Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication.

To bypass a diverse range of fork stalling impediments encountered during genome replication, cells possess a variety of DNA damage tolerance (DDT) mechanisms including translesion synthesis, template switching, and fork reversal. These pathways function to bypass obstacles and allow efficient DNA synthesis to be maintained. In addition, lagging strand obstacles can also be circumvented by downstream priming during Okazaki fragment generation, leaving gaps to be filled post-replication. Whether repriming occurs on the leading strand has been intensely debated over the past half-century. Early studies indicated that both DNA strands were synthesised discontinuously. Although later studies suggested that leading strand synthesis was continuous, leading to the preferred semi-discontinuous replication model. However, more recently it has been established that replicative primases can perform leading strand repriming in prokaryotes. An analogous fork restart mechanism has also been identified in most eukaryotes, which possess a specialist primase called PrimPol that conducts repriming downstream of stalling lesions and structures. PrimPol also plays a more general role in maintaining efficient fork progression. Here, we review and discuss the historical evidence and recent discoveries that substantiate repriming as an intrinsic replication restart pathway for maintaining efficient genome duplication across all domains of life.

Virtual lesion clinic - Evaluation of a teledermatology triage system for referrals for suspected melanoma.

INTRODUCTION: The Virtual Lesion Clinic (VLC) of Waitemata District Health Board (WDHB) improves melanoma assessment and treatment using teledermatology. The VLC is reserved for pigmented lesions referred as suspected melanoma from primary care but indeterminate at the initial triage. OBJECTIVES: To assess the efficacy of the VLC diagnosis of melanoma. METHODS: A retrospective audit of suspected melanoma referrals (1 January 2012 to 31 December 2016) was conducted. Lesions were referred to the VLC if diagnostic uncertainty remained at the initial triage. VLC patients attended MoleMap imaging centres, a dermatologist reviewed history and images remotely and suggested a diagnosis and management plan. Post VLC provisional diagnosis of melanoma, all lesions subsequently excised were reviewed. A positive predictive value (PPV) was calculated using concordance between VLC diagnosis of melanoma and histopathological diagnosis of melanoma. Number needed to excise (NNE) for melanoma was derived, as well as an invasive to in-situ melanoma ratio (IM:MIS) and false negative rate (FNR). RESULTS: The VLC received 1874 referrals for 3546 lesions during the 5-year study period. Six hundred and seventy-nine lesions were recommended excision/biopsy or specialist face-to-face assessment, and 504 lesions were subsequently excised. The PPV was 62%, NNE 1.62 and IM:MIS 0.76 for lesions suspected to be melanoma at VLC assessment. The VLC had a melanoma-specific FNR of 7%. CONCLUSIONS: The VLC is effective in early diagnosis of melanoma with a high positive predictive value, low number needed to excise and low false negative rate amongst lesions referred as suspected melanoma.

E-referrals and teledermatoscopy grading for melanoma: a successful model of care.

BACKGROUND/OBJECTIVES: An e-referral system was developed at a tertiary care hospital in Auckland, New Zealand in 2014 for suspected cutaneous malignancy. E-referrals include patient information, a description of the lesion(s), biopsy results and/or attached photograph(s). Experienced surgical oncologists prioritised the referrals and selected a management option or referred them for a teledermatoscopy opinion. Our aim was to review the efficacy of e-referrals for improving diagnostic accuracy for melanoma. METHODS: Referrals received in 2016 including images and categorisation as confirmed, likely or suspected melanoma by the triage specialist were evaluated. Concordance of the pathological diagnosis with the triage diagnosis and teledermatoscopy diagnosis was determined for each referral. RESULTS: 809 of 3470 e-referrals for skin cancer were categorised as confirmed, likely or suspected melanoma. 230 (28.4%) of these included a referral histopathology confirming melanoma/melanoma in situ. Of the remaining 579 referrals, 315 were sent for urgent diagnostic excision and 264 were referred for teledermatoscopy. 120 of the 315 sent for urgent excision were confirmed as melanoma (53) or melanoma in situ (67) on histopathology: a positive predictive value (PPV) of 38.1% and number needed to excise (NNE) of 2.6. Less than 10% of referrals triaged for teledermatoscopy were confirmed as melanoma (24/264). Almost half of all referrals (374/809, 45.6%) included melanoma/melanoma in situ. The melanoma: melanoma in situ ratio was 1: 1.18. CONCLUSIONS: The e-referral and teledermatoscopy service for suspected melanoma has proven fewer unnecessary excisions of benign lesions than previously reported.

