Gut and bladder fermentation syndromes: a narrative review.

We recently reported the first clinical case of bladder fermentation syndrome (BFS) or urinary auto-brewery syndrome, which caused the patient to fail abstinence monitoring. In BFS, ethanol is generated by Crabtree-positive fermenting yeast Candida glabrata in a patient with poorly controlled diabetes. One crucial characteristic of BFS is the absence of alcoholic intoxication, as the bladder lumen contains transitional epithelium with low ethanol permeability. In contrast, patients with gut fermentation syndrome (GFS) or auto-brewery syndrome can spontaneously develop symptoms of ethanol intoxication even without any alcohol ingestion because of alcoholic fermentation in the gut lumen. In abstinence monitoring, a constellation of laboratory findings with positive urinary glucose and ethanol, negative ethanol metabolites, and the presence of yeast in urinalysis should raise suspicion for BFS, whereas endogenous ethanol production needs to be shown by a carbohydrate challenge test for GFS diagnosis. GFS patients will also likely fail abstinence monitoring because of the positive ethanol blood testing. BFS and GFS are treated by yeast eradication of fermenting microorganisms with antifungals (or antibiotics for bacterial GFS cases) and modification of underlying conditions (diabetes for BFS and gut dysbiosis for GFS). The under-recognition of these rare medical conditions has led to not only harm but also adverse legal consequences for patients, such as driving under the influence (DUI). GFS patients may be at risk of various alcohol-related diseases.

Newly Emerging Drugs of Abuse.

Drug use and the associated overdose deaths have been a serious public health threat in the United States and the world. While traditional drugs of abuse such as cocaine remain popular, recreational use of newer synthetic drugs has continued to increase, but the prevalence of use is likely underestimated. In this review, epidemiology, chemistry, pharmacophysiology, clinical effects, laboratory detection, and clinical treatment are discussed for newly emerging drugs of abuse in the following classes: (1) opioids (e.g., fentanyl, fentanyl analogues, and mitragynine), (2) cannabinoids [THC and its analogues, alkylindole (e.g., JWH-018, JWH-073), cyclohexylphenol (e.g., CP-47,497), and indazole carboxamide (e.g., FUB-AMB, ADB-FUBINACA)], (3) stimulants and hallucinogens [ß-keto amphetamines (e.g., methcathinone, methylone), pyrrolidinophenones (e.g., α-PVP, MDPV), and dimethoxyphenethylamine ("2C" and "NBOMe")], (4) dissociative agents (e.g., 3-MeO-PCP, methoxetamine, 2-oxo-PCE), and (5) sedative-hypnotics (e.g., gabapentin, baclofen, clonazolam, etizolam). It is critically important to coordinate hospital, medical examiner, and law enforcement personnel with laboratory services to respond to these emerging threats.

Accidental intoxications in toddlers: lack of cross-reactivity of vilazodone and its urinary metabolite M17 with drug of abuse screening immunoassays.

BACKGROUND: Vilazodone is an FDA approved medication used to treat major depressive disorder. The authors describe two cases of accidental vilazodone exposure in toddlers who presented with symptoms similar to amphetamine exposure and also with unexplained positive amphetamine urine immunoassay drug screens. Given a lack of published data on cross-reactivity of vilazodone and its metabolites with drug of abuse screening tests, the authors investigated drug of abuse immunoassay cross-reactivity of vilazodone and metabolites using computational and empirical approaches. METHODS: To ascertain the likelihood that vilazodone would cross-react with drug of abuse screening immunoassays, the authors assessed the two-dimensional (2D) similarity of the vilazodone parent molecule and known metabolites to an array of antigenic targets for urine immunoassay drug screens. To facilitate studies of the commercially unavailable M17 metabolite, it was prepared synthetically through a novel scheme. Urine and serum were spiked with vilazodone and M17 into urine (200-100,000 ng/mL) and serum (20-2000 ng/mL) samples and tested for cross-reactivity. RESULTS: Computational analysis using 2D similarity showed that vilazodone and metabolites have generally low similarity to antigenic targets of common drug of abuse screening immunoassays, predicting weak or no cross-reactivity. The M17 metabolite had 2D similarity to amphetamines and tricyclic antidepressants in a range similar to some other compounds exhibiting weak cross-reactivity on these immunoassays. Cross-reactivity testing was therefore performed on two different urine amphetamines immunoassays and a serum tricyclic antidepressant immunoassay. However, actual testing of cross reactivity for vilazodone and the M17 metabolite did not detect cross-reactivity for any urine amphetamines screen at concentrations up to 100,000 ng/mL and for a serum tricyclic antidepressants assays at concentrations up to 2000 ng/mL. CONCLUSION: While the vilazodone metabolite M17 has weak 2D structural similarity to amphetamines and tricyclic antidepressants, the current study did not demonstrate any experimental cross-reactivity with two different urine amphetamines immunoassays and a serum tricyclic antidepressant immunoassay. Vilazodone ingestions in young children present a diagnostic challenge in their similarity to amphetamine ingestions and the lack of routine laboratory tests for vilazodone. Further work is needed to understand the metabolic profile for vilazodone in children versus adults.

