Reaction Mechanisms of H2S Oxidation by Naphthoquinones.

1,4-naphthoquinones (NQs) catalytically oxidize H2S to per- and polysufides and sulfoxides, reduce oxygen to superoxide and hydrogen peroxide, and can form NQ-SH adducts through Michael addition. Here, we measured oxygen consumption and used sulfur-specific fluorophores, liquid chromatography tandem mass spectrometry (LC-MS/MS), and UV-Vis spectrometry to examine H2S oxidation by NQs with various substituent groups. In general, the order of H2S oxidization was DCNQ ~ juglone > 1,4-NQ > plumbagin >DMNQ ~ 2-MNQ > menadione, although this order varied somewhat depending on the experimental conditions. DMNQ does not form adducts with GSH or cysteine (Cys), yet it readily oxidizes H2S to polysulfides and sulfoxides. This suggests that H2S oxidation occurs at the carbonyl moiety and not at the quinoid 2 or 3 carbons, although the latter cannot be ruled out. We found little evidence from oxygen consumption studies or LC-MS/MS that NQs directly oxidize H2S2-4, and we propose that apparent reactions of NQs with inorganic polysulfides are due to H2S impurities in the polysulfides or an equilibrium between H2S and H2Sn. Collectively, NQ oxidation of H2S forms a variety of products that include hydropersulfides, hydropolysulfides, sulfenylpolysulfides, sulfite, and thiosulfate, and some of these reactions may proceed until an insoluble S8 colloid is formed.

Intact and Degenerate Diguanylate Cyclases regulate Shigella Cyclic di-GMP.

The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger c-di-GMP signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but 5 of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the 4 intact DGCs in an S. flexneri strain where these 4 DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these 4 DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN , which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC genes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.

Qualitative and quantitative characteristics of xylazine-associated deaths detected using a post-mortem toxicology testing program.

INTRODUCTION: The United States drug overdose crisis continues to evolve. Xylazine has been increasingly identified as an adulterant in illicit opioid supplies. The incorporation of novel adulterants, like xylazine, into the illicit drug supply adds complexity to post-mortem toxicology testing, public health messaging, substance use mitigation, and the treatment of people who use drugs. METHODS: We assessed trends, decedent characteristics, drug co-detection, and blood concentrations of xylazine-positive post-mortem cases in Michigan. We utilized a toxicology testing program capable of detecting several opioids and non-opioids in post-mortem blood samples within 72 hours. RESULTS: A total of 279 deaths were xylazine-positive between October 2019 and June 2023, with 100 percent positive for fentanyl. Only 30 percent of xylazine-involved samples were positive for naloxone, while 21.2 percent of xylazine-negative and opioid-positive samples were positive for naloxone. The percentage of xylazine-positive deaths increased from 3.2 percent in 2021 to 4.7 percent in January-June 2023. A median of five total drug groups were present among xylazine-positive deaths. Post-mortem xylazine concentrations for 55 decedent blood samples ranged from 5.2 to 200 µg/L. DISCUSSION: Our study demonstrated increases in xylazine detection among post-mortem cases. Our findings are consistent with national trends of increasing xylazine presence among drug-involved deaths. Our range of detected post-mortem xylazine blood concentrations was consistent with what has been reported in previous literature. Fentanyl was detected in 100 percent of xylazine-positive overdose deaths. Naloxone detection was relatively low, highlighting the continued importance of increasing naloxone access and distribution. Deaths associated with xylazine often involved multiple other drugs. Limited human clinical xylazine research precludes accurate interpretation and attribution of causality from these data. CONCLUSIONS: Overdose-related deaths with xylazine detection are increasing in Michigan and across the United States. Further clinical and toxicological research can help contextualize the clinical significance of xylazine in opioid overdose, clarify epidemiologic and clinical research, and inform appropriate public health messaging.

Thirteen Cases of Valeryl Fentanyl in Michigan: A Call for Expanding Opioid Testing.

ABSTRACT: In this report, we describe 13 cases of drug overdose in Michigan in which valeryl fentanyl was found in postmortem blood. Valeryl fentanyl is a schedule I opioid that is rarely found in drug overdoses in the United States. Although little data exist on the mortality and morbidity associated with valeryl fentanyl, its molecular structure indicates that it would be less potent than fentanyl.When analyzing blood samples for valeryl fentanyl, samples from peripheral sites were sometimes negative for quantitative levels; however, samples from central sites in the same decedent were positive. This could indicate unique pharmacokinetics for valeryl fentanyl, which could have implications for other fentanyl analogs. Given the paucity of pharmacodynamic information, the prohibition of its use, the potential to buttress law enforcement efforts in monitoring drug trafficking trends, and to determine the efficacy of current regulations, laboratories should test for valeryl fentanyl. When testing for valeryl fentanyl, and likely other fentanyl analogs, the site of sample collection is important: central sources of blood are preferred to peripheral sources.

