The marker of alkyl DNA base damage, N7-methylguanine, is associated with semen quality in men.

Sperm DNA contains a range of DNA base damage that can arise, in part, from exposure to methylating agents. However, the effects are not fully characterized and so the aim of this study was to investigate associations between semen quality and the levels of N7-methyldeoxyguanosine (N7-MedG), a marker of exposure to methylating agents, and other markers of DNA damage and DNA methylation. Sperm samples were collected from 105 men attending an assisted reproduction clinic as part of a couple undergoing treatment for infertility and semen quality assessed manually according to WHO guidelines. Semen levels of N7-MedG, quantified by immunoslotblot, were significantly higher in men with sperm concentration < 15 × 106/ml (p ≤ 0.01), semen volume < 1.5 ml (p ≤ 0.05) and also in men with any aspect of semen quality below WHO reference levels (p ≤ 0.001). Measures of neutral Comet DNA damage were correlated with semen quality in a univariate analysis but not after adjustment for N7-MedG levels. Sperm concentration was negatively associated with % methylation at the gene for DAZL but no other marker of global or gene-specific DNA methylation. Results support the hypothesis that the known toxic and DNA damaging properties of alkylating agent exposure may have direct deleterious consequences on semen quality.

A randomised phase II trial of hydroxychloroquine and imatinib versus imatinib alone for patients with chronic myeloid leukaemia in major cytogenetic response with residual disease.

In chronic-phase chronic myeloid leukaemia (CP-CML), residual BCR-ABL1+ leukaemia stem cells are responsible for disease persistence despite TKI. Based on in vitro data, CHOICES (CHlorOquine and Imatinib Combination to Eliminate Stem cells) was an international, randomised phase II trial designed to study the safety and efficacy of imatinib (IM) and hydroxychloroquine (HCQ) compared with IM alone in CP-CML patients in major cytogenetic remission with residual disease detectable by qPCR. Sixty-two patients were randomly assigned to either arm. Treatment 'successes' was the primary end point, defined as ≥0.5 log reduction in 12-month qPCR level from trial entry. Selected secondary study end points were 24-month treatment 'successes', molecular response and progression at 12 and 24 months, comparison of IM levels, and achievement of blood HCQ levels >2000 ng/ml. At 12 months, there was no difference in 'success' rate (p = 0.58); MMR was achieved in 80% (IM) vs 92% (IM/HCQ) (p = 0.21). At 24 months, the 'success' rate was 20.8% higher with IM/HCQ (p = 0.059). No patients progressed. Seventeen serious adverse events, including four serious adverse reactions, were reported; diarrhoea occurred more frequently with combination. IM/HCQ is tolerable in CP-CML, with modest improvement in qPCR levels at 12 and 24 months, suggesting autophagy inhibition maybe of clinical value in CP-CML.

Hydatidiform molar pregnancy following assisted reproduction.

INTRODUCTION: The use of assisted reproduction techniques (ART) is increasing; however, reports of molar pregnancy following ART remain scarce. Currently, the Human Fertility and Embryology Authority (HFEA) collates data on the molar pregnancies that have resulted through the use of ART. Recently, they have indicated that they will no longer collect these data. AIM: This paper aimed to examine the incidence of molar pregnancy amongst patients undergoing assisted reproduction. METHODS: We contacted HFEA and placed a request under the Freedom of Information Act (2000) for the number of molar pregnancies that resulted from fresh/frozen embryo transfer since HFEA started collecting data in 1991 to February 2018. We also asked how many patients who had suffered a molar pregnancy went on to have a normal pregnancy and how many had subsequent molar pregnancies, in subsequent treatment cycles. RESULTS: Between 68 and 76 molar pregnancies occurred within this period using ART (n = 274,655). The incidence of molar pregnancy using fresh intracytoplasmic sperm injection (ICSI) (1/4302) and fresh in vitro fertilisation (IVF) (1/4333) was similar. The risk of recurrence of molar pregnancy following a previous molar was higher following ART compared to spontaneous conceptions. CONCLUSION: The use of ICSI should be protective against triploidy; however, the retrospective data suggests that molar pregnancy is not eliminated with the use of ART. It is pertinent to continue to record this data, through the gestational trophoblastic disease centres, in order to ensure no further increase in incidence, appropriate follow-up, and transparency in communication.

