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Objective:Several clinical procedures utilize duodenoscopes, which are processed for reuse after the procedures are completed. However, infection outbreaks due to improper duodenoscope processing occur frequently. To address this, we aimed to assess the contamination rates of duodenoscopes after reprocessing in nonoutbreak settings.Design and setting:Prospective study in 16 clinical sites in the United States.Methods:We sampled and cultured reprocessed duodenoscopes following the FDA/CDC/ASM guideline; “Duodenoscope Surveillance Sampling and Culturing – Reducing the Risks of Infection.” High-concern (HC) organisms were those highly associated with disease, including gram-negative rods, Staphylococcus aureus, Staphylococcus lugdunensis, β-hemolytic Streptococcus, Enterococcus spp, and yeasts. We evaluated duodenoscopes with ≥1 CFU of organisms after reprocessing. The reprocessing environments were also sampled and cultured.Results:We assessed 859 newer-model (NM) duodenoscopes (TJF-Q180V) and 850 older-model (OM) duodenoscopes (TJF-160F/VF); of these, 35 NM samples (4.1%) and 56 OM samples (6.6%) were contaminated with HC organisms. We detected and classified the HC organisms as gastrointestinal (45.4%), human origin (16.7%), environmental (24.1%), waterborne (13.0%), and unidentified (0.9%).Conclusions:We detected an overall HC contamination rate of 5.3% in nonoutbreak settings. Although the relationship between endoscopic contamination and the occurrence of infections remains unclear, attempts should continue to be made to further reduce contamination rates. Additional improvements to the manufacturer’s instructions for use, human factors during the reprocessing procedure, ongoing training programs, cleanliness of reprocessing environments, and the design of the distal end of the duodenoscope should be considered.

International audience; BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation., Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. Here, the authors provide evidence that these hypersensitivities reflect a crucial role for ATM at damaged replication forks being to prevent toxic DNA end-joining leading to chromosome fusions and cell death.

Background: Needle samples may not provide sufficient diagnostic material for the assessment of mediastinal lymph nodes. Objective: The study compared the specimen size and diagnostic performance of a new 19-G endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) needle to that of a standard 22-G EBUS-TBNA needle in a swine model of granulomatous lymphadenopathy. Methods: Granulomatous inflammation was induced in mediastinal lymph nodes (LNs) of 10 domestic swine by injection of talc slurry. The affected LNs were sampled with the 19- and 22-G needles. Collected core tissue area and volume were determined using a specialized software and known needle internal diameter. The sample's quality was assessed using the biopsy core morphology grade (BCMG) as well as the biopsy diagnostic correlation grade (BDCG). Results: There was a significant increase in the average LN size from baseline (11.6 ± 3.2 to 15.2 ± 3.8 mm; p < 0.001) after talc injection. A total of 132 paired samples were collected from 38 LNs. The average mass and volume of the 19-G needle sample were larger than those of the 22-G needle sample: 33.78 ± 47.48 vs. 25.18 ± 32.08 mg (p < 0.002) and 11.40 ± 13.91 vs. 6.91 ± 6.42 mm3 (p < 0.0004), respectively. The pooled needle biopsy samples for the 19- and the 22-G needles had similar BCMG (1.38 ± 0.86 vs. 1.43 ± 0.87, p > 0.2) and BDCG (1.54 ± 0.93 vs. 1.57 ± 0.93, p > 0.2). The 19-G needle samples tended towards less blood contamination (p = 0.057), more often granuloma identification (46 vs. 32%, p = 0.2) and had more cartilage contamination (0.49 ± 1.46 vs. 4.81 ± 16.49% p < 0.003). Conclusion: In experienced hands, the 19- and the 22-G EBUS-TBNA needles have a similar diagnostic yield in the swine model of granulomatous lymphadenopathy. The samples collected by the 19-G needle are larger and may have less blood contamination.

The Notch receptor is necessary for modulating cell fate decisions throughout development, and aberrant activation of Notch signalling has been associated with many diseases, including tumorigenesis. The E3 ligase MDM2 (murine double minute 2) plays a role in regulating the Notch signalling pathway through its interaction with NUMB. In the present study we report that MDM2 can also exert its oncogenic effects on the Notch signalling pathway by directly interacting with the Notch 1 receptor through dual-site binding. This involves both the N-terminal and acidic domains of MDM2 and the RAM [RBP-Jκ (recombination signal-binding protein 1 for Jκ)-associated molecule] and ANK (ankyrin) domains of Notch 1. Although the interaction between Notch1 and MDM2 results in ubiquitination of Notch1, this does not result in degradation of Notch1, but instead leads to activation of the intracellular domain of Notch1. Furthermore, MDM2 can synergize with Notch1 to inhibit apoptosis and promote proliferation. This highlights yet another target for MDM2-mediated ubiquitination that results in activation of the protein rather than degradation and makes MDM2 an attractive target for drug discovery for both the p53 and Notch signalling pathways.