Novel Evaluation of Submandibular Salivary Gland Tissue for Use as an Alternative Postmortem Toxicology Specimen.

ABSTRACT: The collection of blood and tissue provides an opportunity for an objective comparison of autopsy results. Occasionally, a viable tissue sample is not available during autopsy. Expanding upon collected tissues to include a tissue that is accessible, is a possible drug depot, and is amendable to various analytical techniques may complement information obtained from other specimens. Given its absorption of ions, nutrients, and likely drugs via its rich blood supply, we evaluated the use of submandibular salivary gland tissue as an alternative postmortem specimen. The submandibular salivary glands of 52 decedents were excised. The tissue was homogenized, extracted, and analyzed via liquid chromatography tandem mass spectrometry for 43 opioids and 5 nonopioids. Liquid chromatography tandem mass spectrometry salivary tissue results were compared with the decedent's blood results. Results revealed that opioids were detected in salivary gland tissue at a sensitivity and specificity of 94.4% and 94.1%, respectively. Nonopioid drugs were detected at a sensitivity and specificity of 88.2% and 100.0%, respectively. This study suggests a comparable correlation exists between salivary gland tissue and blood results for certain drugs. Further evaluation is warranted. To our knowledge, this is the first report of salivary gland tissue being used for postmortem toxicology testing in humans.

Longitudinal Opioid Surveillance Project Involving Toxicologic Analysis of Postmortem Specimens from 9 Counties in Michigan Suggests the Discovery of New High-Intensity Drug Trafficking Areas.

ABSTRACT: Acetyl fentanyl (AF) is a Schedule I fentanyl analog that has been increasingly seen in heroin and fentanyl polydrug toxicity overdoses in Michigan (MI). Drug users are often unaware of the presence of AF in their drugs because it is often sold mixed into or disguised as heroin. High levels of AF in heroin drug products can cause increased incidence of overdose. This article describes data from a longitudinal opioid surveillance program and details 102 decedents in MI who were found to have evidence of heroin in their postmortem blood. A large portion of these decedents were also found to have evidence of fentanyl and AF. Our data further show significant overlap in incidence rates of AF and heroin-related overdose deaths in several MI counties, suggesting that AF is becoming enmeshed in heroin trafficking. Furthermore, we report unprecedented high incidence rates of AF and heroin-related overdose deaths in Calhoun county, and we propose that it is a high-intensity drug trafficking area. Highways US-131 and US-31 are likely used to transport these drugs. More study is needed into the drug trafficking trends in MI to ascertain drug sources and monitor the ever developing and dangerous polydrug heroin combinations.

Loperamide Abuse and Its Sequelae.

INTRODUCTION: Loperamide is an opioid available over the counter and in prescription form. Loperamide functions as a µ-agonist within the enteric nervous system to slow intestinal motility. Its antidiarrheal properties and primarily peripheral activity make loperamide an important tool in the management of inflammatory bowel disease. CASE REPORT: A 42-year-old man was found unconscious in cardiac arrest, and emergency medical personnel restored normal sinus rhythm. Family reported complaints of abdominal pain and that he "went through a lot" of loperamide. In the emergency department, the patient exhibited symptoms consistent with an opioid overdose. Mental status improved after administration of naloxone, an opioid antagonist. An electrocardiogram revealed a prolonged QTc interval, which progressed into Torsades de Pointes rhythm during admission. The patient succumbed from hypoxic brain injury, and there was evidence of acute pancreatitis at autopsy. Loperamide and desmethylloperamide (loperamide metabolite) were detected in blood samples. Cause of death was ruled loperamide toxicity. DISCUSSION: Because of reduced central nervous system activity and associated euphoria at therapeutic doses, loperamide abuse is rarely reported. This case demonstrates that an overdose on loperamide can occur in patients seeking symptom alleviation, and may mimic the presentation of opioid overdose.

A complicated case of bowel obstruction with sepsis and methamphetamine toxicity in a child with pica.

In this report, a pediatric case of bowel obstruction with sepsis complicated by methamphetamine toxicity is described. The decedent, an eleven-year-old female with a clinical history of pica, was found unresponsive in her home and pronounced dead following unsuccessful resuscitative efforts. Radiologic imaging showed multiple radio-opaque foreign objects in the stomach and bowel. Autopsy revealed a green leafy substance, coins and other metallic items, folded paper, and plastic in her stomach and bowels. Postmortem iliac blood and urine tested positive for amphetamine and methamphetamine. While the decedent's medical history and autopsy findings provided evidence consistent with bowel obstruction with sepsis due to the ingestion of foreign materials, the high methamphetamine concentration was suggestive of concurrent methamphetamine toxicity. Unique complications associated with this case include the phenomenon that methamphetamine toxicity and bowel obstruction can present similarly in children and the reported opinion that accidental drug ingestion is uncommon in children over the age of five. This case emphasizes that the age range for suspected accidental drug ingestion should be expanded for those with pica, as these patients, despite being older, may not be able to differentiate between what they should and should not ingest. Furthermore, when treating a pediatric patient with pica that appears to present with bowel obstruction, unintentional drug ingestion should also be considered, particularly if there is a suspicion that the child lives in a household where drugs are abused, given the prospect that drug toxicity can present similarly.

