Nuclear F-actin and myosins drive relocalization of heterochromatic breaks.

Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, 'safe' repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. Here we show that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes.

Paradoxical androgen receptor regulation by small molecule enantiomers.

Small molecules that target the androgen receptor (AR) are the mainstay of therapy for lethal castration-resistant prostate cancer (CRPC), yet existing drugs lose their efficacy during continued treatment. This evolution of resistance is due to heterogenous mechanisms which include AR mutations causing the identical drug to activate instead of inhibit the receptor. Understanding in molecular detail the paradoxical phenomenon wherein an AR antagonist is transformed into an agonist by structural mutations in the target receptor is thus of paramount importance. Herein, we describe a reciprocal paradox: opposing antagonist and agonist AR regulation determined uniquely by enantiomeric forms of the same drug structure. The antiandrogen BMS-641988, which has (R)-chirality at C-5 encompasses a previously uncharacterized (S)-stereoisomer that is, surprisingly, a potent agonist of AR, as demonstrated by transcriptional assays supported by cell imaging studies. This duality was reproduced in a series of novel compounds derived from the BMS-641988 scaffold. Coupled with in silico modeling studies, the results inform an AR model that explains the switch from potent antagonist to high-affinity agonist in terms of C-5 substituent steric interactions with helix 12 of the ligand binding site. They imply strategies to overcome AR drug resistance and demonstrate that insufficient enantiopurity in this class of AR antagonist can confound efforts to correlate structure with function.

Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates.

Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes.

The Arabidopsis multistress regulator TSPO is a heme binding membrane protein and a potential scavenger of porphyrins via an autophagy-dependent degradation mechanism.

TSPO, a stress-induced, posttranslationally regulated, early secretory pathway-localized plant cell membrane protein, belongs to the TspO/MBR family of regulatory proteins, which can bind porphyrins. This work finds that boosting tetrapyrrole biosynthesis enhanced TSPO degradation in Arabidopsis thaliana and that TSPO could bind heme in vitro and in vivo. This binding required the His residue at position 91 (H91), but not that at position 115 (H115). The H91A and double H91A/H115A substitutions stabilized TSPO and rendered the protein insensitive to heme-regulated degradation, suggesting that heme binding regulates At-TSPO degradation. TSPO degradation was inhibited in the autophagy-defective atg5 mutant and was sensitive to inhibitors of type III phosphoinositide 3-kinases, which regulate autophagy in eukaryotic cells. Mutation of the two Tyr residues in a putative ubiquitin-like ATG8 interacting motif of At-TSPO did not affect heme binding in vitro but stabilized the protein in vivo, suggesting that downregulation of At-TSPO requires an active autophagy pathway, in addition to heme. Abscisic acid-dependent TSPO induction was accompanied by an increase in unbound heme levels, and downregulation of TSPO coincided with the return to steady state levels of unbound heme, suggesting that a physiological consequence of active TSPO downregulation may be heme scavenging. In addition, overexpression of TSPO attenuated aminolevulinic acid-induced porphyria in plant cells. Taken together, these data support a role for TSPO in porphyrin binding and scavenging during stress in plants.

Barriers to and facilitators of paediatric medical device innovation: a scoping review protocol.

INTRODUCTION: The development of paediatric medical devices continues to lag adult medical devices and contributes to issues of inequity, safety, quality and patient outcomes. New legislation and funding mechanisms have been introduced over the past two decades, but the gap remains. Clinical trials have been identified as a pain point, but components of effective clinical research infrastructure are poorly understood. As part of a multimodal research strategy, the Pediatric Device Consortia (PDC) will conduct a scoping review to better understand infrastructural barriers to and facilitators of paediatric medical device clinical research identified in the health sciences literature. METHODS AND ANALYSIS: The following databases will be included for this review: Medline, Embase, Cochrane CENTRAL, Web of Science and IEEE Xplore. Additional grey literature will be sought out through Google Scholar and reviewing the citations of included studies. Included studies will discuss medical devices according to the U.S. Food and Drug Administration classification, focus on the paediatric population (ages 0-21 years) and involve human premarket or postmarket research. All study types that were published in 2007-present in English, Spanish, French or Italian will be included. Using Covidence web-based software, two independent reviewers will screen the resulting titles, abstracts and the full text of potential studies. Conflicts will be resolved by the primary investigator during both phases. REDCap will be used for quantitative and qualitative data charting, generating data tables and narrative synthesis. ETHICS AND DISSEMINATION: This research did not require research ethics board consideration as it does not involve human participants and all data will be collected from published literature. We will share our findings through peer-reviewed manuscripts, clinical and research conference presentations and professional networks available to the PDC. STUDY REGISTRATION: Open Science Framework (https://osf.io/k72bn).

Reduced Isocyanate Release Using a Waterproof, Resin-Based Cast Alternative Relative to Fiberglass Casts.

The effects of occupational isocyanate exposure range from asthma and contact dermatitis to neurotoxicity and cancer. Respiratory sensitization due to orthopedic cast application has been well documented. This study aims to compare the safety of standard-of-care fiberglass casts and a novel waterproof cast alternative by measuring the amount of isocyanate released during off-gassing over time. A 3D-printed arm simulator with comparable casing material amounts was placed in a sealed chamber. An isocyanate-sensing color-changing (SafeAir) tag was used to measure the levels of toxic exposure. Triplicate trials were conducted across all time periods (15 min, 1 h, and 24 h) and conditions. The bare arm simulator and freshly opened tags served as negative controls. Normalized pixel intensity indexes and isocyanate release estimates in ppb were derived from ImageJ-analyzed SafeAir tag photos. Fiberglass casts exhibited greater isocyanate release than both the waterproof alternative (p = 0.0002) and no-cast controls (p = 0.0006), particularly at 24 h. The waterproof alternative and no-cast control did not statistically differ (p = 0.1603). Therefore, the waterproof alternative released less isocyanate than the fiberglass casts. Waterproof cast alternatives may be safer than fiberglass by limiting medical professionals' exposure to toxic isocyanates and, thus, decreasing their risk of suffering occupational asthma.

