Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

RING‐in‐between‐RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub‐conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT‐type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING‐type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub‐binding site on HHARI RING2 important for its recruitment to RING1‐bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.     

Diabetes screening intervals based on risk stratification

Background

Guidelines for frequency of Type 2 diabetes mellitus (DM) screening remain unclear, with proposed screening intervals typically based on expert opinion. This study aims to demonstrate that HbA1c screening intervals may differ substantially when considering individual risk for diabetes.

Methods

This was a multi-institutional retrospective open cohort study. Data were collected between April 1999 to March 2014 from one urban and one rural cohort in Japan. After categorization by age, we stratified individuals based on cardiovascular disease risk (Framingham 10-year cardiovascular risk score) and body mass index (BMI). We adapted a signal-to-noise method for distinguishing true HbA1c change from measurement error by constructing a linear random effect model to calculate signal and noise of HbA1c. Screening interval for HbA1c was defined as informative when the signal-to-noise ratio exceeded 1.

Results

Among 96,456 healthy adults, 46,284 (48.0%) were male; age (range) and mean HbA1c (SD) were 48 (30–74) years old and 5.4 (0.4)%, respectively. As risk increased among those 30–44 years old, HbA1c screening intervals for detecting Type 2 DM consistently decreased: from 10.5 (BMI <18.5) to 2.4 (BMI?>?30) years, and from 8.0 (Framingham Risk Score <10%) to 2.0 (Framingham Risk Score ≥20%) years. This trend was consistent in other age and risk groups as well; among obese 30–44 year olds, we found substantially shorter intervals compared to other groups.

Conclusion

HbA1c screening intervals for identification of DM vary substantially by risk factors. Risk stratification should be applied when deciding an optimal HbA1c screening interval in the general population to minimize overdiagnosis and overtreatment.

     

Defining the identity of mouse embryonic dermal fibroblasts

Embryonic dermal fibroblasts in the skin have the exceptional ability to initiate hair follicle morphogenesis and contribute to scarless wound healing. Activation of the Wnt signaling pathway is critical for dermal fibroblast fate selection and hair follicle induction. In humans, mutations in Wnt pathway components and target genes lead to congenital focal dermal hypoplasias with diminished hair. The gene expression signature of embryonic dermal fibroblasts during differentiation and its dependence on Wnt signaling is unknown. Here we applied Shannon entropy analysis to identify the gene expression signature of mouse embryonic dermal fibroblasts. We used available human DNase‐seq and histone modification ChiP‐seq data on various cell‐types to demonstrate that genes in the fibroblast cell identity signature can be epigenetically repressed in other cell‐types. We found a subset of the signature genes whose expression is dependent on Wnt/β‐catenin activity in vivo. With our approach, we have defined and validated a statistically derived gene expression signature that may mediate dermal fibroblast identity and function in development and disease. genesis 54:415–430, 2016. © 2016 Wiley Periodicals, Inc.     

Monitoring cancer prognosis,diagnosis and treatment efficacy using metabolomics and lipidomics

Introduction

Cellular metabolism is altered during cancer initiation and progression, which allows cancer cells to increase anabolic synthesis, avoid apoptosis and adapt to low nutrient and oxygen availability. The metabolic nature of cancer enables patient cancer status to be monitored by metabolomics and lipidomics. Additionally, monitoring metabolic status of patients or biological models can be used to greater understand the action of anticancer therapeutics.

Objectives

Discuss how metabolomics and lipidomics can be used to (i) identify metabolic biomarkers of cancer and (ii) understand the mechanism-of-action of anticancer therapies. Discuss considerations that can maximize the clinical value of metabolic cancer biomarkers including case–control, prognostic and longitudinal study designs.

Methods

A literature search of the current relevant primary research was performed.

Results

Metabolomics and lipidomics can identify metabolic signatures that associate with cancer diagnosis, prognosis and disease progression. Discriminatory metabolites were most commonly linked to lipid or energy metabolism. Case–control studies outnumbered prognostic and longitudinal approaches. Prognostic studies were able to correlate metabolic features with future cancer risk, whereas longitudinal studies were most effective for studying cancer progression. Metabolomics and lipidomics can help to understand the mechanism-of-action of anticancer therapeutics and mechanisms of drug resistance.

Conclusion

Metabolomics and lipidomics can be used to identify biomarkers associated with cancer and to better understand anticancer therapies.