Reevaluation of Neptunium-Nitric Acid Radiation Chemistry by Multiscale Modeling.

Multiscale modeling has been used to quantitatively reevaluate the radiation chemistry of neptunium in a range of aerated nitric acid solutions (0.1-6.0 mol dm-3). Exact calculation of initial radiolytic yields accounting for changes in radiation track chemistry was found to be crucial for reproducing experimental data. The γ irradiation induces changes in the Np(VI)/Np(V) oxidation-state distribution, predominantly driven by reactions involving HNO2, H2O2, NO2•, and NO3• from the radiolysis of aqueous nitric acid. Oxidation of Np(V) by NO3• (k = 8.1 × 108 dm3 mol-1 s-1) provides the initial increase in Np(VI) concentration, while also delaying net reduction of Np(VI) by consuming HNO2. Reduction of Np(VI) is dominated by thermal reactions with HNO2 (k = 0.7-73 dm3 mol-1 s-1) and H2O2 (k = 1.9 dm3 mol-1 s-1). A steady state is eventually established once the concentration of Np(V) is sufficiently high to be oxidized by NO2• (k = 2.4 × 102-3.1 × 104 dm3 mol-1 s-1). An additional thermal oxidation reaction between Np(V) and HNO3 (k = 2.0 × 103 dm3 mol-1 s-1) is required for nitric acid concentrations >4.0 mol dm-3. For 0.1 mol dm-3 HNO3, the rate of Np(VI) reduction is in excess of that which can be accounted for by radiolytic product mass balance, suggesting the existence of a catalytic-acid-dependent reduction process.

Identification of circulating microRNAs as diagnostic biomarkers for use in multiple myeloma.

BACKGROUND: Multiple myeloma is a plasma cell disorder that is characterised by clonal proliferation of malignant plasma cells in the bone marrow, monoclonal paraprotein in the blood or urine and associated organ dysfunction. It accounts for approximately 1% of cancers and 13% of haematological cancers. Myeloma arises from an asymptomatic proliferation of monoclonal plasma cells termed monoclonal gammopathy of undetermined significance (MGUS). METHODS: MicroRNA expression profiling of serum samples was performed on three patient groups as well as normal controls. Validation of the nine microRNAs detected as promising biomarkers was carried out using TaqMan quantitative reverse transcription PCR. MicroRNA levels in serum were normalised using standard curves to determine the numbers of microRNAs per µl of serum. RESULTS: Three serum microRNAs, miR-720, miR-1308 and miR-1246, were found to have potential as diagnostic biomarkers in myeloma. Use of miR-720 and miR-1308 together provides a powerful diagnostic tool for distinguishing normal healthy controls, as well as patients with unrelated illnesses, from pre-cancerous myeloma and myeloma patients. In addition, the combination of miR-1246 and miR-1308 can distinguish MGUS from myeloma patients. CONCLUSION: We have developed a biomarker signature using microRNAs extracted from serum, which has potential as a diagnostic and prognostic tool for multiple myeloma.

Alkylation of sperm DNA is associated with male factor infertility and a reduction in the proportion of oocytes fertilised during assisted reproduction.

Approximately one-third of IVF cases in the UK are attributed to male factor infertility and in the majority of cases the origin of male infertility is unknown. The integrity of sperm DNA is important both for the success of assisted reproduction and the implications for the off-spring. One type of DNA damage that has not been investigated with respect to fertility outcomes is the adduct N7-methyldeoxyguanosine (N7-MedG), a biomarker for exposure to alkylating agents. A prospective cohort of couples attending for IVF had their N7-MedG levels in sperm measured using an immunoslot blot technique to examine whether sperm N7-MedG levels are associated with male factor infertility, semen quality measures or assisted reproduction outcomes. Sufficient DNA for analysis was obtained from 67/97 couples and N7-MedG was detected in 94% of sperm samples analysed. Men diagnosed with male factor infertility had significantly higher mean levels of N7-MedG in their sperm DNA (P=0.03). Logistic regression analysis showed that N7-MedG levels were significantly negatively associated with the proportion of oocytes successfully fertilised irrespective of the method of fertilisation used (IVF or intra-cytoplasmic sperm injection; ICSI, P<0.001). Therefore exposure to DNA alkylating agents is significantly associated with male infertility and the proportion of oocytes fertilised during assisted reproduction. Reducing such exposure may improve male fertility but further work is required to determine the relative importance of exogenous and endogenous sources of exposure.