Ricin Poisoning after Oral Ingestion of Castor Beans: A Case Report and Review of the Literature and Laboratory Testing.

BACKGROUND: Ricin is a protein toxin derived from the castor bean plant Ricinus communis. Several cases secondary to its consumption have been published and, more recently, its use as a potential bioterrorism agent has also been reported. Oral absorption of ricin is highly erratic, leading to a wide spectrum of symptoms. In addition, conventional urine drug screening tests will not be able to detect this compound, posing a diagnostic challenge. CASE REPORT: A male teenager intended to die by ingesting 200 castor beans after mixing and blending them with juice. Eight hours later, he presented with weakness, light-headedness, nausea, and vomiting and sought medical treatment. The patient was admitted and treated conservatively. An immune-based standard urine toxicology drug screen panel was reported as negative. A comprehensive untargeted urine drug screen test showed the presence of ricinine, a surrogate marker of ricin intoxication. He was transferred to the psychiatric service 3 days after admission. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: This case highlights the importance of knowing the peculiar pharmacokinetic properties of ricin after oral ingestion of castor beans and toxin release through mastication. Emergency physicians should be aware that oral absorption of ricin is dependent on several factors, such type and size of seeds and the geographic harvesting region, making it extremely difficult to estimate its lethality based solely on the number of ingested beans. Finally, comprehensive untargeted urine drug screening testing is highly valuable as a diagnostic tool in this context.

Dilute and shoot approach for toxicology testing.

Toxicology testing is performed in clinical settings, forensic settings, and for controlling doping. Drug screening is a toxicology test to determine if drugs are present in biological samples. The most common specimen type for drug testing is urine, as drugs and/or their metabolites are often more concentrated in the urine, extending the detection window of drugs. The dilute-and-shoot method is a simple procedure used in toxicology testing, where a sample is diluted before being directly injected into the liquid chromatography-mass spectrometry (LC-MS) system. This method is easy, quick, and cost-saving, and can be used for protein-poor liquid specimens such as urine. Thus, it is reasonable and attractive for busy toxicology laboratories to combine the dilute-and-shoot method with high-resolution hyphenated-MS for urine drug screening. This method has several disadvantages, including a suboptimal detection capability for certain analytes, as well as interference from co-eluting matrix components called matrix effects, in which co-eluting matrix molecules alter the ionization efficiency of the analyte molecules at the ionization source in LC-MS, altering (mostly reducing) the analyte detection capability. The matrix effect testing is essential for the validation of LC-MS-based assays. A reasonable approach to addressing these undesirable effects would be to minimize these components. The most straightforward approach is to reduce the amounts of matrix components by using a higher dilution of the specimen and a lower volume for specimen injection. Optimization of the chromatographic separation is another reasonable approach for reducing co-eluting matrix components with the analyte.

Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition.

Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising approach to promote wound healing and tissue regeneration. MSCs expanded in vitro lose early progenitors with differentiation and therapeutic potentials under normoxic condition, whereas hypoxic condition promotes MSC self-renewal through preserving colony forming early progenitors and maintaining undifferentiated phenotypes. Hypoxia inducible factor (HIF) pathway is a crucial signaling pathway activated in hypoxic condition. We evaluated the roles of HIFs in MSC differentiation, colony formation, and paracrine activity under hypoxic condition. Hypoxic condition reversibly decreased osteogenic and adipogenic differentiation. Decrease of osteogenic differentiation depended on HIF pathway; whereas decrease of adipogenic differentiation depended on the activation of unfolded protein response (UPR), but not HIFs. Hypoxia-mediated increase of MSC colony formation was not HIF-dependent also. Hypoxic exposure increased secretion of VEGF, HGF, and basic FGF in a HIF-dependent manner. These findings suggest that HIF has a limited, but pivotal role in enhancing MSC self-renewal and growth factor secretions under hypoxic condition.

