Post mortem blood bromazolam concentrations and co-findings in 96 coronial cases within England and Wales.

Bromazolam is a newly emerging benzodiazepine drug which is not licensed for medicinal use. It may be sourced as a New Psychoactive Substance (NPS) for its desired effects or be consumed unknowingly via counterfeit Xanax® or Valium® preparations. As part of our Coronial workload, we observed an increase in the detection of bromazolam from September 2021 to November 2022. We report a series of 96 cases in which bromazolam was quantitated by high resolution accurate mass - mass spectrometry (HRAM - MS) in post-mortem blood. The mean (SD) post-mortem blood bromazolam concentration from our case series was 64.6 ( ± 79.4) µg/L (range <1-425 µg/L). Routine toxicological screening results have also been reported; the most commonly encountered drugs taken in combination with bromazolam were cocaine, gabapentinoids and diazepam. In 48% of cases at least one further designer benzodiazepine drug was also present (etizolam, flualprazolam, flubromazolam, flubromazepam). It is essential that laboratories providing toxicological investigations are aware of the limitations of their assays; and inclusion of bromazolam within targeted screening panels using LC-MS/MS is encouraged. Bromazolam has not been associated with death in isolation from resulting toxic concentrations; however, it is likely to enhance adverse clinical effects when taken in combination with stimulant and/or centrally-acting depressant drugs (poly-drug deaths). Bromazolam, similar to other benzodiazepines, may also impair cognition and decision making skills.

Sodium nitrite poisoning: A series of 20 fatalities in which post-mortem blood nitrite and nitrate concentrations are reported.

Sodium nitrite has several industrial applications however its accidental or intentional ingestion has been associated with severe toxicity and death. We present a series of 20 cases over 2 years in which evidence of sodium nitrite ingestion was found at the scene and supported by biochemical analysis of post-mortem blood nitrite and nitrate levels. Routine toxicological screening was performed on post-mortem blood samples received at University Hospitals of Leicester (UHL) NHS Trust, including ethanol analysis by headspace gas chromatography-flame ionisation detection (HS GC-FID), drug screening by high resolution accurate mass-mass spectrometry (HRAM-MS) and confirmatory drug quantitation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cases in which the history indicated the possibility of nitrite salts present at the scene, purchase of a suicide kit or a dusky-ash appearance of skin on post-mortem were referred to a specialist laboratory for nitrite and nitrate analysis. Analysis was based upon the gas-phase chemiluminescent reaction between nitric oxide (NO) and ozone; NO levels were determined using an NOA 280A, Sievers NO analyser. Twenty post-mortem cases in which sodium nitrite ingestion was the most probable cause of death were reported between January 2020 and February 2022; mean age was 31 years (range 14-49) with 9/20 (45%) female. 16/20 (80%) of cases had a history of depression and / or mental health issues. In half of the cases, anti-depressant / anti-psychotic drugs were prescribed; these drugs were detected in 8/20 (40%) cases. Ethanol was detected in 4/20 (20%) cases and anti-emetic drugs in 7/20 (35%) cases; anti-emetic drugs may be used to aid retention of sodium nitrite. Illicit drugs (amphetamine, cannabis and cocaine) were present in 3/20 cases (15%). Nitrite was found to be elevated in all but one case (95%), and nitrate was elevated in 17/20 (85%) cases. This paper highlights a surge in numbers of deaths across England and Wales due to sodium nitrite toxicity. Although, nitrite poisoning remains a rare cause of death, it is worthwhile considering its use in individuals with suicidal ideation given its unregulated availability online. The detection and quantitation of nitrite and nitrate requires specialised, highly reliable methodology currently only available in research laboratories. Implication of sodium nitrite ingestion also relies heavily upon circumstantial evidence combined with quantification. The provision of a quantitative nitrite / nitrate analytical service greatly assists in determining the cause of death in these cases.

Reporting Two Fatalities Associated with the Use of 4-Methylethcathinone (4-MEC) and a Review of the Literature.

We report two fatalities that are related to the cathinone 4-methylethcathinone (4-MEC) and review the current knowledge of 4-MEC. Qualitative and quantitative analysis of 4-MEC was performed by validated high performance liquid chromatography-tandem mass spectrometry methods. In the first case a 22-year-old male died in hospital following collapse and seizures after using 4-MEC. Toxicological analysis of postmortem femoral blood revealed the presence of 4-MEC (0.167 mg/L), ethanol (27 mg/100 mL) and paracetamol (5 mg/L). Death was attributed solely to 4-MEC toxicity. The second case involved a 54-year-old man found with a taped plastic bag over his head. Toxicological analysis of postmortem femoral blood revealed the presence of 4-MEC (1.73 mg/L) along with ethanol (229 mg/100 mL), propranolol (0.036 mg/L), venlafaxine (0.284 mg/L) and its metabolite O-desmethylvenlafaxine (0.205 mg/L), and diazepam (<0.005 mg/L) and its metabolite nordiazepam (0.033 mg/L). Death was attributed primarily to asphyxiation. These cases and a review of the current knowledge of 4-MEC pharmacology/toxicology adds to the body of case material for 4-MEC and will assist with interpretation in postmortem toxicology cases in which 4-MEC is detected.

Seven fatalities associated with ethylphenidate.

Ethylphenidate is a stimulant novel psychoactive substance that is an analogue of the prescription drug methylphenidate (Ritalin(®)). Methylphenidate is used commonly for the treatment of attention deficit hyperactivity disorder. Due to its stimulant effects ethylphenidate is being abused. There is a single case report of a death associated with ethylphenidate in Germany, and a case series of 19 deaths in the East of Scotland, but otherwise, the contribution of ethylphenidate to death is poorly documented. We report the analytical results of 7 cases (between February 2013 and January 2015) in which ethylphenidate was detected and quantitated with a validated liquid chromatography tandem mass spectrometry method (LC-MS/MS). The individuals (all male) ranged in age from 23 to 49 years (median 25 years). The concentration of ethylphenidate in the cases ranged from 0.026mg/L to 2.18mg/L in unpreserved post-mortem femoral blood. Only one case had ethylphenidate present as a sole drug. All other cases had at least 2 other drug classes present (benzodiazepines, heroin, methadone antipsychotics, other new psychoactive compounds). Ethylphenidate toxicity was the sole contribution to the cause of death in one case. Hanging was the cause of death in 2 cases, with the other 4 cases being reported as having occurred due to mixed drug toxicity. These data will further help with the interpretation of post-mortem ethylphenidate levels.

Morphological development and characterization of aromatase and estrogen receptors alpha and beta in fetal ovaries of cattle from days 110 to 250.

To better understand the role of estradiol-17beta in fetal ovarian development, presence and localization of cytochrome P450 aromatase (P450arom) and estrogen receptors alpha (ERalpha) and beta (ERbeta) proteins were characterized in fetal ovaries of cattle using immunohistochemistry. Fetal cattle ovaries were collected from an abattoir and sorted into fetal age groups (days 110, 130, 150, 170, 190, 210, 230, 250+) based on crown-rump length. In addition to immunohistochemistry, morphological analysis of ovarian and follicular formation was made. Ovaries appeared lobular at day 110, but by the end of gestation (day 250+) ovaries were oval-shaped similar to those found in adult animals. Ovarian structures within different lobes appeared to be at different developmental stages. At day 110, oocytes and pre-granulosa cells were observed in ovigerous cords that were still open to the surface epithelium. Most ovigerous cords appeared to be closed to the surface epithelium on day 130, all closed by day 150 and were no longer present at day 210. Ovarian follicles were classified as follows: Type 1(primordial): single layer of flattened granulosa cells, Type 1a (transitory): single layer of mixed flattened and cuboidal granulosa cells, Type 2 (primary): at least one but less than two layers of cuboidal granulosa cells, Type 3 (small preantral): two to three layers of granulosa cells, Type 4 (large preantral): four to six layers of granulosa, and the theca layer is forming around the follicle, Type 5 (antral): contain greater than six layers of granulosa cells, several layers of theca cells and the antrum has formed. Type 1 follicles were observed in day 110 ovaries. Follicle Types 1a and 2 were first observed on day 130. Type 3 follicles were first observed on day 150 and Types 4 and 5 were first observed on day 170. P450arom protein was localized in granulosa cells of follicle Types 2-5 and cells of rete tubules throughout the experimental period. There was punctate expression within stroma and rete masses. There was ERalpha protein localization in pre-granulosa cells and germ cells of ovigerous cords and all surface epithelial cells. There was also localization in granulosa cells and oocytes of all follicle types and cells of rete tubules. There was punctate ERalpha protein expression in stroma and rete masses. ERbeta protein was localized in pre-granulosa cells and germ cells of ovigerous cords. Expression was also localized to granulosa cells of all follicle types and cells of rete tubules. ERbeta protein was punctate in oocytes of follicles, surface epithelial cells, stroma and rete masses. Thus, the fetal ovary of cattle has the steroidogenic enzyme (P450arom) to convert androgens to estradiol-17beta, and estrogen receptors alpha and beta to facilitate an estrogen response within the fetal ovary.

Resilience: resistance factor for depressive symptom.

The purpose was to explore influence of resilience on the willingness of African Americans aged 65 and over to seek mental health care for depressive symptoms. Specifically, the study examined relationships between personal resilience and willingness of undiagnosed, community dwelling older adults to seek mental health care for depressive symptoms. A cross-sectional, correlational, causal modelling design was used to study older African Americans (N= 158; 121 women and 37 men) recruited from churches, retirement organizations and senior nutrition centres. Participants completed study instruments to measure depressive symptoms, resilience, willingness to seek mental health care, and general demographics information. Descriptive statistics and multiple regression analyses were preformed. Depressive symptoms and resilience accounted for 15.4% of the willingness to seek mental health care variance; extraction of resilience lowered variance to 0.9%. A direct, predictive relationship between resilience and willingness to seek mental health care was documented. Understanding resilience and willingness to seek mental health care supports future research for interventions that bolster resilience in older adults. Identifying the influence of resilience on such willingness may provide direction for developing interventions for older African Americans and may be applicable to vulnerable, marginalized and minority older adults worldwide.

Interaction of epithelial ion channels with the actin-based cytoskeleton.

The interaction of ion channels with the actin-based cytoskeleton in epithelial cells not only maintains the polarized expression of ion channels within specific membrane domains, it also functions in the intracellular trafficking and regulation of channel activity. Initial evidence supporting an interaction between epithelial ion channels and the actin-based cytoskeleton came from patch-clamp studies examining the effects of cytochalasins on channel activity. Cytochalasins were shown to either activate or inactivate epithelial ion channels. An interaction between the actin-based cytoskeleton and epithelial ion channels was further supported by the fact that the addition of monomeric or filamentous actin to excised patches had an effect on channel activity comparable to that of cytochalasins. Through the recent application of molecular and proteomic approaches, we now know that the interactions between epithelial ion channels and actin can either be direct or indirect, the latter being mediated through scaffolding or actin-binding proteins that serve as links between the channels and the actin-based cytoskeleton. This review discusses recent advances in our understanding of the interactions between epithelial ion channels and the actin-based cytoskeleton, and the roles these interactions play in regulating the cell surface expression, activity, and intracellular trafficking of epithelial ion channels.

Postoperative pulmonary complications after gynecologic surgery.

OBJECTIVE: Investigate the frequency of, and risks for postoperative pulmonary complications after surgery for non-malignant gynecologic disorders. METHOD: A retrospective component included medical record data for one year. A prospective component enrolled 300 patients consecutively who were scheduled for gynecologic surgeries. RESULT: Postoperative pulmonary complications occurred in 1.22% of 328 open abdominal procedures in the retrospective study, and 2.16% of 232 in the prospective study. Pooling the data yielded a frequency estimate of 1.61%. Mean hospital length of stay (pooled data) increased 1.75 days in those with postoperative pulmonary complications. Smoking was the only significant risk factor (relative risk=3.9 using pooled data). CONCLUSION: Postoperative pulmonary complications after surgery for non-malignant gynecologic disorders are infrequent but increase hospital length of stay. Smokers are at increased risk.

Gallbladder Na+/H+ exchange activity is up-regulated prior to cholesterol crystal formation.

BACKGROUND: Gallbladder Na+ and H2O absorption are increased prior to gallstone formation and may promote cholesterol nucleation. Na+/H+ exchange (NHE) isoforms NHE2 and NHE3 are involved in gallbladder Na+ transport in prairie dogs. We examined whether increased gallbladder Na+ absorption observed during early gallstone formation is the result of NHE up-regulation. MATERIALS AND METHODS: Native gallbladder and primary cultures of gallbladder epithelial cells (GBECs) harvested from prairie dogs fed nonlithogenic (CON) or 1.2% cholesterol diet for varying lengths of time to induce cholesterol-saturated bile (PreCRYS), cholesterol crystals (CRYS), or gallstones (GS) were used. NHE activity was assessed by measuring dimethylamiloride-inhibitable 22Na+ uptake under H+ gradient in primary GBECs. HOE-694 was used to determine NHE2 and NHE3 contributions. NHE protein and mRNA expression were examined by Western and Northern blots, respectively. RESULTS: Gallbladder total NHE activity was 25.1 +/- 1.3 nmol mg protein(-1) min(-1) in the control and increased during gallstone formation peaking at the PreCRYS stage (98.4 +/- 3.9 nmol mg protein(-1) min(-1)). There was a shift in NHE activity from NHE2 to NHE3 as the animals progressed from no stones through the PreCRYS and CRYS stages to gallstones. The increase in NHE activity was partly caused by an increased Vmax without any change in K(Na)m. Both NHE2 and NHE3 protein increased moderately during the PreCRYS stage without increases in mRNA expression. CONCLUSIONS: Increased gallbladder Na+ absorption observed prior to crystal formation is in part caused by an increase NHE activity which is not fully accounted for by an increase in NHE proteins and mRNA levels but may be explained by enhanced localization in the membranes and/or altered regulation of NHE.

In-line assessment of pulmonary drug delivery using light obscuration.

A candidate optical technology for characterisation of the value and quality of the drug dose delivered to a patient from an inhaler is examined. The theoretical reasoning behind the design of this technology is presented with reference to the optical scattering of the drug cloud. A technical implementation is presented; based upon the light obscuration signature, observed in the delivery path during drug delivery. Dose evaluation studies are reported, performed on different types of drug formulations using regulatory testing procedures and the proposed optical sensing system. Further applications of this technology are discussed in relation to the identification and evaluation of inhaler-drug formulation combinations, which are most suitable for particular patients.

Increasing active student participation in histology.

As have many institutions, the New York University School of Medicine (NYUSM) has instituted curriculum revisions that allow for increased independent study but impose significant time constraints on laboratory courses. Challenged by decreased laboratory time, we sought creative solutions for our histology course, "Cell Biology of Tissues and Organs," using computer- and Web-based materials. While these solutions have facilitated independent study, they have not fostered the subtler lessons learned in the laboratory, such as communication skills and team approaches to learning. To retain these elements and enrich our course, we focused on increasing student participation in the laboratory through unit reviews presented by students, student-generated comprehensive reviews, and practice exams. The active engagement of students in the learning process has resulted in a more exciting laboratory experience for all.

