Hydrogen Bonding in Amorphous Indomethacin.

Amorphous Indomethacin has enhanced bioavailability over its crystalline forms, yet amorphous forms can still possess a wide variety of structures. Here, Empirical Potential Structure Refinement (EPSR) has been used to provide accurate molecular models on the structure of five different amorphous Indomethacin samples, that are consistent with their high-energy X-ray diffraction patterns. It is found that the majority of molecules in amorphous Indomethacin are non-bonded or bonded to one neighboring molecule via a single hydrogen bond, in contrast to the doubly bonded dimers found in the crystalline state. The EPSR models further indicate a substantial variation in hydrogen bonding between different amorphous forms, leading to a diversity of chain structures not found in any known crystal structures. The majority of hydrogen bonds are associated with the carboxylic acid group, although a significant number of amide hydrogen bonding interactions are also found in the models. Evidence of some dipole-dipole interactions are also observed in the more structurally ordered models. The results are consistent with a distribution of Z-isomer intramolecular type conformations in the more disordered structures, that distort when stronger intermolecular hydrogen bonding occurs. The findings are supported by 1H and 2H NMR studies of the hydrogen bond dynamics in amorphous Indomethacin.

Set up to 'fail'? Implementing contamination minimisation procedures and environmental monitoring in a Sexual Assault Referral Centre.

There are 47 Sexual Assault Referral Centres (SARCs) in the UK positioned regionally to provide specialist medical and forensic services to the victims of rape and sexual assault. The centres are managed and operated through multiagency agreements bringing together medical and forensic expertise to provide the necessary care for the victim and support the recovery of forensic evidence. A principal factor in preserving the evidence integrity and mitigating the risk of miscarriages of justice is reducing the risk from background DNA contamination to the samples and evidence recovered. This paper outlines a study reviewing the implementation of contamination reduction procedures at one SARC, providing a longitudinal examination of the outcomes of environmental monitoring procedures over a 12-month period. The outcomes show a myriad of factors affecting the process that need continual review and modification of the operating procedures to counteract these risks, as they arise. The Forensic Regulator's "Code of Practice for Forensic Medical Examinations - FSR-C-116", published in May 2020, provides an essential and useful mandate of activities, but this study shows further consideration is required in regard to SARC cleaning procedures and measuring the extent of background DNA contamination. Considerable investment, and further research is needed to implement new and more effective approaches within 'real-world' and local constraints.

The prevalence and characterisation of TRAF3 and POT1 mutations in canine B-cell lymphoma.

The genetic and mutational basis of canine lymphoma remains poorly understood. Several genes, including TRAF3 and POT1, are mutated in canine B-cell lymphoma (cBCL), and are likely involved in the pathogenesis of this disease. The purpose of this study was to assess the prevalence of TRAF3 and POT1 mutations in a cohort of dogs with cBCL, compared to dogs with non-cBCL diseases (including four dogs with T-cell lymphoma [cTCL]). Forty-nine dogs were included (n = 24 cBCL; n = 25 non-cBCL). Eleven dogs had matched non-tumour DNA assessed to determine if mutations were germline or somatic. All dogs had TRAF3 and POT1 assessed by Sanger sequencing. The prevalence of deleterious TRAF3 and POT1 mutations in cBCL was 36% and 17%, respectively. A deleterious TRAF3 mutation was suspected to be germline in 1/5 cases with matched non-tumour DNA available for comparison. Deleterious mutations were not found in specimens from the non-cBCL group. Several synonymous variants were identified in both genes in cBCL and non-cBCL samples, which likely represent polymorphisms. These results indicate TRAF3 and POT1 mutations are common in cBCL. Deleterious TRAF3 and POT1 mutations were only identified in dogs with cBCL, and not in dogs with non-cBCL diseases, suggesting they are important in the pathogenesis of cBCL. Future studies to investigate the prognostic and therapeutic implications of these mutations are required.