Workload measurement in subspecialty dermatopathology.

AIM: To measure pathologist workload in subspecialty dermatopathology. METHODS: Three subspecialty dermatopathologists, working in a university-affiliated laboratory, participated in a time-motion study during which they reported 2891 consecutive skin cases received from community-based dermatologists. All pathology reports were retrospectively reviewed and workload measured using the Royal College of Pathologists (RCPath) guidelines and the level 4 equivalent (L4E) method. RESULTS: The majority of dermatopathology cases were scored as low (32%) or intermediate (52%) complexity using the RCPath matrix. Only 16% of cases were considered high or very high complexity. The mean RCPath score per case was 2.68 units. Using L4E complexity levels, 83% of specimens were level 3, 15% were level 4, and only 2% were higher complexity (levels 5 and 6). Mean values for specimens/case, blocks/case, and slides/case were 1.31, 1.52, and 2.92, respectively. Time-motion analysis demonstrated a mean workload per hour of 16.3 cases, 21.3 specimens, 45.1 slides, 43.0 RCPath units, and 12.2 L4E. All three dermatopathologists reported >35 RCPath units per hour. CONCLUSIONS: The RCPath histopathology workload guidelines underestimate the workload achievable by an experienced dermatopathologist, and thus are not directly applicable to subspecialty dermatopathology practice. Hourly work rates 3-4 times that recommended by the RCPath workload matrix are routinely achievable, but extrapolation to yearly workload estimates requires detailed knowledge of practice pattern and time required for non-clinical duties such as teaching, research and administration.

Implantation, clinical pregnancy and miscarriage rates after introduction of ultrasound-guided embryo transfer.

The purpose of this study was to compare the reproductive outcome of ultrasound-guided (USG) embryo transfers versus clinical touch embryo transfers. A statistically powered retrospective analysis of women undergoing fresh and frozen embryo transfers in a National Health Service-based tertiary referral centre in the Department of Reproductive Medicine, St Mary's Hospital, Manchester was carried out. A total of 1723 embryo transfers were included in the analysis. The implantation rate was significantly higher in the USG embryo transfer group compared with the non-USG embryo transfer group (fresh: 19.9 versus 9.5%, P < 0.0001; frozen: 13.1 versus 7.3%, P < 0.0004). The clinical pregnancy rate was also significantly higher in the former group (fresh: 26.9 versus 12.5%, P < 0.0001; frozen: 15.6 versus 8.9%, P < 0.0015). For the frozen embryos, the miscarriage rate was significantly elevated among the USG embryo transfer group [unadjusted rate ratio (RR) = 1.65, 95% CI: 1.04, 2.62], but this was of borderline significance when the model was adjusted for the potential confounders (adjusted RR = 1.56, 95% CI: 0.997, 2.45). There was no difference in the ectopic pregnancy rates between the two groups. The findings of this study show that the practice of USG embryo transfer is associated with statistically higher implantation and clinical pregnancy rates in IVF.

Comparative analysis of epicuticular lipid profiles of sympatric and allopatric field populations of Anopheles gambiae s.s. molecular forms and An. arabiensis from Burkina Faso (West Africa).

We analysed by gas chromatography-mass spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID) the epicuticular lipid profiles of field females of the major Afro-tropical malaria vector, Anopheles gambiae. The samples were collected in three villages in Burkina Faso (West Africa), where An. gambiae M and S molecular forms and An. arabiensis live sympatrically. The aim was to compare the cuticular hydrocarbon (CHC) composition of individual field specimens of these three taxa, to highlight possible differences among them. All the samples analysed by GC-MS (55 individuals and eight pools) were characterized by the same 48 CHCs and 10 oxygenated compounds. The 19 most abundant CHCs were quantified in 174 specimens by GC-FID: quantitative intra-taxon differences were found between allopatric populations of both An. arabiensis and S-form. Inter-taxa quantitative differences in the relative abundances of some hydrocarbons between pairs of sympatric taxa were also found, which appear to be mainly linked to local situations, with the possible exception of diMeC(35) between An. arabiensis and S-form. Moreover, MeC(29) shows some degree of differentiation between S- and M-form in all three villages. Possible causes of these differences are discussed.