Potential benefits of dietary seaweeds as protection against COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic in Japan is not as disastrous as it is in other Western countries, possibly because of certain lifestyle factors. One such factor might be the seaweed-rich diet commonly consumed in Japan. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which binds to angiotensin-converting enzyme 2 (ACE2) on the cell surface and downregulates ACE2, likely elevating the ratio of angiotensin-converting enzyme (ACE) to ACE2. The overreaction of the immune system, combined with the cytokine storm and ACE dominance, is purported to cause the condition of COVID-19 patients to deteriorate rapidly. Dietary seaweeds contain numerous components, including ACE inhibitory peptides, soluble dietary fibers (eg, fucoidan, porphyran), omega-3 fatty acids, fucoxanthin, fucosterol, vitamins D3 and B12, and phlorotannins. These components exert antioxidant, anti-inflammatory, and antiviral effects directly as well as indirectly through prebiotic effects. It is possible that ACE inhibitory components could minimize the ACE dominance caused by SARS-CoV-2 infection. Thus, dietary seaweeds might confer protection against COVID-19 through multiple mechanisms. Overconsumption of seaweeds should be avoided, however, as seaweeds contain high levels of iodine.

Advances in drugs of abuse testing.

Drugs of abuse testing is widely used clinically and forensically. Urine is the preferred type of specimen for drugs of abuse screening, but saliva, sweat, hair, and meconium are emerging types of specimens. GC-MS has been used as a gold standard for confirmatory drug testing, but LC-tandem-MS can analyze more diverse types of analytes than GC-MS. Thus, LC-tandem-MS becomes a new gold standard for confirmatory drug testing. Unlike GC-MS, LC-tandem-MS is not suited for non-targeted comprehensive drug screening. But with the advent of high-resolution-MS such as Tof-MS, which can discriminate the compounds of similar molecular masses but with different formulas, LC-hybrid-Tof-MS is usable for non-targeted comprehensive drug screening. Another technical advancement is the advent of miniature ambient ionization MS, which can analyze biological specimens including urine within one minute. Thus these mass spectrometers are promising for rapid drugs of abuse testing in a POC setting.

Synthetic drugs of abuse.

Synthetic drugs of abuse contain various psychoactive substances. These substances have recently emerged as novel drugs of abuse in public; thus, they are known as novel psychoactive substances (NPS). As these compounds are artificially synthesized in a laboratory, they are also called designer drugs. Synthetic cannabinoids and synthetic cathinones are the two primary classes of NPS or designer drugs. Synthetic cannabinoids, also known as "K2" or "Spice," are potent agonists of the cannabinoid receptors. Synthetic cathinones, known as "Bath salts," are beta-keto amphetamine derivatives. These compounds can cause severe intoxication, including overdose deaths. NPS are accessible locally and online. NPS are scheduled in the US and other countries, but the underground chemists keep modifying the chemical structure of these compounds to avoid legal regulation; thus, these compounds have been evolving rapidly. These drugs are not detectable by traditional drug screening, and thus, these substances are mainly abused by young individuals and others who wish to avoid drug detection. These compounds are analyzed primarily by mass spectrometry.

Interferon Type I Regulates Inflammasome Activation and High Mobility Group Box 1 Translocation in Hepatocytes During Ehrlichia-Induced Acute Liver Injury.

Inflammasomes are an important innate immune host defense against intracellular microbial infection. Activation of inflammasomes by microbial or host ligands results in cleavage of caspase-1 (canonical pathway) or caspase-11 (noncanonical pathway), release of interleukin (IL)-1ß, IL-18, high mobility group box 1 (HMGB1), and inflammatory cell death known as pyroptosis. Ehrlichia are obligate, intracellular, gram-negative bacteria that lack lipopolysaccharide but cause potentially life-threatening monocytic ehrlichiosis in humans and mice that is characterized by liver injury followed by sepsis and multiorgan failure. Employing murine models of mild and fatal ehrlichiosis caused by infection with mildly and highly virulent Ehrlichia muris (EM) and Ixodes ovatus Ehrlichia (IOE), respectively, we have previously shown that IOE infection triggers type I interferon (IFN-I) response and deleterious caspase-11 activation in liver tissues, which promotes liver injury and sepsis. In this study, we examined the contribution of IFN-I signaling in hepatocytes (HCs) to Ehrlichia-induced liver injury. Compared to EM infection, we found that IOE enter and replicate in vitro cultured primary murine HCs and induce secretion of IFNß and several chemokines, including regulated upon activation, normal T-cell expressed, and secreted (RANTES), monocyte chemoattractant protein 1 (MCP1), monokine induced by gamma (MIG)/chemokine (C-X-C motif) ligand 9 (CXCL9), macrophage inflammatory protein 1 alpha (MIP1α), keratinocyte-derived chemokine (KC), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Notably, in vitro stimulation of uninfected and Ehrlichia-infected HCs with recombinant IFNß triggered activation of caspase-1/11, cytosolic translocation of HMGB1, and enhanced autophagy and intracellular bacterial replication. Secretion of HMGB1 by IOE-infected HCs was dependent on caspase-11. Primary HCs from IOE- but not EM-infected mice also expressed active caspase-1/11. Conclusion: HC-specific IFN-I signaling may exacerbate liver pathology during infection with obligate intracellular Ehrlichia by promoting bacterial replication and detrimental caspase-11-mediated inflammasome activation.