The visualisation of fingermarks on Pangolin scales using gelatine lifters.

Recent media reports document the plight of the Pangolin and its current position as "the most trafficked mammal in the world". They are described by some as scaly anteaters as all species are covered in hard keratinous tissue in the form of overlapping scales acting as a "flexible dermal armour". It is estimated that between 2011 and 2013, 117,000-234,000 pangolins were slaughtered, but the seizures may only represent as little as 10% of the true volume of pangolins being illegally traded. In this paper, methods to visualise fingermarks on Pangolin scales using gelatine lifters is presented. The gelatine lifters provide an easy to use, inexpensive but effective method to help wildlife crime rangers across Africa and Asia to disrupt the trafficking. The gelatine lifting process visualised marks producing clear ridge detail on 52% of the Pangolin scales examined, with a further 30% showing the impression of a finger with limited ridge detail. The paper builds on an initial sociotechnical approach to establishing requirement, then it focuses on the methods and outcomes relating to lifting fingermarks off Pangolin scales using gelatine lifters, providing an evaluation of its use in practice.

Plastic and Non-plastic Debris Ingestion in Three Gull Species Feeding in an Urban Landfill Environment.

Plastic debris is recognized as a widespread, common and problematic environmental pollutant. An important consequence of this pollution is the ingestion of plastic debris by wildlife. Assessing the degree to which different species ingest plastics, and the potential effects of these plastics on their health are important research needs for understanding the impacts of plastic pollution. We examined debris (plastic and other types) ingestion in three sympatric overwintering gull species (Herring gulls Larus smithsonianus, Great Black-backed Gulls Larus marinus, and Iceland Gulls Larus glaucoides) to understand how debris ingestion differs among species, age classes and sexes in gulls. We also assessed how plastic burdens were associated with body condition to investigate how gulls may be affected by debris ingestion. There were no differences among the species, age classes or sexes in the incidence of debris ingestion (plastic or otherwise), the mass or number of debris pieces ingested. We found no correlation between ingested plastics burdens and individual condition. Gulls ingested plastic debris, but also showed high levels of other debris types as well, including metal, glass and building materials, including a metal piece of debris found within an abscess in the stomach. Thus, when the health effects of debris ingestion on gulls, and other species that ingest debris, is of interest, either from a physical or chemical perspective, it may be necessary to consider all debris types and not just plastic burdens as is often currently done for seabirds.

Apoptosis inhibition of Atlantic salmon (Salmo salar) peritoneal macrophages by Piscirickettsia salmonis.

To improve the understanding of the piscirickettsiosis pathogenesis, the in vivo apoptosis modulation of peritoneal macrophages and lymphocytes was studied in juvenile Salmo salar intraperitoneally injected with Piscirickettsia salmonis. Five fish were sampled at post-exposure days 1, 5, 8 (preclinical), 20 (clinical) and 40 (post-clinical period of the disease), and the leucocytes of their coelomic washings were analysed by flow cytometry (using the JC-1 cationic dye), TUNEL and cytology to detect apoptotic cells. A selective and temporal pattern of apoptosis modulation by P. salmonis infection was observed. Apoptosis in lymphocytes was not affected, whereas it was inhibited in macrophages but only during the preclinical stage of the induced piscirickettsiosis. Hence, it is postulated that P. salmonis inhibits macrophage apoptosis at the beginning of the disease development to survive, multiply and probably be transported inside these phagocytes; once this process is complete, macrophage apoptosis is no longer inhibited, thus facilitating the exit of the bacteria from the infected cells for continuing their life cycle.

Effect of simvastatin on vascular tone in porcine coronary artery: Potential role of the mitochondria.