Medical optimisation can reduce morbidity and mortality associated with elective aortic aneurysm repair.

OBJECTIVES: Patients with aortic aneurysms have significant comorbidities which influence outcome. Our practice includes comprehensive assessment to identify comorbidities, allowing subsequent medical optimisation prior to aneurysm repair. The aim of this study was to assess this process and to identify any factors predictive of outcome. DESIGN: Prospective observational study. MATERIALS: Medical case notes of 200 patients referred with aortic pathology. METHODS: Data analysed included preoperative, perioperative and postoperative factors. Multiple logistic regression analysis was performed to identify predictors of outcome. RESULTS: Following assessment 17 patients (8.5%) were found to be unfit for intervention and 165 patients (82.5%) proceeded to aneurysm repair. In this group assessment uncovered previously undiagnosed cardiac, respiratory and renal comorbidity in 19%, 57% and 29% of patients respectively. Multiple logistic regression analysis indicated that optimisation by a renal physician reduced post-operative renal impairment (OR 0.12, 95% CI 0.03-0.45, P=0.002) while optimisation by a cardiologist reduced respiratory complications (OR 0.7, 95% CI 0.05-0.99, P=0.049). An abnormal echocardiogram was associated with pneumonia (OR 6.9, 95% CI 1.6-29, P=0.01) and death (OR 7.9, 95% CI 1.15-54, P=0.036). CONCLUSION: Pre-operative assessment identifies previously undiagnosed comorbidity in a significant proportion of patients. Subsequent medical optimisation may reduce post-operative morbidity and mortality.

Multimodal immunosuppressant therapy in steroid-refractory common variable immunodeficiency sprue: a case report complicating cytomegalovirus infection.

Immunodeficient patients can develop malabsorption, mimicking celiac disease clinically and histologically. Such individuals may also occasionally require immunosuppressive therapy for autoimmune disorders. We have identified a patient with common variable immunodeficiency (CVID)-associated sprue complicated by duodenal cytomegalovirus (CMV) infection following corticosteroid and ancillary immunomodulatory therapy. Ganciclovir and a modification of the immunosuppressant regimen improved both clinical symptoms and villous atrophy. To our knowledge, this is original documentation of duodenal CMV infection secondary to immunomodulatory therapy for steroid-refractory CVID-sprue.

Live birth with sperm cryopreserved for 21 years prior to cancer treatment: case report.

Advances in cancer treatment have led to significant improvements in the likelihood of reaching remission and long-term survival for men. Chemo- and radiotherapy-induced infertility are significant treatment side effects. Cryopreservation before the start of treatment enables sperm to be stored, thereby preserving the man's potential fertility. Here, we describe the successful use (with ICSI) of sperm cryopreserved prior to cancer treatment, for a total of 21 years. We believe this to be the longest period of sperm cryopreservation, resulting in a live birth, so far reported in the literature.

The chemical characterization of the epicuticular hydrocarbons of Aedes aegypti (Diptera: Culicidae).

The chemical characterization of the hydrocarbon fraction of the epicuticular lipids of the vector mosquito Aedes aegypti (Linnaeus) was performed using gas chromatography (GC) and gas chromatography-electron impact mass spectrometry (GC-MS). Seventy eight compounds were detected in purified hexane extracts and of these, 42 hydrocarbons were identified and several of the remaining compounds were partially characterized. The hydrocarbon classes present were n-alkanes, monomethylalkanes, dimethylalkanes and alkenes and the results were similar to those published for other Aedes species. Quantitative comparisons of cuticular hydrocarbon profiles were made between males and females, different age groups and between a standard laboratory strain and a recently colonized strain of A. aegypti. These results provide baseline data for further studies on the possible role of mosquito cuticular hydrocarbons in the modification of mating behaviour.