Effects of pH manipulation, CatSper stimulation and Ca2+-store mobilization on [Ca2+]i and behaviour of human sperm.

STUDY QUESTION: How do the alkaline pH, progesterone and 4-aminopyridine interact in their effects on human sperm? SUMMARY ANSWER: Behaviour of human sperm (proportion of hyperactivated cells and motility kinematics) were related directly to [Ca2+]i irrespective of pH or the agonist applied. WHAT IS KNOWN ALREADY?: CatSper channels of human sperm, which are central to generation of sperm [Ca2+]i signals and induction of hyperactivated motility, are activated by intracellular alkalinization and progesterone. Progesterone (P4) is much less effective than 4-aminopyridine (4-AP) (which mobilizes stored Ca2+ but also raises pHi) as an inducer of hyperactivation. STUDY DESIGN, SIZE, DURATION: This was a laboratory study, spanning ~18 months that used 15 sperm donors and involved more than 100 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors and patients were recruited in accordance with local ethics approval (ERN_12-0570R). [Ca2+]i responses of suspended cell populations were examined by fluorimetric recording and motility parameters assessed by computer-assisted sperm analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Increasing pHo from 7.4 to 8.5 raised pHi (from 6.9 to 7.2) and significantly increased both [Ca2+]i and the proportion of hyperactivated cells. Stimulation of cells with P4 (1 nM-20 µM) induced a biphasic (transient and plateau) increase in [Ca2+]i. The [Ca2+]i increase was of similar amplitude and dose-dependency at pHo = 7.4 and pHo = 8.5. 4-aminopyridine (0.2-5 mM) induced a biphasic [Ca2+]i increase that was dose-dependent across the entire range tested and was strongly enhanced at pH 8.5. Motility was assessed 300 s post-stimulation, during the plateau phase of the progesterone and 4-AP-induced [Ca2+]i responses. Progesterone had only a small effect on hyperactivated motility even at the highest dose used (20 µM; < 5% increase in the proportion of cells classified as hyperactivated) which was insensitive to pHo. 4-Aminopyridine potently stimulated hyperactivated motility, this effect being dose-dependent and greatly enhanced at pHo = 8.5. The relationship between [Ca2+]i (fluorescence of fluo4) and proportion of hyperactivated cells, irrespective of pHo, agonist or dose, was fitted by a single curve (second order polynomial; R2 = 0.96). Similar analysis of curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) showed a linear relationship to [Ca2+]i (R2 > 0.9). LIMITATIONS, REASONS FOR CAUTION: This was an in-vitro study and caution must be taken when extrapolating these results to in vivo regulation of sperm. Though controls indicate that saturation of fluo4 did not affect the findings, at the highest doses of progesterone the true amplitude of the [Ca2+]i transient may not have been reported by the dye. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that (i) activation of human sperm CatSper by progesterone (and presumably other ligands that act similarly) and consequent acquisition of hyperactivated motility is not significantly enhanced by intracellular alkalinization; (ii) VCL, ALH and hyperactivation are directly related to [Ca2+]i, irrespective of the mechanism by which Ca2+ is mobilized, and the ability of stimuli to induce prolonged [Ca2+]i elevation (as occurs upon mobilization of stored Ca2+) determines the observed effect on cell behaviour. STUDY FUNDING/COMPETING INTEREST(S): CA was supported by the Nigerian government (Tertiary Education Trust (TET) Fund). The authors have no conflicts of interest.

Mucormycosis in Burns: A Review.

Mucormycosis is a rare fungal infection with a high mortality rate. It presents with scattered black/necrotic ulcers, white fungal elements, and progression of wounds despite seemingly adequate debridement. Diagnosis is confirmed on wound histology; however, this is often delayed. There is currently no comprehensive review of burn-related mucormycosis within the literature, making this the first article to provide evidence-based treatment guidance. We performed a review of publications from 1946 to the present. There were 151 cases of mucormycosis complicating burns. The mortality rate was 54.5%, and there was a significant increase in mortality with axial body site involvement compared with isolated peripheral involvement. The standard treatment was prompt and radical debridement. Utilization of frozen section to guide debridement aided in clinical decision making. No systemic treatment reached statistical significance; however, amphotericin B trended toward significance. Although there is no strong evidence for topical amphotericin B or hyperbaric oxygen, there may be benefit in some cases. This study recommends early radical debridement in conjunction with the European Confederation of Medical Mycology guidelines of IV liposomal/lipid complex amphotericin B more than 5mg/kg/day, with posaconazole 800 mg daily in divided doses as a salvage or oral step-down.

