Novel Mouse Model Reveals That Serine Phosphorylation of L-Plastin Is Essential for Effective Splenic Clearance of Pneumococcus.

Asplenia imparts susceptibility to life-threatening sepsis with encapsulated bacteria, such as the pneumococcus. However, the cellular components within the splenic environment that guard against pneumococcal bacteremia have not been defined. The actin-bundling protein L-plastin (LPL) is essential for the generation of marginal zone B cells and for anti-pneumococcal host defense, as revealed by a mouse model of genetic LPL deficiency. In independent studies, serine phosphorylation of LPL at residue 5 (S5) has been described as a key "switch" in regulating LPL actin binding and subsequent cell motility, although much of the data are correlative. To test the importance of S5 phosphorylation in LPL function, and to specifically assess the requirement of LPL S5 phosphorylation in anti-pneumococcal host defense, we generated the "S5A" mouse, expressing endogenous LPL bearing a serine-to-alanine mutation at this position. S5A mice were bred to homozygosity, and LPL was expressed at levels equivalent to wild-type, but S5 phosphorylation was absent. S5A mice exhibited specific impairment in clearance of pneumococci following i.v. challenge, with 10-fold-higher bacterial bloodstream burden 24 h after challenge compared with wild-type or fully LPL-deficient animals. Defective bloodstream clearance correlated with diminished population of marginal zone macrophages and with reduced phagocytic capacity of multiple innate immune cells. Development and function of other tested leukocyte lineages, such as T and B cell motility and activation, were normal in S5A mice. The S5A mouse thus provides a novel system in which to elucidate the precise molecular control of critical immune cell functions in specific host-pathogen defense interactions.

Contrasting biogeographical patterns in Margarella (Gastropoda: Calliostomatidae: Margarellinae) across the Antarctic Polar Front.

Members of the trochoidean genus Margarella (Calliostomatidae) are broadly distributed across Antarctic and sub-Antarctic ecosystems. Here we used novel mitochondrial and nuclear gene sequences to clarify species boundaries and phylogenetic relationships among seven nominal species distributed on either side of the Antarctic Polar Front (APF). Molecular reconstructions and species-delimitation analyses recognized only four species: M. antarctica (the Antarctic Peninsula), M. achilles (endemic to South Georgia), M. steineni (South Georgia and Crozet Island) and the morphologically variable M. violacea (=M. expansa, M. porcellana and M. pruinosa), with populations in southern South America, the Falkland/Malvinas, Crozet and Kerguelen Islands. Margarella violacea and M. achilles are sister species, closely related to M. steineni, with M. antarctica sister to all these. This taxonomy reflects contrasting biogeographic patterns on either side of the APF in the Southern Ocean. Populations of Margarella north of the APF (M. violacea) showed significant genetic variation but with many shared haplotypes between geographically distant populations. By contrast, populations south of the APF (M. antarctica, M. steineni and M. achilles) exhibited fewer haplotypes and comprised three distinct species, each occurring across a separate geographical range. We hypothesize that the biogeographical differences may be the consequence of the presence north of the APF of buoyant kelps - potential long-distance dispersal vectors for these vetigastropods with benthic-protected development - and their near-absence to the south. Finally, we suggest that the low levels of genetic diversity within higher-latitude Margarella reflect the impact of Quaternary glacial cycles that exterminated local populations during their maxima.

Recurrent hyperactive ESR1 fusion proteins in endocrine therapy-resistant breast cancer.

Background: Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. Patients and methods: To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287-395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. Results: We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3' partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Conclusions: Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6-7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.

Imaging of palatal lumps.

Palatal tumours are relatively rare and of variable aetiology, rendering radiological evaluation a daunting process for many. A systematic approach to the imaging of a palatal lump is therefore essential. The hard and soft palates are oral cavity and oropharyngeal structures, respectively. They have different tissue compositions, and therefore, lesions occur with different frequencies at each site. The hard palate has the highest concentration of minor salivary glands in the upper aerodigestive tract and most tumours here are salivary in origin, whereas most tumours at the soft palate are epithelial in origin, i.e., squamous cell carcinomas, in line with other oropharyngeal subsites. The most common malignant tumours of the palate, after squamous cell carcinoma, are minor salivary gland tumours, predominantly adenoid cystic and mucoepidermoid carcinomas. These tumours have a propensity to spread perineurally; understanding the anatomy and imaging features of perineural spread is vital, as it can have significant implications for patient management and tumour resectability. When confronted with a palatal lump, it is important to consider the following: its location on the hard or soft palate; whether it is mucosal or submucosal; the frequently occurring lesions at that site; the most suitable imaging techniques (ultrasound, computed tomography, magnetic resonance imaging); whether there are typical imaging features for any of the common lesions; and whether there are aggressive features, such as bone erosion or perineural spread. This approach allows the radiologist to narrow the differential diagnosis and assist the clinicians with planning treatment.

Can acclimation of thermal tolerance, in adults and across generations, act as a buffer against climate change in tropical marine ectotherms?

Thermal acclimation capacity was investigated in adults of three tropical marine invertebrates, the subtidal barnacle Striatobalanus amaryllis, the intertidal gastropod Volegalea cochlidium and the intertidal barnacle Amphibalanus amphitrite. To test the relative importance of transgenerational acclimation, the developmental acclimation capacity of A. amphitrite was investigated in F1 and F2 generations reared at a subset of the same incubation temperatures. The increase in CTmax (measured through loss of key behavioural metrics) of F0 adults across the incubation temperature range 25.4-33.4°C was low: 0.00°C (V. cochlidium), 0.05°C (S. amaryllis) and 0.06°C (A. amphitrite) per 1°C increase in incubation temperature (the acclimation response ratio; ARR). Although the effect of generation was not significant, across the incubation temperature range of 29.4-33.4°C, the increase in CTmax in the F1 (0.30°C) and F2 (0.15°C) generations of A. amphitrite was greater than in the F0 (0.10°C). These correspond to ARR's of 0.03°C (F0), 0.08°C (F1) and 0.04°C (F2), respectively. The variability in CTmax between individuals in each treatment was maintained across generations, despite the high mortality of progeny. Further research is required to investigate the potential for transgenerational acclimation to provide an extra buffer for tropical marine species facing climate warming.

Klebsiella pneumoniae FimK Promotes Virulence in Murine Pneumonia.

Klebsiella pneumoniae, a chief cause of nosocomial pneumonia, is a versatile and commonly multidrug-resistant human pathogen for which further insight into pathogenesis is needed. We show that the pilus regulatory gene fimK promotes the virulence of K. pneumoniae strain TOP52 in murine pneumonia. This contrasts with the attenuating effect of fimK on urinary tract virulence, illustrating that a single factor may exert opposing effects on pathogenesis in distinct host niches. Loss of fimK in TOP52 pneumonia was associated with diminished lung bacterial burden, limited innate responses within the lung, and improved host survival. FimK expression was shown to promote serum resistance, capsule production, and protection from phagocytosis by host immune cells. Finally, while the widely used K. pneumoniae model strain 43816 produces rapid dissemination and death in mice, TOP52 caused largely localized pneumonia with limited lethality, thereby providing an alternative tool for studying K. pneumoniae pathogenesis and control within the lung.

Transfusion in children: epidemiology and 10-year survival of transfusion recipients.

OBJECTIVE: To describe the epidemiology of blood transfusion in children: including the incidence of transfusion, the diagnoses leading to transfusion, donor exposure (DE) and post-transfusion survival. STUDY DESIGN AND METHODS: The Epidemiology and Survival of Transfusion Recipients (EASTR) Study was a multi-centre epidemiological study with prospective survival monitoring. Cross-sectional sampling of adult and paediatric transfusion recipients in 29 hospitals was used to select three separate cohorts of red cell (RBC), platelet (PLT) and fresh frozen plasma (FFP) recipients between October 2001 and September 2002. This paper presents the analysis of results for children <16 years. RESULTS: Children <16 years comprised 449 (5%) of the RBC, 362 (9%) of the FFP and 452 (13%) of the PLT recipients. In children 54% of RBC, 63% FFP and 45% PLT recipients were under 1 year of age and 57% RBC, 60% FFP and 52% PLT were male. Median (IQR) DEduring the study year was 3(2-8); 5(2-13) and 11(6-21) in the RBC, FFP and PLT cohorts, respectively. A total of 20% of RBC, 31% of FFP and 54% of PLT recipients had been exposed to >10 donors. Perinatal conditions were the commonest indication for transfusion in the RBC (36%) and FFP (44%) cohorts and comprised 31% of the PLT cohort. Medical conditions (48%), predominantly malignancy (33%), were the most frequent indication in the PLT cohort. The 10 year (95% CI) survival rates were 81% (77-85%), 72% (67-76%) and 71% (66-75%)for RBC, FFP and PLT cohorts, respectively. CONCLUSIONS: Around half of paediatric transfusion recipients are under 1 year of age. Exposure to components from multiple donors is common. At least 70% of paediatric recipients are long survivors and are at risk for late complications of transfusion.

Transfusion in adults: 10-year survival of red cell, plasma and platelet recipients following transfusion.

OBJECTIVE: To determine the long-term survival of adult recipients (>16 years) transfused with red blood cells (RBC), platelets (PLT) and fresh frozen plasma (FFP) in England and Wales. STUDY DESIGN AND METHODS: The EASTR study (Epidemiology and Survival of Transfusion Recipients) was a national multi-centre epidemiological study with cross-sectional sampling from 29 representative hospitals in England supplied by NHS Blood and Transplant (NHSBT). Three separate groups of RBC (n = 9142), FFP (n = 4232) and PLT (3584) recipients were sampled over 1 year (1 October 2001-30 September 2002), with prospective survival monitoring for 10 years. This study presents the data for adult recipients (>16 years of age). RESULTS: The median age interquartile range (IQR) of adult transfusion recipients was RBC 70 (54-79), FFP 66 (51-76), PLT 62 (48-72). The 10-year survival for adult RBC, FFP and PLT recipients was highest for RBC recipients at 36% confidence interval (CI 35-37%, n = 8675), compared with 30% for both FFP (CI 29-32%, n = 3849) and PLT (CI 28-30%, n = 3110) recipients. In all groups, post-transfusion survival decreased with age, and a risk-adjusted analysis showed that reason for transfusion, transfusion type (surgical or medical) and cancer diagnosis (presence or absence) were all significantly associated with survival. Older patients with cancer receiving a medical rather than surgical transfusion had the highest hazard of death. CONCLUSION: This study shows that survival following transfusion in England is broadly similar to that reported in other wealthy nations. More than 70% of recipients die within 10 years of transfusion, but long-term survival is common in younger patients (>80% 10-year survival in RBC recipients aged 16-39 years).

Salivary gland FNA cytology: role as a triage tool and an approach to pitfalls in cytomorphology.

OBJECTIVE: To highlight the importance of salivary gland fine needle aspiration (FNA) cytology as a triage tool for surgery and to determine its sensitivity and specificity. To discuss the diagnostic pitfalls and potential role of ancillary techniques in diagnosis and prognosis. METHODS: The study included a total of 920 cases of salivary gland FNAs received in the cytopathology department of University College London Hospital during December 2004 to December 2012. The cases with known histological outcomes were analysed to determine the sensitivity and specificity. RESULTS: Surgery was carried out on 180 (19.6%) of 920 patients. Excluding nine with inadequate/non-diagnostic cytology, the sensitivity of FNA cytology for a malignant outcome was 89% (33/37) and the specificity was 97% (130/134). Diagnostic pitfalls are discussed with respect to eight FNAs with discrepant histology. Histological outcome was not available for 740 cases (80.4%): excluding 88 non-diagnostic FNAs, 324 (49.7%) had non-neoplastic diagnoses (not indicating surgery) and 328 (50.3%) had neoplastic diagnoses, which included recurrences/metastases of known tumours. Patients with other neoplasms on FNA were lost to follow-up and may have had surgery elsewhere. Cases with clinical concerns were discussed at weekly multidisciplinary meetings. CONCLUSION: Salivary gland FNA is crucial in the distinction of non-neoplastic from neoplastic lesions, emphasizing the fact that FNA is an excellent triage tool for surgery. Salivary gland FNA has a high sensitivity and specificity. However, it is important to interpret the cytological diagnoses in the light of clinical findings and imaging. Diagnostic pitfalls are seen in a minority of cases and could potentially be overcome with the help of recently described diagnostic and prognostic markers.

rDock: a fast, versatile and open source program for docking ligands to proteins and nucleic acids.

Identification of chemical compounds with specific biological activities is an important step in both chemical biology and drug discovery. When the structure of the intended target is available, one approach is to use molecular docking programs to assess the chemical complementarity of small molecules with the target; such calculations provide a qualitative measure of affinity that can be used in virtual screening (VS) to rank order a list of compounds according to their potential to be active. rDock is a molecular docking program developed at Vernalis for high-throughput VS (HTVS) applications. Evolved from RiboDock, the program can be used against proteins and nucleic acids, is designed to be computationally very efficient and allows the user to incorporate additional constraints and information as a bias to guide docking. This article provides an overview of the program structure and features and compares rDock to two reference programs, AutoDock Vina (open source) and Schrödinger's Glide (commercial). In terms of computational speed for VS, rDock is faster than Vina and comparable to Glide. For binding mode prediction, rDock and Vina are superior to Glide. The VS performance of rDock is significantly better than Vina, but inferior to Glide for most systems unless pharmacophore constraints are used; in that case rDock and Glide are of equal performance. The program is released under the Lesser General Public License and is freely available for download, together with the manuals, example files and the complete test sets, at http://rdock.sourceforge.net/

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.

Extinction and recolonization of maritime Antarctica in the limpet Nacella concinna (Strebel, 1908) during the last glacial cycle: toward a model of Quaternary biogeography in shallow Antarctic invertebrates.

Quaternary glaciations in Antarctica drastically modified geographical ranges and population sizes of marine benthic invertebrates and thus affected the amount and distribution of intraspecific genetic variation. Here, we present new genetic information in the Antarctic limpet Nacella concinna, a dominant Antarctic benthic species along shallow ice-free rocky ecosystems. We examined the patterns of genetic diversity and structure in this broadcast spawner along maritime Antarctica and from the peri-Antarctic island of South Georgia. Genetic analyses showed that N. concinna represents a single panmictic unit in maritime Antarctic. Low levels of genetic diversity characterized this population; its median-joining haplotype network revealed a typical star-like topology with a short genealogy and a dominant haplotype broadly distributed. As previously reported with nuclear markers, we detected significant genetic differentiation between South Georgia Island and maritime Antarctica populations. Higher levels of genetic diversity, a more expanded genealogy and the presence of more private haplotypes support the hypothesis of glacial persistence in this peri-Antarctic island. Bayesian Skyline plot and mismatch distribution analyses recognized an older demographic history in South Georgia. Approximate Bayesian computations did not support the persistence of N. concinna along maritime Antarctica during the last glacial period, but indicated the resilience of the species in peri-Antarctic refugia (South Georgia Island). We proposed a model of Quaternary Biogeography for Antarctic marine benthic invertebrates with shallow and narrow bathymetric ranges including (i) extinction of maritime Antarctic populations during glacial periods; (ii) persistence of populations in peri-Antarctic refugia; and (iii) recolonization of maritime Antarctica following the deglaciation process.

Phase IIa randomized, placebo-controlled study of antimicrobial photodynamic therapy in bacterially colonized, chronic leg ulcers and diabetic foot ulcers: a new approach to antimicrobial therapy.