Disruption of apical–basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation. Affinity purification/mass spectrometry revealed a critical role of DLG5 in the formation of protein assemblies containing core Hippo kinases mammalian ste20 homologs 1/2 (MST1/2) and Par-1 polarity proteins microtubule affinity-regulating kinases 1/2/3 (MARK1/2/3). Consistent with this finding, Hippo signaling is markedly hyperactive in mammalian Dlg5−/− tissues and cells in vivo and ex vivo and in Drosophila upon dlg5 knockdown. Conditional deletion of Mst1/2 fully rescued the phenotypes of brain-specific Dlg5 knockout mice. Dlg5 also interacts genetically with Hippo effectors Yap1/Taz. Mechanistically, we show that DLG5 inhibits the association between MST1/2 and large tumor suppressor homologs 1/2 (LATS1/2), uses its scaffolding function to link MST1/2 with MARK3, and inhibits MST1/2 kinase activity. These data reveal a direct connection between cell polarity proteins and Hippo, which is essential for proper development of multicellular organisms.

Introduction: Endobronchial Ultrasound guided Trans-bronchial Needle Aspiration, (EBUS-TBNA) is used for the diagnosis and/or staging of cancer and other disorders. A novel 19G EBUS-TBNA needle ([Olympus Respiratory America], Redmond, WA, USA) with a larger diameter was developed to collect larger samples. Aims and Objectives: Evaluations were done in patients to assess the performance of the new 19G EBUS-TBNA needle. Methods: Product performance evaluations were conducted in 4 countries by 13 physicians experienced with EBUS-TBNA from Sept 2015 to Nov 2015. Physicians in each country were provided with 20 needles and asked to evaluate their performance in patients by completing evaluation forms. The forms asked physicians to rate the needles9 absolute (stand-alone) and relative performance (with respect to their experience of using current commercially available 21G and 22G needles) in the areas of penetration, visibility, and sample collection. Eighty completed evaluation forms were returned and analyzed. Results: For relative performance, physicians rated the 19G needle as comparable or better on 97.5% of the forms for sample collection, 92.5% for penetration, and 95% for visibility. For absolute performance, physicians rated the ability of the device to collect samples, its penetration, and its visibility as acceptable 98.75%, 97.5%, and 97.5% of the time respectively. One subject in Poland experienced bleeding after puncturing of the right hilum. Conclusions: Product performance evaluations of the 19G needle suggest that its performance is equivalent to or better than currently commercially available needles. However, additional clinical evaluations are warranted.

Mutations in the head and tail domains of the motor protein myosin VIIA (MYO7A) cause deaf-blindness (Usher syndrome type 1B, USH1B) and nonsyndromic deafness (DFNB2, DFNA11). The head domain binds to F-actin and serves as the MYO7A motor domain, but little is known about the function of the tail domain. In a genetic screen, we have identifiedpolkamice, which carry a mutation (c.5742 + 5G > A) that affects splicing of the MYO7A transcript and truncates the MYO7A tail domain at the C-terminal FERM domain. In the inner ear, expression of the truncated MYO7A protein is severely reduced, leading to defects in hair cell development. In retinal pigment epithelial (RPE) cells, the truncated MYO7A protein is expressed at comparative levels to wild-type protein but fails to associate with and transport melanosomes. We conclude that the C-terminal FERM domain of MYO7A is critical for melanosome transport in RPE cells. Our findings also suggest thatMYO7Amutations can lead to tissue-specific effects on protein levels, which may explain why some mutations inMYO7Alead to deafness without retinal impairment.

Deafness is the most common form of sensory impairment in the human population and is frequently caused by recessive mutations. To obtain animal models for recessive forms of deafness and to identify genes that control the development and function of the auditory sense organs, we performed a forward genetics screen in mice. We identified 13 mouse lines with defects in auditory function and six lines with auditory and vestibular defects. We mapped several of the affected genetic loci and identified point mutations in four genes. Interestingly, all identified genes are expressed in mechanosensory hair cells and required for their function. One mutation maps to thepejvakingene, which encodes a new member of the gasdermin protein family. Previous studies have described two missense mutations in the humanpejvakingene that cause nonsyndromic recessive deafness (DFNB59) by affecting the function of auditory neurons. In contrast, thepejvakinallele described here introduces a premature stop codon, causes outer hair cell defects, and leads to progressive hearing loss. We also identified a novel allele of the humanpejvakingene in an Iranian pedigree that is afflicted with progressive hearing loss. Our findings suggest that the mechanisms of pathogenesis associated withpejvakinmutations are more diverse than previously appreciated. More generally, our findings demonstrate that recessive screens in mice are powerful tools for identifying genes that control the development and function of mechanosensory hair cells and cause deafness in humans, as well as generating animal models for disease.

Ubiquitylation is crucial for proper cellular responses to DNA double-strand breaks (DSBs). If unrepaired, these highly cytotoxic lesions cause genome instability, tumourigenesis, neurodegeneration or premature ageing. Here, we conduct a comprehensive, multilayered screen to systematically profile all human ubiquitin E2-enzymes for impacts on cellular DSB responses. Applying a widely applicable approach, we use an exemplary E2 family, UBE2Ds, to identify ubiquitylation-cascade components downstream of E2s. Thus, we uncover the nuclear E3-ligase RNF138 as a key homologous recombination (HR)-promoting factor that functions with UBE2Ds in cells. Mechanistically, UBE2Ds and RNF138 accumulate at DNA-damage sites and act at early resection stages by promoting CtIP ubiquitylation and accrual. This work supplies insights into regulation of DSB repair by HR. Moreover, it provides a rich information resource on E2s that can be exploited by follow-on studies.