USP1-trapping lesions as a source of DNA replication stress and genomic instability.

The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by "USP1-trapping". We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.

A New Smartphone-Based Optic Nerve Head Biometric for Verification and Change Detection.

Purpose: Lens adapted smartphones are being used regularly instead of ophthalmoscopes. The most common causes of preventable blindness in the world, which are glaucoma and diabetic retinopathy, can develop asymptomatic changes to the optic nerve head (ONH) especially in the developing world where there is a dire shortage of ophthalmologists but ubiquitous mobile phones. We developed a proof-of-concept ONH biometric (application [APP]) to use as a routine biometric on a mobile phone. The unique blood vessel pattern is verified if it maps on to a previously enrolled image. Methods: The iKey APP platform comprises three deep neural networks (DNNs) developed from anonymous ONH images: the graticule blood vessel (GBV) and the blood vessel specific feature (BVSF) DNNs were trained on unique blood vessel vectors. A non-feature specific (NFS) baseline ResNet50 DNN was trained for comparison. Results: Verification reached an accuracy of 97.06% with BVSF, 87.24% with GBV and 79.8% using NFS. Conclusions: A new ONH biometric was developed with a hybrid platform of ONH algorithms for use as a verification biometric on a smartphone. Failure to verify will alert the user to possible changes to the image, so that silent changes may be observed before sight threatening disease progresses. The APP retains a history of all ONH images. Future longitudinal analysis will explore the impact of ONH changes to the iKey biometric platform. Translational Relevance: Phones with iKey will host ONH images for biometric protection of both health and financial data. The ONH may be used for automatic screening by new disease detection DNNs.

Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells.

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.

In a Class of Its Own: A New Family of Deubiquitinases Promotes Genome Stability.

Several proteins are ubiquitylated in response to genotoxic stress; however, the roles of deubiquitinases (DUBs) in reversing these modifications are less well characterized. Two independent studies by Kwasna et al. (2018) and Haahr et al. (2018) identify a new type of cysteine protease DUB called ZUFSP, which cleaves K63-linked polyubiquitin chains at DNA damage sites to promote genome stability.

SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin-RING ubiquitin ligase function.

NEDD8 is a ubiquitin-like modifier most well-studied for its role in activating the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). While many non-cullin neddylation substrates have been proposed over the years, validation of true NEDD8 targets has been challenging, as overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery. Here, we developed a deconjugation-resistant form of NEDD8 to stabilize the neddylated form of cullins and other non-cullin substrates. Using this strategy, we identified Ubc12, a NEDD8-specific E2 conjugating enzyme, as a substrate for auto-neddylation. Furthermore, we characterized SENP8/DEN1 as the protease that counteracts Ubc12 auto-neddylation, and observed aberrant neddylation of Ubc12 and other NEDD8 conjugation pathway components in SENP8-deficient cells. Importantly, loss of SENP8 function contributes to accumulation of CRL substrates and defective cell cycle progression. Thus, our study highlights the importance of SENP8 in maintaining proper neddylation levels for CRL-dependent proteostasis.

Racial Differences in Selective Laser Trabeculoplasty Efficacy.

AIM: Sub-Saharan Africa has a population of 1 billion, with one ophthalmologist per million people. Basic ophthalmic support services are virtually absent for all but a few urban populations. Minimally invasive laser treatment may help. This study reports our initial experience using selective laser trabeculoplasty (SLT) in a mixed-racial population of adult glaucoma patients in Durban, South Africa. STUDY DESIGN: Institution Review Board approved the 5-year chart review. MATERIALS AND METHODS: Consecutive glaucomatous adults underwent SLT (Lumenis Selecta) on one or both eyes applying 360° treatment of 120 to 140 closely spaced burns (400 urn spot size for 3 ns; range 1.1-1.4 mJ). Significance of change in intraocuar pressure (IOP) from baseline at 1, 3, 6, and 12 months was assessed by two-tailed paired t-test. RESULTS: Among 148 eyes of 84 patients (60 African, 21 Indian, 3 Caucasian), 69 had already undergone glaucoma therapy, and 15 untreated (de novo). Among all eyes, mean IOP was reduced by >32% with mean IOP < 15 mm Hg from baseline at all four study intervals (p < 0.0001). A 20% reduction in IOP was sustained at 12 months in 90% of African eyes but in only 50% of Indian eyes. CONCLUSION: Selective laser trabeculoplasty was effective in producing clinically significant IOP reduction among South African adults with or without prior medical or surgical anti-glaucoma therapy. Socioeconomically comparable individuals of Indian ancestry showed good therapeutic responses, but significantly less efficacious than those observed among Black subjects. Programs to provide first-line SLT management of glaucoma in Africa, where 90% of patients are unable to sustain prescribed medical therapy, appear to be a very appropriate option. HOW TO CITE THIS ARTICLE: Goosen E, Coleman K, Visser L, Sponsel WE. Racial Differences in Selective Laser Trabeculoplasty Efficacy. J Curr Glaucoma Pract 2017;11(1):22-27.