Statins induce acute vasorelaxation which may contribute to the overall benefits of statins in the treatment of cardiovascular disease. The mechanism underlying this relaxation is unknown. As statins have been shown to alter mitochondrial function, in this study we investigated the role of mitochondria in the relaxation to simvastatin. Relaxation of porcine coronary artery segments by statins was measured using isolated tissue baths. Mitochondrial activity was determined by measuring changes in rhodamine 123 fluorescence. Changes in intracellular calcium levels were determined in freshly isolated smooth muscle cells with Fluo-4 using standard epifluorescent imaging techniques. Simvastatin, but not pravastatin, produced a slow relaxation of the coronary artery, which was independent of the endothelium. The relaxation was attenuated by the mitochondrial complex I inhibitor rotenone (10µM) and the complex III inhibitor myxothiazol (10µM), or a combination of the two. The complex III inhibitor antimycin A (10µM) produced a similar time-dependent relaxation of the porcine coronary artery, which was attenuated by rotenone. Changes in rhodamine 123 fluorescence showed that simvastatin (10µM) depolarized the membrane potential of mitochondria in both isolated mitochondria and intact blood vessels. Simvastatin and antimycin A both inhibited calcium-induced contractions in isolated blood vessels and calcium influx in smooth muscle cells and this inhibition was prevented by rotenone. In conclusion, simvastatin produces an endothelium-independent relaxation of the porcine coronary artery which is dependent, in part, upon effects on the mitochondria. The effects on the mitochondria may lead to a reduction in calcium influx and hence relaxation of the blood vessel.

Calbindin-D-28K like immunoreactivity in superficial dorsal horn neurons and effects of sciatic chronic constriction injury.

The neuropathic pain that results from peripheral nerve injury is associated with alterations in the properties of neurons in the superficial spinal laminae. Chronic constriction injury (CCI) of the rat sciatic nerve increases excitatory synaptic drive to excitatory neurons in the substantia gelatinosa while limiting that to inhibitory neurons. Since the calcium-binding protein calbindin D-28K has been associated with excitatory neurons, we examined whether CCI altered the properties of neurons expressing calbindin-like immunoreactivity (Cal+). These account for 30% of the neurons in lamina I and II. Calbindin did not co-localize with any particular electrophysiological phenotype of neuron; in substantia gelatinosa, it was found in some tonic, delay, irregular, phasic and transient firing neurons and in some cells that displayed central, radial or vertical morphology. When neuronal phenotype was defined more precisely in terms of both morphology and electrophysiological properties, no strong correlation with calbindin expression was found. The frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSC) in calbindin negative (Cal-) neurons was greater than that in Cal+ neurons. CCI did not alter the proportion of Cal+ neurons in the dorsal horn. Although CCI promoted a fourfold increase in sEPSC frequency in Cal+ neurons, sEPSC amplitude was reduced by 22% and charge transfer per second was unchanged. Since synaptic drive to Cal+ neurons is weak and there is no firm correlation between neuronal phenotype and calbindin expression, it is doubtful whether these neurons play a major role in the generation of central sensitization.

Cesarean delivery is not associated with decreased at-birth fracture rates in osteogenesis imperfecta.

PURPOSE: Osteogenesis imperfecta (OI) predisposes to recurrent fractures. Patients with the moderate to severe forms of OI present with antenatal fractures, and the mode of delivery that would be safest for the fetus is not known. METHODS: We conducted systematic analyses of the largest cohort of individuals with OI (n = 540) enrolled to date in the OI Linked Clinical Research Centers. Self-reported at-birth fracture rates were compared among individuals with OI types I, III, and IV. Multivariate analyses utilizing backward-elimination logistic regression model building were performed to assess the effect of multiple covariates, including method of delivery, on fracture-related outcomes. RESULTS: When accounting for other covariates, at-birth fracture rates did not differ based on whether delivery was by vaginal route or by cesarean delivery (CD). Increased birth weight conferred higher risk for fractures irrespective of the delivery method. In utero fracture, maternal history of OI, and breech presentation were strong predictors for choosing CD. CONCLUSION: Our study, the largest to analyze the effect of various factors on at-birth fracture rates in OI, shows that CD is not associated with decreased fracture rate. With the limitation that the fracture data were self-reported in this cohort, these results suggest that CD should be performed only for other maternal or fetal indications, not for the sole purpose of fracture prevention in OI.Genet Med 18 6, 570-576.