PLK1 regulates the PrimPol damage tolerance pathway during the cell cycle.

Replication stress and DNA damage stall replication forks and impede genome synthesis. During S phase, damage tolerance pathways allow lesion bypass to ensure efficient genome duplication. One such pathway is repriming, mediated by Primase-Polymerase (PrimPol) in human cells. However, the mechanisms by which PrimPol is regulated are poorly understood. Here, we demonstrate that PrimPol is phosphorylated by Polo-like kinase 1 (PLK1) at a conserved residue between PrimPol's RPA binding motifs. This phosphorylation is differentially modified throughout the cell cycle, which prevents aberrant recruitment of PrimPol to chromatin. Phosphorylation can also be delayed and reversed in response to replication stress. The absence of PLK1-dependent regulation of PrimPol induces phenotypes including chromosome breaks, micronuclei, and decreased survival after treatment with camptothecin, olaparib, and UV-C. Together, these findings establish that deregulated repriming leads to genomic instability, highlighting the importance of regulating this damage tolerance pathway following fork stalling and throughout the cell cycle.

Contemporary management of civilian penetrating cervicothoracic arterial injuries.

BACKGROUND: The management of arterial injury at the thoracic outlet has long hinged on the fundamental principles of extensile exposure and vascular anastomosis. Nonetheless, treatment options for such injuries have evolved to include both endovascular stent placement and temporary vascular shunts. The purpose of this study was to evaluate our recent experience with penetrating cervicothoracic arterial injuries in light of these developments in trauma care. METHODS: Patients with penetrating injuries to the innominate, carotid, subclavian, or axillary arteries managed at a single civilian trauma center between 2000 and 2013 were categorized as the modern era (ME) cohort. The management strategies and outcomes pertaining to the ME group were compared to those of previously reported experience (PE) concerning injuries to the innominate, carotid, subclavian, or axillary arteries at the same institution from 1974 to 1988. RESULTS: Over the two eras, there were 202 patients: 110 in the ME group and 92 in the PE group. Most of the injuries in both groups were managed with primary repair (45% vs. 46%; p = 0.89). A similar proportion of injuries in each group was managed with anticoagulation alone (14% vs. 10%; p = 0.40). In the ME group, two cases were managed with temporary shunt placement, and endovascular stent placement was performed in 12 patients. Outcomes were similar between the groups (bivariate comparison): mortality (ME, 15% vs. PE, 14%; p = 0.76), amputation following subclavian or axillary artery injury (ME, 5% vs. PE, 4%; p = 0.58), and posttreatment stroke following carotid injury (ME, 2% vs. PE, 6%; p = 0.57). CONCLUSIONS: Experience with penetrating arterial cervicothoracic injuries at a high-volume urban trauma center remained remarkably similar with respect to both anatomic distribution of injury and treatment. Conventional operative exposure and repair remain the cornerstone of treatment for most civilian cervicothoracic arterial injuries. LEVEL OF EVIDENCE: Therapeutic study, level V.

Intravitreal injection of lipoamino acid-modified connexin43 mimetic peptide enhances neuroprotection after retinal ischemia.

Optic neuropathy is associated with retinal ganglion cell (RGC) loss leading to optic nerve damage and visual impairment. Recent research has shown that transient block of connexin43 (Cx43) hemichannels by a Cx43 mimetic peptide (MP), Peptide5, delivered systemically or by intravitreal injection after retinal ischemia inhibits uncontrolled hemichannel opening to provide significantly reduced vessel leak and inflammation as well as significantly enhanced RGC survival. We have previously shown, in vitro, that a chemically modified C12-C12-Cx43 MP has a twofold greater half-life in bovine vitreous (ex vivo) than the native peptide. The present study investigated the ability of intravitreally injected, chemically modified C12-C12-Cx43 MP to further enhance RGC survival in a rat retinal ischemia-reperfusion model. Intravitreally injected native Cx43 MP or C12-C12-Cx43 MP both minimized vessel leak, reduced inflammation, and protected RGC after ischemic injury. However, the modified C12-C12-Cx43 MP, with its prolonged vitreous stability, showed significantly lower levels of Cx43 expression post-injury, with a trend towards a greater reduction in vessel leak and further RGC protection, suggesting that these molecules may be a clinically relevant neuroprotective tool in the treatment of optic neuropathy.

Crystal structures of three classes of non-steroidal anti-inflammatory drugs in complex with aldo-keto reductase 1C3.

Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts.