Examining Interventions and Adverse Events After Nonfatal Opioid Overdoses in Veterans.

Background: Veterans are twice as likely to experience a fatal opioid overdose compared with their civilian counterparts. Recognition has increased that effective opioid overdose prevention likely requires a holistic approach that addresses the biopsychosocial factors contributing to opioid-related morbidity and mortality. Methods: This retrospective descriptive study includes veterans who were administered naloxone for treatment of opioid overdose in the emergency department at Veterans Affairs San Diego Healthcare System from July 1, 2013 through April 1, 2017. Subjects were excluded if they received palliative/hospice care or were lost to follow-up, if there was documented lack of response to naloxone administration, and if overdose occurred secondary to inpatient administration of opioids. Data were collected via chart review. Results: Thirty-five patients were included in this study. At the time of nonfatal opioid overdose, 29 (82.9%) had an active opioid prescription, and the mean morphine equivalent daily dose (MEDD) was 117 mg. Thirty-three (94.3%) had comorbid psychiatric disorders and 20 (57.1%) had substance use disorders. Within 6 months following overdose, subjects received care from mental health (45.5%), addiction treatment services (50.0%), and pain management (40.0%). Documented repeat overdose occurred in 4 patients. Conclusions: This study may aid in the identification of potential areas for improvement in the prevention of opioid overdose and opioid-related mortality among veterans. Interventions designed to improve access to, engagement, and retention in effective care are pivotal for addressing the opioid epidemic as it evolves.

Identification of potent small molecule inhibitors of SARS-CoV-2 entry.

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

Drug repurposing screens identify chemical entities for the development of COVID-19 interventions.

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.

Skeletal Muscle Energetics and Mitochondrial Function Are Impaired Following 10 Days of Bed Rest in Older Adults.

BACKGROUND: Older adults exposed to periods of inactivity during hospitalization, illness, or injury lose muscle mass and strength. This, in turn, predisposes poor recovery of physical function upon reambulation and represents a significant health risk for older adults. Bed rest (BR) results in altered skeletal muscle fuel metabolism and loss of oxidative capacity that have recently been linked to the muscle atrophy program. Our primary objective was to explore the effects of BR on mitochondrial energetics in muscle from older adults. A secondary objective was to examine the effect of ß-hydroxy-ß-methylbuturate (HMB) supplementation on mitochondrial energetics. METHODS: We studied 20 older adults before and after a 10-day BR intervention, who consumed a complete oral nutritional supplement (ONS) with HMB (3.0 g/d HMB, n = 11) or without HMB (CON, n = 9). Percutaneous biopsies of the vastus lateralis were obtained to determine mitochondrial respiration and H2O2 emission in permeabilized muscle fibers along with markers of content. RNA sequencing and lipidomics analyses were also conducted. RESULTS: We found a significant up-regulation of collagen synthesis and down-regulation of ribosome, oxidative metabolism and mitochondrial gene transcripts following BR in the CON group. Alterations to these gene transcripts were significantly blunted in the HMB group. Mitochondrial respiration and markers of content were both reduced and H2O2 emission was elevated in both groups following BR. CONCLUSIONS: In summary, 10 days of BR in older adults causes a significant deterioration in mitochondrial energetics, while transcriptomic profiling revealed that some of these negative effects may be attenuated by an ONS containing HMB.

Identification of plicamycin, TG02, panobinostat, lestaurtinib, and GDC-0084 as promising compounds for the treatment of central nervous system infections caused by the free-living amebae Naegleria, Acanthamoeba and Balamuthia.

The free-living amebae Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis. Acanthamoeba can also cause chronic keratitis and both Balamuthia and Acanthamoeba can cause skin and systemic infections. There are minimal drug development pipelines for these pathogens despite a lack of available treatment regimens and high fatality rates. To identify anti-amebic drugs, we screened 159 compounds from a high-value repurposed library against trophozoites of the three amebae. Our efforts identified 38 compounds with activity against at least one ameba. Multiple drugs that bind the ATP-binding pocket of mTOR and PI3K are active, highlighting these compounds as important inhibitors of these parasites. Importantly, 24 active compounds have progressed at least to phase II clinical studies and overall 15 compounds were active against all three amebae. Based on central nervous system (CNS) penetration or exceptional potency against one amebic species, we identified sixteen priority compounds for the treatment of meningoencephalitis caused by these pathogens. The top five compounds are (i) plicamycin, active against all three free-living amebae and previously U.S. Food and Drug Administration (FDA) approved, (ii) TG02, active against all three amebae, (iii and iv) FDA-approved panobinostat and FDA orphan drug lestaurtinib, both highly potent against Naegleria, and (v) GDC-0084, a CNS penetrant mTOR inhibitor, active against at least two of the three amebae. These results set the stage for further investigation of these clinically advanced compounds for treatment of infections caused by the free-living amebae, including treatment of the highly fatal meningoencephalitis.

The ReFRAME library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors.

Several mammarenaviruses, chiefly Lassa virus (LASV) in Western Africa and Junín virus (JUNV) in the Argentine Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The lack of licensed mammarenavirus vaccines and partial efficacy of current anti-mammarenavirus therapy limited to an off-label use of the nucleoside analog ribavirin underscore an unmet need for novel therapeutics to combat human pathogenic mammarenavirus infections. This task can be facilitated by the implementation of "drug repurposing" strategies to reduce the time and resources required to advance identified antiviral drug candidates into the clinic. We screened a drug repurposing library of 11,968 compounds (Repurposing, Focused Rescue and Accelerated Medchem [ReFRAME]) and identified several potent inhibitors of LCMV multiplication that had also strong anti-viral activity against LASV and JUNV. Our findings indicate that enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis, the pro-viral MCL1 apoptosis regulator, BCL2 family member protein and the mitochondrial electron transport complex III, play critical roles in the completion of the mammarenavirus life cycle, suggesting they represent potential druggable targets to counter human pathogenic mammarenavirus infections.

The ReFRAME library as a comprehensive drug repurposing library and its application to the treatment of cryptosporidiosis.

The chemical diversity and known safety profiles of drugs previously tested in humans make them a valuable set of compounds to explore potential therapeutic utility in indications outside those originally targeted, especially neglected tropical diseases. This practice of "drug repurposing" has become commonplace in academic and other nonprofit drug-discovery efforts, with the appeal that significantly less time and resources are required to advance a candidate into the clinic. Here, we report a comprehensive open-access, drug repositioning screening set of 12,000 compounds (termed ReFRAME; Repurposing, Focused Rescue, and Accelerated Medchem) that was assembled by combining three widely used commercial drug competitive intelligence databases (Clarivate Integrity, GVK Excelra GoStar, and Citeline Pharmaprojects), together with extensive patent mining of small molecules that have been dosed in humans. To date, 12,000 compounds (∼80% of compounds identified from data mining) have been purchased or synthesized and subsequently plated for screening. To exemplify its utility, this collection was screened against Cryptosporidium spp., a major cause of childhood diarrhea in the developing world, and two active compounds previously tested in humans for other therapeutic indications were identified. Both compounds, VB-201 and a structurally related analog of ASP-7962, were subsequently shown to be efficacious in animal models of Cryptosporidium infection at clinically relevant doses, based on available human doses. In addition, an open-access data portal (https://reframedb.org) has been developed to share ReFRAME screen hits to encourage additional follow-up and maximize the impact of the ReFRAME screening collection.

Ex Vivo Cell-Based Screening Platform for Modulators of Hepatosteatosis.

Nonalcoholic fatty liver disease (NAFLD) is the result of the ectopic accumulation of lipids in hepatic cells and is the early stage of liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma. While some mechanisms of aberrant lipid storage are understood, unbiased phenotypic drug screening holds the potential to identify new therapeutic small molecule mechanisms that reverse lipid accumulation in hepatic cells and prevent disease progression. Immortalized hepatocyte cell lines are often used as in vitro models of hepatocyte function, including in the study of lipid accumulation. However, mechanisms and therapeutic agents studied in these systems suffer from poor translation to primary cells and animal models of disease. Herein, we report an ex vivo high-throughput screening platform using primary mouse hepatocytes with a physiologically relevant lipid-laden phenotype isolated from mice that are administered a choline-methionine deficient diet. This screening platform using primary diseased hepatocytes may help to overcome a major hurdle in liver disease drug discovery and could lead to the development of new therapeutics for hepatosteatosis.

The Complexity of Pain Management in Patients with Erythromelalgia.

A 15-year-old girl diagnosed with erythromelalgia was admitted to the hospital with severe pain in her feet associated with burning, pruritus, erythema, and swelling. She had not responded to conventional management and received some relief only from cold bath immersions, which resulted in chronic blistering and multiple episodes of superinfection. After a successful trial of spinal cord stimulation, she had a permanent implantation procedure. The spinal cord stimulator relieved her pain and improved function but not the sensation of burning pain. However, this pain resolved after she started daily mexiletine. This case demonstrates that erythromelalgia sometimes can be managed successfully with a combination of pharmacologic and interventional procedures.

Using the RosettaSurface algorithm to predict protein structure at mineral surfaces.