A cross-sectional multicenter study of osteogenesis imperfecta in North America - results from the linked clinical research centers.

Osteogenesis imperfecta (OI) is the most common skeletal dysplasia that predisposes to recurrent fractures and bone deformities. In spite of significant advances in understanding the genetic basis of OI, there have been no large-scale natural history studies. To better understand the natural history and improve the care of patients, a network of Linked Clinical Research Centers (LCRC) was established. Subjects with OI were enrolled in a longitudinal study, and in this report, we present cross-sectional data on the largest cohort of OI subjects (n = 544). OI type III subjects had higher prevalence of dentinogenesis imperfecta, severe scoliosis, and long bone deformities as compared to those with OI types I and IV. Whereas the mean lumbar spine area bone mineral density (LS aBMD) was low across all OI subtypes, those with more severe forms had lower bone mass. Molecular testing may help predict the subtype in type I collagen-related OI. Analysis of such well-collected and unbiased data in OI can not only help answering questions that are relevant to patient care but also foster hypothesis-driven research, especially in the context of 'phenotypic expansion' driven by next-generation sequencing.

The heart-rate-reducing agent, ivabradine, reduces mechanical allodynia in a rodent model of neuropathic pain.

BACKGROUND: Peripheral nerve injury increases the excitability of primary sensory neurons. This triggers the onset of neuropathic pain and maintains its persistence. Because changes in hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are implicated in this process, we examined the action of the heart-rate-reducing agent, ivabradine, a clinically approved HCN blocker, in the rat chronic constriction injury (CCI) model of neuropathic pain. METHODS: The effects of ivabradine on mechanical allodynia were assessed using von Frey filaments, and the effects on cardiovascular parameters were monitored by telemetry. Ivabradine block of HCN channels in dorsal root ganglion neurons was confirmed by whole-cell recording. RESULTS: In rats subject to CCI, ivabradine (6 mg/kg by gavage twice a day) significantly reduced mechanical allodynia. Cumulative effects were seen with twice daily oral administration over a 4-day period. Allodynia returned 4 days after the final drug dose. Mean arterial pressure was maintained and only a 15% pharmacological reduction in heart rate was observed. There was no cumulative effect of ivabradine on cardiovascular parameters. CONCLUSION: Because ivabradine is effective at an oral dose that produces only moderate pharmacological heart rate reduction, and this is known to be well tolerated in a clinical context, these results underline its possible use in neuropathic pain management.

A novel long-acting glucose-dependent insulinotropic peptide analogue: enhanced efficacy in normal and diabetic rodents.

AIM: Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that is released from intestinal K cells in response to nutrient ingestion. We aimed to investigate the therapeutic potential of the novel N- and C-terminally modified GIP analogue AC163794. METHODS: AC163794 was synthesized by solid-phase peptide synthesis. Design involved the substitution of the C-terminus tail region of the dipeptidyl peptidase IV (DPP-IV)-resistant GIP analogue [d-Ala(2) ]GIP(1-42) with the unique nine amino acid tail region of exenatide. The functional activity and binding of AC163794 to the GIP receptor were evaluated in RIN-m5F ß-cells. In vitro metabolic stability was tested in human plasma and kidney membrane preparations. Acute insulinotropic effects were investigated in isolated mouse islets and during an intravenous glucose tolerance test in normal and diabetic Zucker fatty diabetic (ZDF) rats. The biological actions of AC163794 were comprehensively assessed in normal, ob/ob and high-fat-fed streptozotocin (STZ)-induced diabetic mice. Acute glucoregulatory effects of AC163794 were tested in diet-induced obese mice treated subchronically with AC3174, the exendatide analogue [Leu(14) ] exenatide. Human GIP or [d-Ala(2) ]GIP(1-42) were used for comparison. RESULTS: AC163794 exhibited nanomolar functional GIP receptor potency in vitro similar to GIP and [d-Ala(2) ]GIP(1-42). AC163794 was metabolically more stable in vitro and displayed longer duration of insulinotropic action in vivo versus GIP and [d-Ala(2) ]GIP(1-42). In diabetic mice, AC163794 improved HbA1c through enhanced insulinotropic action, partial restoration of pancreatic insulin content and improved insulin sensitivity with no adverse effects on fat storage and metabolism. AC163794 provided additional baseline glucose-lowering when injected to mice treated with AC3174. CONCLUSIONS: These studies support the potential use of a novel GIP analogue AC163794 for the treatment of type 2 diabetes.