Semen cryopreservation, utilisation and reproductive outcome in men treated for Hodgkin's disease.

Between 1978 and 1990, 122 men underwent semen analysis before starting sterilising chemotherapy for Hodgkin's disease. Eighty-one (66%) had semen quality within the normal range, 25 were oligospermic (<20 x 10(6) sperm per ml) and five were azoospermic (no sperm in the ejaculate). Semen from 115 men was cryopreserved and after a median follow-up time of 10.1 years, 33 men have utilised stored semen (actuarial rate 27%) and nine partners have become pregnant resulting in 11 live births and one termination for foetal malformation. Actuarial 10 year rates of destruction of semen before death or utilisation and death before utilisation are 19% and 13% respectively. This retrospective cohort study demonstrates that approximately one-quarter of men utilising cryopreserved semen after treatment for Hodgkin's disease obtain a live birth. The high non-utilisation rate is intriguing and warrants further investigation.

Hip fracture incidence in New Zealand, revisited.

AIM: Earlier predictions of the incidence of hip fractures in the older adult population suggested that by 2011 the rate would rise to epidemic proportions. The purpose of this study was to compare the number hip fractures occuring in New Zealand from 1988 to 1999 with the hip fracture rate predicted in 1990 by Rockwood, Horne and Cryer. METHODS: Data on the number of patients admitted to New Zealand hospitals with a diagnosis of fractured neck of femur were obtained, and compared with weighted regression and baseline predictions of Rockwood et al. RESULTS: The numbers of hip fractures for females, from 1988 to 1993, were similar to the number predicted, yet have been significantly lower than stated predictions since 1995. For males, hip fracture numbers since 1995 were less than the weighted regressions predicted (NS). CONCLUSIONS: Numbers of hip fractures since 1995 have been fewer than predicted. Possible reasons for maintaining the rates of hospitalisation due to fractured neck of femur at pre-1995 levels, are discussed.

Structural determinants of adenophostin A activity at inositol trisphosphate receptors.

Adenophostin A is the most potent known agonist of inositol 1,4,5-trisphosphate (InsP(3)) receptors. Ca(2+) release from permeabilized hepatocytes was 9.9 +/- 1.6-fold more sensitive to adenophostin A (EC(50), 14.7 +/- 2.4 nM) than to InsP(3) (145 +/- 10 nM), consistent with the greater affinity of adenophostin A for hepatic InsP(3) receptors (K(d) = 0.48 +/- 0.06 and 3.09 +/- 0.33 nM, respectively). Here, we systematically modify the structures of the glucose, ribose, and adenine moieties of adenophostin A and use Ca(2+) release and binding assays to define their contributions to high-affinity binding. Progressive trimming of the adenine of adenophostin A reduced potency, but it fell below that of InsP(3) only after complete removal of the adenine. Even after substantial modifications of the adenine (to uracil or even unrelated aromatic rings, retaining the beta-orientation), the analogs were more potent than InsP(3). The only analog with an alpha-ribosyl linkage had massively decreased potency. The 2'-phosphate on the ribose ring of adenophostin A was essential and optimally active when present on a five-membered ring in a position stereochemically equivalent to its location in adenophostin A. Xylo-adenophostin, where xylose replaces the glucose ring of adenophostin A, was only slightly less potent than adenophostin A, whereas manno-adenophostin (mannose replacing glucose) had similar potency to InsP(3). These results are consistent with the relatively minor role of the 3-hydroxyl of InsP(3) (the equivalent is absent from xylo-adenophostin) and greater role of the equatorial 6-hydroxyl (the equivalent is axial in manno-adenophostin). This is the first comprehensive analysis of all the key structural elements of adenophostin A, and it provides a working model for the design of related high-affinity ligands of InsP(3) receptors.

Synthesis of the enantiomers of 6-deoxy-myo-inositol 1,3,4,5-tetrakisphosphate, structural analogues of myo-inositol 1,3,4,5-tetrakisphosphate.

