion bodies, cell death and metabolic burden are associated with non-suitable (too high or too low) levels of recombinant protein expression. Tunable expression systems allow adjusting the recombinant protein expression using process technological means. This enables to exploit the cell's metabolic capacities to a maximum. Aims: In this thesis, tunable recombinant protein expression in E. coli is reviewed thoroughly from a biological and process technological point of view. In a consequent step expression tuning is applied to produce vascular endothelial growth factor-A165 (VEGF-A165), a pharmacologically relevant key player in angiogenesis, in correctly folded and active form in E. coli periplasm. Therefore the well-established system for expression tuning, the E. coli pBAD mixed feed platform, is investigated for the development of an upstream production process. Material and Methods: An E. coli C41 strain with intact L-arabinose metabolism was used in a mixed feed environment with D-glucose as main substrate and L-arabinose as inducing substrate. Following Quality by Design (QbD) principles, the three most promising critical process parameters (CPPs) namely the specific growth rate, specific inducer uptake rate and temperature were investigated in a design of experiments. A 23 factorial design (3 factors at 2 levels) for GIII and 22 factorial design in the case of DsbA signal sequence were conducted, each with a set of 3 center points. Results: So far expression tuning was only addressed in a few studies. For the first time these studies were reviewed with respect to latest findings on induction kinetics and mechanistics. According to the current level of knowledge some promoter system were successfully for expression tuning, in some cases analytical evidence on single cell level is still pending and some attempts did only influence protein expression on population level. For the first time, a promising mixed feed system was applied for tunable protein expression in the periplasm of E. coli. Beside of the observation of quality and quantity dependencies on the investigated CPPs it was demonstrated that the product transcription rate could indirectly be included in an experimental design by the successful use of a tunable promoter system in E. coli. Conclusion: In summary, the use of a tunable expression system was successfully applied in the development of an upstream process for the production of VEGF-A165 in the periplasm of E. coli. We anticipate that expression tuning is able to tackle further issues caused by inappropriate transcription levels and therefore is not only a major benefit for process development, but can pave the way for continuous production of biopharmaceuticals by the issues of constant product quality and culture long term stability14