BACKGROUND: With increasing problems of antibiotic resistance, photodynamic therapy (PDT) is being developed as a novel antimicrobial treatment. Following light activation, cationic photosensitizer PPA904 [3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide] kills a broad spectrum of bacteria in vitro and this has a variety of potential clinical applications. OBJECTIVES: To determine if PDT in bacterially colonized chronic leg ulcers and chronic diabetic foot ulcers can reduce bacterial load, and potentially lead to accelerated wound healing. METHODS: Sixteen patients with chronic leg ulcers and 16 patients with diabetic foot ulcers (each eight active treatment/eight placebo) were recruited into a blinded, randomized, placebo-controlled, single-treatment, Phase IIa trial. All patients had ulcer duration > 3 months, bacterially colonized with > 10 colony-forming units cm . After quantitatively assessing pretreatment bacterial load via swabbing, PPA904 or placebo was applied topically to wounds for 15 min, followed immediately by 50 J cm of red light and the wound again sampled for quantitative microbiology. The wound area was measured for up to 3 months following treatment. RESULTS: Treatment was well tolerated with no reports of pain or other safety issues. In contrast to placebo, patients on active treatment showed a reduction in bacterial load immediately post-treatment (P < 0·001). After 3 months, 50% (four of eight) of patients with actively treated chronic leg ulcer showed complete healing, compared with 12% (one of eight) of patients on placebo. CONCLUSIONS: This first controlled study of PDT in chronic wounds demonstrated significant reduction in bacterial load. An apparent trend towards wound healing was observed; further study of this aspect with larger patient numbers is indicated.

Psychological approaches to chronic pain management: evidence and challenges.

Psychological interventions are a mainstay of modern pain management practice and a recommended feature of a modern pain treatment service. Systematic reviews for the evidence of psychological interventions are reviewed in this article. The evidence for effectiveness is strongest for cognitive behavioural therapy with a focus on cognitive coping strategies and behavioural rehearsal. Most evidence is available for treatments of adult pain, although adolescent chronic pain treatments are also reviewed. It is clear that treatment benefit can be achieved with cognitive behavioural methods. It is possible to effect change in pain, mood, and disability, changes not achieved by chance or by exposure to any other treatment. However, the overall effect sizes of treatments for adults, across all trials, are modest. Reasons for the relatively modest treatment effects are discussed within the context of all treatments for chronic pain being disappointing when measured by the average. Suggestions for improving both trials and evidence summaries are made. Finally, consideration is given to what can be achieved by the pain specialist without access to specialist psychology resource.

Missing links: Weber-Cockayne keratin mutations implicate the L12 linker domain in effective cytoskeleton function.

We have identified mutations in keratins K5 (Arg331Cys) and K14 (Val270Met) in two kinships affected by the dominantly-inherited skin blistering disease, Weber-Cockayne epidermolysis bullosa simplex (EBS-WC). Linkage analysis, DNA sequencing and clinical and ultrastructural analysis are combined to provide the first detailed description of classical EBS-WC. Both phenotypes show similar blistering on trauma, indicating that both mutations compromise the structural resilience of the basal keratinocytes by affecting the keratin cytoskeleton. The location of these mutations in the L12 linker, which bisects the alpha-helical rod region of intermediate filament proteins, identifies another keratin mutation cluster leading to hereditary skin fragility syndromes.

Keratin 16 and keratin 17 mutations cause pachyonychia congenita.

Pachyonychia congenita (PC) is a group of autosomal dominant disorders characterized by dystrophic nails and other ectodermal aberrations. A gene for Jackson-Lawler PC was recently mapped to the type I keratin cluster on 17q. Here, we show that a heterozygous missense mutation in the helix initiation motif of K17 (Asn92Asp) co-segregates with the disease in this kindred. We also show that Jadassohn-Lewandowsky PC is caused by a heterozygous missense mutation in the helix initiation peptide of K16 (Leu130Pro). The known expression patterns of these keratins in epidermal structures correlates with the specific abnormalities observed in each form of PC.

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.