     

Development of a core outcome set for clinical trials in rosacea: study protocol for a systematic review of the literature and identification of a core outcome set using a Delphi survey

BackgroundRosacea is a chronic inflammatory disorder affecting millions of individuals worldwide. Diagnosis is based on signs and symptoms with management and treatment aimed to suppress inflammatory lesions, erythema, and telangiectasia. While many clinical trials of rosacea exist, the lack of consensus in outcome reporting across all trials poses a concern. Proper evaluation and comparison of treatment modalities is challenging. In order to address the inconsistencies present, this project aims to determine a core set of outcomes which should be evaluated in all clinical trials of rosacea.Methods/designThis project will utilize a methodology similar to previous core outcome set research. A long list of outcomes will be extracted over four phases: (1) systematic literature review, (2) patient interviews, (3) other published sources, and (4) stakeholder involvement. Potential outcomes will be examined by the Steering Committee to provide further insight. The Delphi process will then be performed to prioritize and condense the list of outcomes generated. Two homogenous groups of physicians and patients will participate in two consecutive rounds of Delphi surveys. A consensus meeting, composed of physicians, patients, and stakeholders, will be conducted after the Delphi exercise to further select outcomes, taking into account participant scores. By the end of the meeting, members will vote and decide on a final recommended set of core outcomes. For the duration of the study, we will be in collaboration with both the Core Outcome Measures in Effectiveness Trials (COMET) and Cochrane Skin Group - Core Outcome Set Initiative (CSG-COUSIN).DiscussionThis study aims to develop a core outcome set to guide assessment in clinical trials of rosacea. The end-goal is to improve the reliability and consistency of outcome reporting, thereby allowing sufficient evaluation of treatment effectiveness and patient satisfaction.

Electronic supplementary material

The online version of this article (doi:10.1186/s13063-016-1554-3) contains supplementary material, which is available to authorized users.     

Twist and Degrade—Impact of Molecular Structure on the Photostability of Nonfullerene Acceptors and Their Photovoltaic Blends

Nonfullerene acceptors (NFAs) dominate organic photovoltaic (OPV) research due to their promising efficiencies and stabilities. However, there is very little investigation into the molecular processes of degradation, which is critical to guiding design of novel NFAs for long‐lived, commercially viable OPVs. Here, the important role of molecular structure and conformation in NFA photostability in air is investigated by comparing structurally similar but conformationally different promising NFAs: planar O‐IDTBR and nonplanar O‐IDFBR. A three‐phase degradation process is identified: i) initial photoinduced conformational change (i.e., torsion about the core–benzothiadiazole dihedral), induced by noncovalent interactions with environmental molecules, ii) followed by photo‐oxidation and fragmentation, leading to chromophore bleaching and degradation product formation, and iii) finally complete chromophore bleaching. Initial conformational change is a critical prerequisite for further degradation, providing fundamental understanding of the relative stability of IDTBR and IDFBR, where the already twisted IDFBR is more prone to degradation. When blended with the donor polymer poly(3‐hexylthiophene), both NFAs exhibit improved photostability while the photostability of the polymer itself is significantly reduced by the more miscible twisted NFA. The findings elucidate the important role of NFA molecular structure in photostability of OPV systems, and provide vital insights into molecular design rules for intrinsically photostable NFAs.     

High efficiency Agrobacterium‐mediated site‐specific gene integration in maize utilizing the FLP‐FRT recombination system

An efficient Agrobacterium‐mediated site‐specific integration (SSI) technology using the flipase/flipase recognition target (FLP/FRT) system in elite maize inbred lines is described. The system allows precise integration of a single copy of a donor DNA flanked by heterologous FRT sites into a predefined recombinant target line (RTL) containing the corresponding heterologous FRT sites. A promoter‐trap system consisting of a pre‐integrated promoter followed by an FRT site enables efficient selection of events. The efficiency of this system is dependent on several factors including Agrobacterium tumefaciens strain, expression of morphogenic genes Babyboom (Bbm) and Wuschel2 (Wus2) and choice of heterologous FRT pairs. Of the Agrobacterium strains tested, strain AGL1 resulted in higher transformation frequency than strain LBA4404 THY‐ (0.27% vs. 0.05%; per cent of infected embryos producing events). The addition of morphogenic genes increased transformation frequency (2.65% in AGL1; 0.65% in LBA4404 THY‐). Following further optimization, including the choice of FRT pairs, a method was developed that achieved 19%–22.5% transformation frequency. Importantly, >50% of T0 transformants contain the desired full‐length site‐specific insertion. The frequencies reported here establish a new benchmark for generating targeted quality events compatible with commercial product development.     

Tumor-infiltrating lymphocytes: Streamlining a complex manufacturing process

Adoptive cell therapy of tumor-infiltrating lymphocytes has shown promise for treatment of refractory melanoma and other solid malignancies; however, challenges to manufacturing have limited its widespread use. Traditional manufacturing efforts were lengthy, cumbersome and used open culture systems. We describe changes in testing and manufacturing that decreased the process cycle time, enhanced the robustness of critical quality attribute testing and facilitated a functionally closed system. These changes have enabled export of the manufacturing process to support multi-center clinical trials.