How often are late preterm births the result of non-evidence based practices: analysis from a retrospective cohort study at two tertiary referral centres in a nationalised healthcare system.

OBJECTIVE: To determine the proportion, characteristics, and predictors of late preterm birth (LPTB) in relation to evidence-based (EB) and non-evidence based (NEB) indications. DESIGN: Retrospective cohort study. SETTING: Two Canadian tertiary referral centres. POPULATION: All live singleton LPTBs over 1 year from 2010 to 2011, excluding major congenital anomalies. METHODS: Indications for LPTB were classified a priori as EB (i.e. based on practice guidelines or on evidence from randomised controlled trials) or NEB. Data were abstracted from maternal antenatal and labour records. Univariate analyses were completed using Fischer's exact, Pearson's chi-square, or analysis of variance (anova) F-tests. Logistic regression included gestation at birth, delivery provider, previous stillbirth, previous caesarean section, corticosteroid administration, and previous preterm birth as predictors for NEB LPTB. MAIN OUTCOME MEASURES: The proportion, characteristics, and predictors of women with NEB versus EB LPTBs. RESULTS: Of 524 LPTBs, 25.2% (n = 132) were NEB. Logistic regression revealed that NEB LPTBs were less likely if patients were delivered by their own doctor or their doctor's practice partner (OR 0.53, 95% CI 0.34-0.83). However, NEB LPTBs were more likely in women who had experienced a previous stillbirth (OR 2.57, 95% CI 1.20-5.49). CONCLUSIONS: Approximately one-quarter of LPTBs are NEB. Further research is needed to see if a review of the indications for LPTB, and subsequent reduction in NEB LPTBs, translates into improved neonatal outcomes and cost savings.

Evidence of exotoxin secretion of Piscirickettsia salmonis, the causative agent of piscirickettsiosis.

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial-free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE-214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.

Suicide attempt by self-poisoning: characteristics of suicide attempters seen at the Emergency Room at the University Hospital of the West Indies.

OBJECTIVE: To document the characteristics of self-poisoning suicide attempters who were brought to the University Hospital of the West Indies (UHWI) Emergency Room and to outline the type of drug used in the attempt. METHOD: This was a retrospective study conducted over the period 2005-2009. Data were gathered from patients' case records, log books and the hospital records using a questionnaire developed for this study. The questionnaire examined demographics, parameters of the drug ingested, patient's disposition, and reasons for attempt, final outcome and the type of discharge of patients who reported to the UHWI Emergency Room due to a suicide attempt by self-poisoning. RESULTS: Over the five-year period, 127 cases of suicide attempt by self-poisoning were reported. Significantly more females than males presented to the hospital due to self-poisoning (3:1, chi2 = 33.37; p < 0.001). Of this amount, 96 cases (75.6%) were females and 31 (24.4%) were males. The age group most recorded was 16-30 years (70.8%). The most common reason for the suicide attempt was an interpersonal conflict (52%). The drug category most often used in self-poisoning was analgesics (52%) with acetaminophens being the most common (26.2%). CONCLUSION: These findings are consistent with global suicide trends and indicate an urgent need to develop and implement national preventative and treatment measures for groups known to be at risk of suicidal attempts.