Frequency and Detection of Insulin Infusion Site Failure in the Type 1 Diabetes Exchange Online Community.

Insulin infusion site (IIS) failures are a weakness in insulin pump therapy. We examined experience with IIS failures among U.S. individuals with diabetes on insulin pump through survey distributed to the T1D Exchange Online Community. Demographic factors, IIS characteristics, and diabetes-related perceptions were assessed by logistic regression to determine odds of higher (≥1 per month) or lower (<1 per month) reported IIS failure frequency. IIS failures were common; 41.4% reported ≥1 per month. IIS failure is usually detected through development of hyperglycemia rather than pump alarm. No assessed demographic factor or IIS characteristic was predictive; however, higher odds of ≥1 failure per month were associated with feelings of burnout (odds ratios [OR] 1.489 [1.024, 2.165]) and considering pump discontinuation (OR 2.233 [1.455, 3.427]). IIS failures are frequent and unpredictable, typically require hyperglycemia for detection, and are associated with negative perceptions. More should be done toward preventing IIS failures and/or detecting them sooner.

A novel maturity index for assessing medical device startups.

Background: Startup companies in the healthcare sector often fail because they lack sufficient entrepreneurial, regulatory, and business development expertise. Maturity models provide useful frameworks to assess the state of business elements more systematically than heuristic assessments. However, previous models were developed primarily to characterize the business state of larger nonmedical companies. A maturity index designed specifically for startup companies in the medical product sector could help to identify areas in which targeted interventions could assist business development. Methods: A novel MedTech Startup Maturity Index (SMI) was developed by a collaborative team of academic and industry experts and refined through feedback from external stakeholders. Pediatric medical device startups associated with the West Coast Consortium for Technology & Innovation in Pediatrics (CTIP) were scored and ranked according to the SMI following semi-structured interviews. The CTIP executive team independently ranked the maturity of each company based on their extensive experiences with the same companies. Results: SMI scores for 16 companies ranged from 1.2 to 3.8 out of 4. These scores were well aligned with heuristic CTIP rankings for 14 out of 16 companies, reflected by strong correlations between the two datasets (Spearman's rho = 0.721, P = 0.002, and Kendall's tau-b = 0.526, P = 0.006). Conclusions: The SMI yields maturity scores that correlate well with expert rankings but can be assessed without prior company knowledge and can identify specific areas of concern more systematically. Further research is required to generalize and validate the SMI as a pre-/post-evaluation tool.

Peristalsis in the junction region of the Drosophila larval midgut is modulated by DH31 expressing enteroendocrine cells.

BACKGROUND: The underlying cellular and molecular mechanisms that coordinate the physiological processes in digestion are complex, cryptic, and involve the integration of multiple cellular and organ systems. In all intestines, peristaltic action of the gut moves food through the various stages of digestion from the anterior end towards the posterior, with the rate of flow dependent on signals, both intrinsic and extrinsic to the gut itself. RESULTS: We have identified an enteroendocrine cell type that regulates gut motility in the Drosophila melanogaster larval midgut. These cells are located at the junction of the anterior and the acidic portions of the midgut and are a group of enteroendocrine cells that express the peptide hormone Diuretic Hormone 31 in this region of the gut. Using cell ablation and ectopic activation via expression of the Chlamydomonas reinhardtii blue light-activated channelopsin, we demonstrate that these enteroendocrine cells are both necessary and sufficient for the peristalsis in the junction region of the midgut and require the Diuretic Hormone 31 to affect normal peristalsis in this region. Within the same junction region of the midgut, we have also identified morphological features suggesting that this region acts as a valve that regulates the transit of food from the anterior midgut into the acidic portion of the gut. CONCLUSIONS: We have characterized and described a set of enteroendocrine cells called the Midgut Junction DH31 expressing cells that are required for peristaltic movement in the junction region between the anterior portion and acidic region of the larval midgut of Drosophila melanogaster. We have shown that the Midgut Junction DH31 expressing cells are necessary and sufficient for motility and that the peptide hormone DH31 is required for peristalsis in the junction region of the midgut. The Drosophila model system will allow for a further dissection of the digestion process and provide a better understanding of the mechanisms that regulate digestion in all organisms.

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair.

DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells.

And yet, it moves: nuclear and chromatin dynamics of a heterochromatic double-strand break.

Heterochromatin is mostly composed of repeated DNA sequences prone to aberrant recombination. How cells maintain the stability of these sequences during double-strand break (DSB) repair has been a long-standing mystery. Studies in Drosophila cells revealed that faithful homologous recombination repair of heterochromatic DSBs relies on the striking relocalization of repair sites to the nuclear periphery before Rad51 recruitment and repair progression. Here, we summarize our current understanding of this response, including the molecular mechanisms involved, and conserved pathways in mammalian cells. We will highlight important similarities with pathways identified in budding yeast for repair of other types of repeated sequences, including rDNA and short telomeres. We will also discuss the emerging role of chromatin composition and regulation in heterochromatin repair progression. Together, these discoveries challenged previous assumptions that repair sites are substantially static in multicellular eukaryotes, that heterochromatin is largely inert in the presence of DSBs, and that silencing and compaction in this domain are obstacles to repair.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

Concentrating pre-mRNA processing factors in the histone locus body facilitates efficient histone mRNA biogenesis.

The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3' processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3' end processing with transcription termination.