How SUMOylation Fine-Tunes the Fanconi Anemia DNA Repair Pathway.

Fanconi anemia (FA) is a rare human genetic disorder characterized by developmental defects, bone marrow failure and cancer predisposition, primarily due to a deficiency in the repair of DNA interstrand crosslinks (ICLs). ICL repair through the FA DNA repair pathway is a complicated multi-step process, involving at least 19 FANC proteins and coordination of multiple DNA repair activities, including homologous recombination, nucleotide excision repair and translesion synthesis (TLS). SUMOylation is a critical regulator of several DNA repair pathways, however, the role of this modification in controlling the FA pathway is poorly understood. Here, we summarize recent advances in the fine-tuning of the FA pathway by small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligases (STUbLs) and other SUMO-related interactions, and discuss the implications of these findings in the design of novel therapeutics for alleviating FA-associated condition, including cancer.

Sequential replication-coupled destruction at G1/S ensures genome stability.

Timely ubiquitin-mediated protein degradation is fundamental to cell cycle control, but the precise degradation order at each cell cycle phase transition is still unclear. We investigated the degradation order among substrates of a single human E3 ubiquitin ligase, CRL4(Cdt2), which mediates the S-phase degradation of key cell cycle proteins, including Cdt1, PR-Set7, and p21. Our analysis of synchronized cells and asynchronously proliferating live single cells revealed a consistent order of replication-coupled destruction during both S-phase entry and DNA repair; Cdt1 is destroyed first, whereas p21 destruction is always substantially later than that of Cdt1. These differences are attributable to the CRL4(Cdt2) targeting motif known as the PIP degron, which binds DNA-loaded proliferating cell nuclear antigen (PCNA(DNA)) and recruits CRL4(Cdt2). Fusing Cdt1's PIP degron to p21 causes p21 to be destroyed nearly concurrently with Cdt1 rather than consecutively. This accelerated degradation conferred by the Cdt1 PIP degron is accompanied by more effective Cdt2 recruitment by Cdt1 even though p21 has higher affinity for PCNA(DNA). Importantly, cells with artificially accelerated p21 degradation display evidence of stalled replication in mid-S phase and sensitivity to replication arrest. We therefore propose that sequential degradation ensures orderly S-phase progression to avoid replication stress and genome instability.

CDK1-dependent inhibition of the E3 ubiquitin ligase CRL4CDT2 ensures robust transition from S Phase to Mitosis.

Replication-coupled destruction of a cohort of cell cycle proteins ensures efficient and precise genome duplication. Three proteins destroyed during replication via the CRL4(CDT2) ubiquitin E3 ligase, CDT1, p21, and SET8 (PR-SET7), are also essential or important during mitosis, making their reaccumulation after S phase a critical cell cycle event. During early and mid-S phase and during DNA repair, proliferating cell nuclear antigen (PCNA) loading onto DNA (PCNA(DNA)) triggers the interaction between CRL4(CDT2) and its substrates, resulting in their degradation. We have discovered that, beginning in late S phase, PCNA(DNA) is no longer sufficient to trigger CRL4(CDT2)-mediated degradation. A CDK1-dependent mechanism that blocks CRL4(CDT2) activity by interfering with CDT2 recruitment to chromatin actively protects CRL4(CDT2) substrates. We postulate that deliberate override of replication-coupled destruction allows anticipatory accumulation in late S phase. We further show that (as for CDT1) de novo SET8 reaccumulation is important for normal mitotic progression. In this manner, CDK1-dependent CRL4(CDT2) inactivation contributes to efficient transition from S phase to mitosis.

Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface.