Functional characterization of Na(+)/H(+) exchangers in primary cultures of prairie dog gallbladder.

Gallbladder Na(+) absorption is linked to gallstone formation in prairie dogs. We previously reported Na(+)/H(+) exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I(sc), (11.6 +/- 0.5 microA. cm(-2)), potential differences, V(t) (2.1 +/- 0.2 mV), and resistance, R(t) (169 +/- 12 omega. cm(2)). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable (22)Na(+) uptake under a H(+) gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of (22)Na(+) uptake was 21.4 +/- 1.3 nmol x mg prot(-1) x min(-1), of which 9.5 +/- 0.7 (approximately 45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for approximately 6%, approximately 66% and approximately 28% of GBECs' total NHE activity, respectively. GBECs exhibited saturable NHE kinetics ( V(max) 9.2 +/- 0.3 nmol x mg prot(-1) x min(-1); K(m) 11.4 +/- 1.4 m M Na(+)). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na(+) transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.

Change of place, change of pace, change of status: rural community training for junior doctors, does it influence choices of training and career?

INTRODUCTION: The Rural and Remote Area Placement Program (RRAPP) began in April 2000 to provide a rural community practice training term for junior doctors and to increase the opportunities for junior doctors to experience training outside the hospital setting. Recent research into the community-based training and experience for junior doctors in Australia suggests that such experience contributes to their decision-making about future training and career. METHODS: A structured national survey was undertaken of all 107 junior doctors who had participated in RRAPP prior to October 2003 and included semi-structured interviews of 54 participants from prior to October 2002. RESULTS: Data indicated that rural and community experience influenced the choice of further rural and general practice training and also provided a useful setting for junior doctors to reflect on, and confirm, future training plans. This study provided evidence of the positive influence of RRAPP on the career choices of junior doctors, with greater than 70% of participants confirming RRAPP's influence on their plans. This study also provided insight into the process of these career decisions. Decision-making was precipitated by taking junior doctors 'outside their comfort zone' of the tertiary hospital and providing a different perspective on both the present and the future. CONCLUSION: In addition to the contrast in setting and the expansion of knowledge about rural community practice, RRAPP junior doctors identified the change of place, the change of pace, and the change of status as instrumental in their decision-making about future training and careers.

What diagnostic tests are needed for community-acquired pneumonia?

Diagnostic tests play an important part in the evaluation and management of patients with CAP. Tests have key roles in diagnosing the presence of CAP and in assessing severity. An ideal test for microbiologic diagnosis in CAP is not yet available, and initial antimicrobial therapy usually is empiric. Nonetheless, when appropriately applied and correctly performed, tests for the identification of pathogens in CAP are useful and cost-effective.

Neutralization of human papillomavirus type 11 (HPV-11) by serum from women vaccinated with yeast-derived HPV-11 L1 virus-like particles: correlation with competitive radioimmunoassay titer.

Neutralization of human papillomavirus type 11 (HPV-11) has been demonstrated using serum and cervical secretions from primates vaccinated with virus-like particles (VLPs). Theoretically, neutralizing antibodies could protect women from HPV infection. The immunogenicity of a yeast-derived HPV-11 L1 VLP vaccine was tested in women. Serum specimens were evaluated for HPV-11 titer by competitive radioimmunoassay (cRIA) and for neutralization by use of the athymic mouse xenograft system. Analysis of serum from 104 subjects showed a dose response in HPV-11 cRIA titers and neutralization. Overall, 68 (82.9%) of 82 postimmunization serum specimens from VLP recipients were 100% neutralizing when used in the assay at a 1:50 dilution. Of 69 serum specimens, 63 (91.3%) with cRIA titers >200 milliMerck units per milliliter were neutralizing. Immunization with HPV VLPs elicits a vigorous serum immune response in a high percentage of women. The HPV-11 cRIA titer appears to be a surrogate marker for neutralization.