D-myo-Inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] is produced rapidly from the established second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P4] in stimulated cells. Despite extensive investigations, in particular concerning its potential role in mediating cellular Ca2+ influx, no exact cellular function has been described for this inositol phosphate; however, binding sites have been identified in a number of tissues and it has been shown to act synergistically with Ins(1,4,5)P3. To assist in the elucidation of the mechanism of action and structural requirements within the Ins(1,3,4,5)P4 moiety that are necessary for recognition and activation of the receptor, structural analogues of this tetrakisphosphate are required. Routes for the synthesis of racemic 6-deoxy-myo-inositol 1,3,4,5-tetrakisphosphate [6-deoxy-DL-Ins(1,3,4,5)P4] and the chiral antipodes D- and L-6-deoxy-myo-inositol 1,3,4,5-tetrakisphosphate are described here. The racemic tetrakisphosphate was synthesised from DL-1,2-O-isopropylidene-myo-inositol in eight steps. Deoxygenation at C-6 was achieved following the Barton-McCombie procedure. Both chiral tetrakisphosphates were synthesised through resolution of racemic cis-diol 6-deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-inositol with the chiral auxiliary (S)-(+)-O-acetylmandelic acid. Absolute configuration was confirmed by synthesis of the known D-6-deoxy-myo-inositol. Both D-6-deoxy-Ins(1,3,4,5)P4 and its enantiomer will be useful tools to unravel the enigmatic role of Ins(1,3,4,5)P4 in the polyphosphoinositide pathway of signal transduction.

Achieving pregnancy against the odds: successful implantation of frozen-thawed embryos generated by ICSI using spermatozoa banked prior to chemo/radiotherapy for Hodgkin's disease and acute leukaemia.

Two cases are reported of successful pregnancies following long-term semen banking prior to chemotherapy and radiotherapy for malignancy. With the first case, the patient banked semen at the age of 20 years prior to chemotherapy for Hodgkin's disease; 11 years later the thawed semen was used for IVF with intracytoplasmic sperm injection (ICSI), resulting in twins being born following the transfer of frozen-thawed embryos. In the second case, the patient banked semen at the age of 17 years prior to chemotherapy and radiotherapy for acute myeloid leukaemia; 8 years later it was used for ICSI, resulting in triplets being born following the transfer of frozen-thawed embryos. These cases support long-term semen banking for men whose future fertility may be compromised by suppression of spermatogenesis secondary to administration of chemo/radiotherapy treatment. The advent of successful ICSI combined with embryo cryopreservation has increased the chance of thawed cryopreserved semen achieving fertilization. Banking of a single ejaculate prior to commencement of chemotherapy/radiotherapy treatment may preserve potential fertility without compromising the oncology treatment.

Total synthesis of nucleobase-modified adenophostin A mimics.

The adenophostins exhibit approximately 10-100 times higher receptor binding and Ca2+ mobilising potencies in comparison with the natural second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Despite many synthetic attempts to determine the minimal structural requirement for this unusual behaviour of the adenophostins, few related simplified analogues displaying higher activity than that of Ins(1,4,5)P3 have been reported. However, biological evaluation of such analogues has revealed that one of the key factors for the enhanced biological activity is the adenine moiety. To further understand the effect that the adenine base has upon the activity of the adenophostins, congeners in which this functionality is replaced by uracil, benzimidazole, 2-methoxynaphthalene, 4-methylanisole and 4-methylnaphthalene using the common intermediate 1,2-di-O-acetyl-5-O-benzyl-3-O-(3,4-di-O-acetyl-2,6-di-O-benzyl-alpha-D-glucopyranosyl)-ribofuranose have been synthesised using a base replacement strategy. The synthesis of the uracil and benzimidazole analogues was achieved using the Vorbrüggen condensation procedure. The 1'-C-glycosidic analogues were prepared using Friedel-Crafts type C-aryl glycosidation reactions. Phosphate groups were introduced using the phosphoramidite method with subsequent removal of all-benzyl protecting groups by catalytic hydrogenation or catalytic hydrogen transfer. Apart from one analogue with an alpha-glycosidic linkage all compounds were more potent than Ins(1,4,5)P3 and most tended more towards adenophostin in activity. These analogues will be valuable tools to unravel the role that the adenine moiety plays in the potent activity of the adenophostins and demonstrate that this strategy is effective at producing highly potent ligands.