A vaccine capable of stimulating protective antiviral antibody responses is needed to curtail the global AIDS epidemic caused by HIV-1. Although rarely elicited during the course of natural infection or upon conventional vaccination, the membrane-proximal ectodomain region (MPER) of the HIV-1 glycoprotein of M(r) 41,000 (gp41) envelope protein subunit is the target of 3 such human broadly neutralizing antibodies (BNAbs): 4E10, 2F5, and Z13e1. How these BNAbs bind to their lipid-embedded epitopes and mediate antiviral activity is unclear, but such information might offer important insight into a worldwide health imperative. Here, EPR and NMR techniques were used to define the manner in which these BNAbs differentially recognize viral membrane-encrypted residues configured within the L-shaped helix-hinge-helix MPER segment. Two distinct modes of antibody-mediated interference of viral infection were identified. 2F5, like 4E10, induces large conformational changes in the MPER relative to the membrane. However, although 4E10 straddles the hinge and extracts residues W672 and F673, 2F5 lifts up residues N-terminal to the hinge region, exposing L669 and W670. In contrast, Z13e1 effects little change in membrane orientation or conformation, but rather immobilizes the MPER hinge through extensive rigidifying surface contacts. Thus, BNAbs disrupt HIV-1 MPER fusogenic functions critical for virus entry into human CD4 T cells and macrophages either by preventing hinge motion or by perturbing MPER orientation. HIV-1 MPER features important for targeted vaccine design have been revealed, the implications of which extend to BNAb targets on other viral fusion proteins.

Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347.

NEMO is an essential regulatory component of the IkappaB kinase (IKK) complex, which controls activation of the NF-kappaB signaling pathway. Herein, we show that NEMO exists as a disulfide-bonded dimer when isolated from several cell types and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Treatment of cells with hydrogen peroxide (H(2)O(2)) induces further formation of NEMO dimers. Disulfide bond-mediated formation of NEMO dimers requires Cys54 and Cys347. The ability of these residues to form disulfide bonds is consistent with their location in a NEMO dimer structure that we generated by molecular modeling. We also show that pretreatment with H(2)O(2) decreases TNFalpha-induced IKK activity in NEMO-reconstituted cells, and that TNFalpha has a diminished ability to activate NF-kappaB DNA binding in cells reconstituted with NEMO mutant C54/347A. This study implicates NEMO as a target of redox regulation and presents the first structural model for the NEMO protein.

Preparation for cancer care: perceptions of newly qualified health care professionals.

The present paper is derived from a larger survey which examined the perceptions of recently qualified health care professionals' experience on evidence-based practice, team working and cancer care. This study reports solely on the findings relating to cancer care. The perceptions of recently qualified professionals in relation to their initial educational input on issues such as confidence, anxiety, communication skills and practice in cancer care as well as adequacy of support, professional supervision and use of reflection were gathered using a cross-sectional postal survey design. A total of 50 graduates from each professional category in nursing, occupational therapy, physiotherapy, and social work were sampled yielding a total sample of 200. Eighty-five questionnaires were returned yielding a response rate of 43%. Twenty-eight (33%) respondents stated that they were currently involved in working with people with cancer. These were as follows: 5 nurses, 8 physiotherapists, 9 occupational therapists and 6 social workers. Despite the low response rate, the findings suggest that health care professionals' educational input and experiences of working with people with cancer were overall positive; for example, in the respondents' confidence, communication skills, decrease in anxiety and application of knowledge gained in classroom to professional practice. Moreover, most respondents learnt about caring for cancer patients through practice rather than classroom teaching. A high percentage (i.e. 64%;18) across all groups felt supported when caring for people with cancer and reported receiving professional supervision as well as being able to actively reflect on their practice. The implications for education and practice were discussed particularly as there have been few studies conducted in relation to the specific needs and collaborative learning of these health care professional groups.

Preparing for professional practice: how well does professional training equip health and social care practitioners to engage in evidence-based practice?

This paper reports on the findings of a study that aimed to explore how relevant initial training is in relation to evidence-based practice, and explore the perceptions of recently qualified practitioners about their confidence to engage in evidence-based practice. A cross-sectional postal survey was used to ascertain the views of nurses, social workers, occupational therapists and physiotherapists who had been qualified no longer than two years prior to the survey, and had qualified at one of three London Universities. Fifty questionnaires were sent out to each professional group (a sample of 200 overall) and there was a 43% response rate achieved. The results show a clear discrepancy between what are generally positive attitudes towards evidence-based practice and the value of research evidence and the infrequency with which they actually do make use of research resources and engage in evidence-based practice. A number of constraints to engagement in accessing and utilising evidence were identified.