Evaluation of the Elecsys Syphilis Immunoassay for Detection of Syphilis in Populations at Risk of Disease in the US and Argentina.

BACKGROUND: The Elecsys® syphilis immunoassay is an automated, qualitative immunoassay that uses a double-antigen sandwich format to detect antibodies to Treponema pallidum in human serum and plasma. We aimed to validate performance of the immunoassay in various populations at risk for syphilis infection in the US and Argentina. METHODS: Samples were obtained for a number of study cohorts, including participants from routine syphilis testing at high or low risk for syphilis, HIV-positive patients, pregnant women, and patients in various stages of syphilis infection. The primary objective was to validate the Elecsys syphilis immunoassay by comparing it with a composite testing algorithm using US Food and Drug Administration (FDA)-approved tests, including the predicate IMMULITE 2000 syphilis screening assay, the rapid plasma reagin, and the T. pallidum particle agglutination assay. RESULTS: Complete algorithm testing was performed on all 2660 collected samples. Acceptable precision was demonstrated in all samples. Comparison of the Elecsys syphilis immunoassay with the final syphilis status for all samples yielded a diagnostic sensitivity of 99.5% (95% CI, 98.21-99.94) and a diagnostic specificity of 99.2% (95% CI, 98.69-99.49). Overall, the lower limit of the 95% CIs for sensitivity and specificity met the expected performance of ≥95%. CONCLUSION: This is the first study that confirms the high sensitivity and specificity of the Elecsys syphilis immunoassay in US and Argentinian cohorts and highlights the assay's usefulness as an alternative to current tests for the diagnosis of syphilis infection in a broad range of participant cohorts.

The effect of chronic renal failure on the benzoylecgonine blood level: a case report.

Chronic renal failure results in reduced elimination of a variety of substances within the blood, including numerous drugs and their metabolites. This report describes a case of a man who died in jail, after less than 48 hours of being incarcerated, wherein postmortem toxicology testing revealed a blood benzoylecgonine level of 0.25 mg/L with no cocaine detected, suggesting possible recent cocaine use in jail. Autopsy and investigation revealed severe underlying cardiovascular disease and dialysis-dependent CRF, thus accounting for the elevated benzoylecgonine levels and allaying concerns that the man obtained and used cocaine in jail.

Accidental death via intravaginal absorption of methamphetamine.

In this paper a drug fatality that involved an unintended drug delivery route is described. The decedent, a 23-year-old female in custody in a county jail on suspicion of a felony drug offense, was discovered in a holding cell unconscious and unresponsive. Following unsuccessful cardiopulmonary resuscitation attempts she was pronounced dead at the scene. At autopsy a wad of multiple small loosely wrapped plastic packages held together with another layer of clear plastic was found in the decedent's vagina. The smaller plastic packages contained an off-white pasty substance that was later identified as methamphetamine. Toxicological testing of specimens collected during autopsy revealed methamphetamine in the decedent's subclavian blood, vitreous fluid, and urine at extremely high concentrations (42.6, 20.1, and 771 mg/L, respectively). Amphetamine, the active metabolite of methamphetamine, was also present in the subclavian blood, vitreous fluid, and urine at significant concentrations (1.3, 0.5, and 20.4 mg/L, respectively). The cause of death was attributed to toxic effects of methamphetamine and the manner of death was ruled accidental. This report suggests that lethal concentrations of methamphetamine may be distributed to the systemic circulation via intravaginal absorption.

Complicated suicide versus autoeroticism?: a case involving multiple drugs and a porta-potty.

In this report, a unique and bizarre case of complicated suicide is presented. The decedent was found dead in the basin of a porta-potty, wearing women's pantyhose, jewelry, and makeup. The initial investigation was suspect for homicide. Although an autoerotic accidental death cannot be excluded, the patient's medical history and autopsy results provided evidence for suicide, including several substances positive in his serum. Tramadol was quantified to be 140 mg/L, approximately 470 times the therapeutic range. Moreover, formaldehyde was also present, presumably absorbed from the contents of the chemical toilet. An exhaustive search could not reveal similar circumstances of suicide in a porta-potty or with the levels of tramadol found in the decedent.