Hydrogen gas treatment prolongs replicative lifespan of bone marrow multipotential stromal cells in vitro while preserving differentiation and paracrine potentials.

Cell therapy with bone marrow multipotential stromal cells/mesenchymal stem cells (MSCs) represents a promising approach in the field of regenerative medicine. Low frequency of MSCs in adult bone marrow necessitates ex vivo expansion of MSCs after harvest; however, such a manipulation causes cellular senescence with loss of differentiation, proliferative, and therapeutic potentials of MSCs. Hydrogen molecules have been shown to exert organ protective effects through selective reduction of hydroxyl radicals. As oxidative stress is one of the key insults promoting cell senescence in vivo as well as in vitro, we hypothesized that hydrogen molecules prevent senescent process during MSC expansion. Addition of 3% hydrogen gas enhanced preservation of colony forming early progenitor cells within MSC preparation and prolonged the in vitro replicative lifespan of MSCs without losing differentiation potentials and paracrine capabilities. Interestingly, 3% hydrogen gas treatment did not decrease hydroxyl radical, protein carbonyl, and 8-hydroxydeoxyguanosine, suggesting that scavenging hydroxyl radical might not be responsible for these effects of hydrogen gas in this study.

Epidermal growth factor (EGF) treatment on multipotential stromal cells (MSCs). Possible enhancement of therapeutic potential of MSC.

Adult bone marrow multipotential stromal cells (MSCs) hold great promise in regenerative medicine and tissue engineering. However, due to their low numbers upon harvesting, MSCs need to be expanded in vitro without biasing future differentiation for optimal utility. In this concept paper, we focus on the potential use of epidermal growth factor (EGF), prototypal growth factor for enhancing the harvesting and/or differentiation of MSCs. Soluble EGF was shown to augment MSC proliferation while preserving early progenitors within MSC population, and thus did not induce differentiation. However, tethered form of EGF was shown to promote osteogenic differentiation. Soluble EGF was also shown to increase paracrine secretions including VEGF and HGF from MSC. Thus, soluble EGF can be used not only to expand MSC in vitro, but also to enhance paracrine secretion through drug-releasing MSC-encapsulated scaffolds in vivo. Tethered EGF can also be utilized to direct MSC towards osteogenic lineage both in vitro and in vivo.

Fabrication and characterization of prosurvival growth factor releasing, anisotropic scaffolds for enhanced mesenchymal stem cell survival/growth and orientation.

Scaffolds that not only mimic the mechanical and structural properties of the target tissue but also support cell survival/growth are likely necessary for the development of mechanically functional cardiovascular tissues. To reach these goals, we have generated scaffolds that are elastic to approximate soft tissue mechanical properties, are nanofibrous to mimic fibrous nature of extracellular matrix (ECM), have aligned structure to guide cellular alignment, and are capable of releasing insulin-like growth factor (IGF-1) to administrate cellular growth and survival. We have developed a technique that can quickly fabricate (<3 h) such scaffolds by simultaneously electrospinning elastase-sensitive polyurethaneurea nanofibers, encapsulating IGF-1 into poly(lactide-co-glycolide) (PLGA) microspheres and assembling them into scaffolds. Scaffold morphology, mechanical properties, degradation with or without elastase, and bioactivity of the released IGF-1 were assessed. The scaffolds had degree of alignment approximately 70%. They were flexible and relatively strong, with tensile strengths of 3.4-11.1 MPa, elongations at break of 71-88%, and moduli of 2.3-7.9 MPa at the alignment direction. IGF-1 release profile and bioactivity were dependent on PLGA content and molecular weight and IGF-1 loading. The released IGF-1 remained bioactive for 4 weeks. The fabricated nanofibers were elastase-sensitive with weight remaining <59% after a 4-week degradation in the presence of elastase. Mesenchymal stem cells (MSCs) were seeded on the scaffolds and cultured either under normal culture conditions (21% O(2), 5% CO(2), and 20% fetal bovine serum (FBS)) or hypoxia/nutrient starvation conditions (5% O(2), 5% CO(2), and 1% FBS) to evaluate the effect of IGF-1 loading on cell growth and survival. Under normal culture conditions, MSCs were found to align on the scaffolds with a degree of alignment matching that of the scaffold. The IGF-1 loaded scaffolds enhanced MSC growth during a 7-day culture period, with higher IGF-1 content showing better stimulus effect. Under hypoxia/nutrient starvation conditions, the IGF-1 loaded scaffolds were found to significantly improve MSC survival.

Newly Emerging Drugs of Abuse and Their Detection Methods: An ACLPS Critical Review.

OBJECTIVES: Illicit drug abuse has reached an epidemic level in the United States. Drug overdose has become the leading cause of injury-related deaths since 2008 due to the recent surge of opioid overdose by heroin, controlled prescription drugs, and nonmethadone synthetic opioids. Synthetic designer drugs such as synthetic cathinones ("bath salts") and synthetic cannabinoids ("Spice" and "K2") continue to emerge and attract recreational users. METHODS: The emergence of new drugs of abuse poses a steep challenge for clinical toxicology laboratories. Limited information about the emerging drugs and their metabolism, "rebranding" of the illicit drugs, and a lack of Food and Drug Administration-approved screening methods for these drugs contribute to this difficulty. Here we review detection methods that can aid in identifying emerging drugs of abuse. RESULTS: One promising approach is the utilization of untargeted drug screening by mass spectrometry. Historically, gas chromatography-mass spectrometry has been the gold standard. CONCLUSIONS: Liquid chromatography-tandem mass spectrometry and liquid chromatography-high-resolution mass spectrometry offer improved detection capability of new drugs with simplified sample preparation, making it the new standard.

Spheroid Culture of Mesenchymal Stem Cells.

Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.

Soft Elasticity-Associated Signaling and Bone Morphogenic Protein 2 Are Key Regulators of Mesenchymal Stem Cell Spheroidal Aggregates.

Cell therapy with adult mesenchymal stem cells (MSCs) is a promising approach to regenerative medicine and autoimmune diseases. There are various approaches to improve the efficacy of MSC-based therapeutics, and MSC preparation as spheroidal aggregates, or MSC spheroids, is a novel preparatory and delivery method. Spheroid formation induces a dramatic change in the gene expression profile of MSCs. Self-activation of interleukin-1 (IL1) signaling was shown to be upstream of both pro- and anti-inflammatory genes in MSC spheroids, but the molecular pathways that initiate IL1 signaling remain unknown. As bone morphogenic protein (BMP)2 upregulation precedes that of IL1B expression during spheroid formation, we hypothesized that BMP2 signaling triggers IL1 signaling in MSC spheroids. Contrary to expectations, BMP2 signaling decreased expression of IL1B and downstream genes in a SMAD6-dependent manner. Conversely, IL1B signaling enhanced BMP2 expression. Another major difference between two-dimensional (2D) monolayer culture and three-dimensional (3D) spheroid culture is the Young's elasticity modulus, or stiffness, of the materials surrounding the cells, as there is a million-fold difference between a plastic surface for standard 2D culture (GPa) and 3D spheroidal aggregates (0.1 kPa). We tested another hypothesis that soft elasticity-associated mechano-signaling initiates the gene expression change during spheroid formation. Results showed that both BMP2 expression and inflammatory signaling are upregulated in an elasticity-associated signaling-dependent manner in MSCs. Lastly, BMP2 signaling enhanced cell survival and cell spreading of MSC spheroids. In summary, our study suggests that soft elasticity and BMP2 signaling are critical for MSC spheroids.

Improving Cell Engraftment in Cardiac Stem Cell Therapy.

Myocardial infarction (MI) affects millions of people worldwide. MI causes massive cardiac cell death and heart function decrease. However, heart tissue cannot effectively regenerate by itself. While stem cell therapy has been considered an effective approach for regeneration, the efficacy of cardiac stem cell therapy remains low due to inferior cell engraftment in the infarcted region. This is mainly a result of low cell retention in the tissue and poor cell survival under ischemic, immune rejection and inflammatory conditions. Various approaches have been explored to improve cell engraftment: increase of cell retention using biomaterials as cell carriers; augmentation of cell survival under ischemic conditions by preconditioning cells, genetic modification of cells, and controlled release of growth factors and oxygen; and enhancement of cell survival by protecting cells from excessive inflammation and immune surveillance. In this paper, we review current progress, advantages, disadvantages, and potential solutions of these approaches.