Determination of protein structure on mineral surfaces is necessary to understand biomineralization processes toward better treatment of biomineralization diseases and design of novel protein-synthesized materials. To date, limited atomic-resolution data have hindered experimental structure determination for proteins on mineral surfaces. Molecular simulation represents a complementary approach. In this chapter, we review RosettaSurface, a computational structure prediction-based algorithm designed to broadly sample conformational space to identify low-energy structures. We summarize the computational approaches, the published applications, and the new releases of the code in the Rosetta 3 framework. In addition, we provide a protocol capture to demonstrate the practical steps to employ RosettaSurface. As an example, we provide input files and output data analysis for a previously unstudied mineralization protein, osteocalcin. Finally, we summarize ongoing challenges in energy function optimization and conformational searching and suggest that the fusion between experiment and calculation is the best route forward.

Anti-metastatic activity of biologically synthesized gold nanoparticles on human fibrosarcoma cell line HT-1080.

Plants are exploited as a potential source for the large-scale production of noble gold nanoparticles in the recent years owing to their various potential applications in nanobiotechnology and nanomedicine. The present work describes green biosynthetic procedures for the production of gold nanoparticles for the first time by using an aqueous extract of the Dysosma pleiantha rhizome. The biosynthesized gold nanoparticles were confirmed and characterized by ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and scanning electron microscopy equipped with energy dispersive spectroscopy. The results revealed that aqueous extract of D. pleiantha rhizome has potential to reduce chloroauric ions into gold nanoparticles and the synthesized gold nanoparticles were showed spherical in shape with an average of 127nm. Further, we investigated the anti-metastatic activity of biosynthesized gold nanoparticles against human fibrosarcoma cancer cell line HT-1080. The results showed that the biosynthesized gold nanoparticles were non-toxic to cell proliferation and, also it can inhibit the chemo-attractant cell migration of human fibrosarcoma cancer cell line HT-1080 by interfering the actin polymerization pathway. Thus, the usage of gold nanoparticles biosynthesized from D. pleiantha rhizome can be used as a potential candidate in the drug and gene delivery to metastatic cancer.

Activation tagging in Salvia miltiorrhiza can cause increased leaf size and accumulation of tanshinone I and IIA in its roots.

BACKGROUND: Salvia miltiorrhiza Bunge (Danshen), an important herb in traditional Chinese medicine, is commonly used for treatment of cardiovascular diseases. One of the major bioactive constituents of Danshen, diterpenoid tanshinone, has been proved with pharmacological properties and have the potential to be a new drug candidate against various diseases. In our previous study, we have established an activation tagging mutagenesis (ATM) population of callus lines of S. miltiorrhiza Bunge by Agrobacterium- mediated transformation. RESULTS: In the present study, we have identified ATM transgenic Salvia plant (SH41) with different leaf morphology and more tanshinones in its roots. The transgenic background of SH41 was identified by PCR (using hpt II primers) and Southern blots. PCR analysis showed a single band of hpt II gene and Southern blot analysis showed single insertion in SH41. External appearance of ATM transgenic SH41 was observed with broader leaves comparing to non-transformed plants. More healthy trichomes as well as bigger and wobbly guard cells and stomata were observed in SH41 by scanning electron microscopy (SEM). Quantitative analysis of active compounds in SH41 roots revealed a significant increase in tanshinone I (3.7 fold) and tanshinone IIA (2 fold) contents as compared to the wild plant. CONCLUSIONS: We have generated an activation tagged transgenic Salvia plant (SH41) with different leaf morphology and high diterpenes content in its roots. The increased amount of tanshinones in SH41 will definitely offer a route for maximizing the benefits of this plant in traditional Chinese herbal medicines. The present report may also facilitate the application of ATM for genetic manipulation of other medicinal crops and subsequent improved metabolite contents.

Comparative analysis among three Taiwan-specific Gentiana species and Chinese medicinal plant Gentiana scabra.

BACKGROUND: The root of Gentiana scabra is commonly known as Longdan in Chinese herbal medicines and has been used in the treatment of inflammation, anorexia, indigestion and gastric infections for over 2000 years. High market demand had made G. scabra (GS) plants not to be the only source of Longdan in China, other Gentiana spp., G. triflora, G. manshurica and G. rigescens, were also recognized as Longdan in China now. RESULTS: In this study, we identified three Taiwan-specific Gentiana spp., G. davidii var. formosana (GDF) and G. arisanensis (GA) and G. scabrida var. punctulata (GSP) that are phylogenetically different from GS (main source of Longdan). However, the active compounds of Longdan, gentiopicroside and swertiamari, were found in GSP and GDF showed higher antioxidant ability and free radical scavenging activities than Chinese Longdan. This discovery might explore the medicinal potential of GDF. Meanwhile, another Taiwan-specific Gentiana spp., GSP, was found to have the strongest antioxidant ability and free radical scavenging activities which might suggest a possible use of GSP as a source of natural antioxidant agents for industrial purpose. CONCLUSIONS: The finding of this study indicated that ITS analysis can be used to identify Taiwan-specific Gentiana spp. Also the Taiwan-specific Gentiana spp. which has strongest antioxidant and free radical scavenging activities among others could be a better choice for industrial purpose.

Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures­as typified by DNA methylation or histone modification at silent pluripotency loci­and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming.

Wild bitter gourd improves metabolic syndrome: a preliminary dietary supplementation trial.

BACKGROUND: Bitter gourd (Momordica charantia L.) is a common tropical vegetable that has been used in traditional or folk medicine to treat diabetes. Wild bitter gourd (WBG) ameliorated metabolic syndrome (MetS) in animal models. We aimed to preliminarily evaluate the effect of WBG supplementation on MetS in Taiwanese adults. METHODS: A preliminary open-label uncontrolled supplementation trial was conducted in eligible fulfilled the diagnosis of MetS from May 2008 to April 2009. A total of 42 eligible (21 men and 21 women) with a mean age of 45.7 ± 11.4 years (23 to 63 years) were supplemented with 4.8 gram lyophilized WBG powder in capsules daily for three months and were checked for MetS at enrollment and follow-up monthly. After supplementation was ceased, the participants were continually checked for MetS monthly over an additional three-month period. MetS incidence rate were analyzed using repeated-measures generalized linear mixed models according to the intention-to-treat principle. RESULTS: After adjusting for sex and age, the MetS incidence rate (standard error, p value) decreased by 7.1% (3.7%, 0.920), 9.5% (4.3%, 0.451), 19.0% (5.7%, 0.021), 16.7% (5.4%, 0.047), 11.9% (4.7%, 0.229) and 11.9% (4.7%, 0.229) at visit 2, 3, 4, 5, 6, and 7 compared to that at baseline (visit 1), respectively. The decrease in incidence rate was highest at the end of the three-month supplementation period and it was significantly different from that at baseline (p = 0.021). The difference remained significant at end of the 4th month (one month after the cessation of supplementation) (p = 0.047) but the effect diminished at the 5th and 6th months after baseline. The waist circumference also significantly decreased after the supplementation (p < 0.05). The WBG supplementation was generally well-tolerated. CONCLUSION: This is the first report to show that WBG improved MetS in human which provides a firm base for further randomized controlled trials to evaluate the efficacy of WBG supplementation.

Pain Management Needs Assessment: A Survey of Radiation Therapists at a Large Academic Comprehensive Cancer Centre.

OBJECTIVE: To determine the knowledge of pain management among the radiation therapists (RTs) at the Odette Cancer Centre (OCC) to aid in the development of a formalized education strategy. METHODS: A needs assessment survey comprising eight topics pertaining to pain management was distributed to 130 RTs at the OCC. Survey topics were ranked using a 4-point Likert scale based on preference for further education, familiarity with the topic, and relevance to practice. RESULTS: RTs rated topics pertaining to the undertreatment, pathophysiology, assessment, diagnosis, and treatment of pain as the most relevant topics requiring further education. RTs were most unfamiliar with topics concerning opioids and addiction, but did not find a need for further education. They also felt that breakthrough cancer pain was the most significant topic for further education. CONCLUSION: Implementation of an educational intervention for RTs to more effectively and efficiently address pain management for their patient population is needed. Topics of most clinical relevance include: undertreatment of pain, pathophysiology of pain, assessment and diagnosis of acute and chronic pain as well as its treatment.

A comparison of the arthritis foundation self-help program across three race/ethnicity groups.

OBJECTIVE: Despite high prevalence rates of pain among older adults, relatively few studies have examined the impact of the Arthritis Foundation Self-Help Program (ASHP) in this age group, particularly older minority groups. DESIGN: This study compared the effects of the ASHP on groups of Hispanic, African American and non-Hispanic White older adults. SETTING: Three senior centers in New York City. PARTICIPANTS: Data are presented for 112 (37 African American, 38 Hispanic and 37 non-Hispanic White) participants (mean age= 75 years) age 60 and over with diverse noncancer pain disorders. INTERVENTION: Participants enrolled in the 6-week Arthritis Self Help Course. MAIN OUTCOME MEASURES: Participants were surveyed before and after course completion (in person) and at 18 weeks (by telephone). Demographic and clinical data were collected at baseline; outcomes included pain, mood, self-efficacy, and number of days per week spent exercising. RESULTS: All three groups experienced significant decreases in pain intensity (P< or =.05). Significant improvements were also found in mood scores for non-Hispanic White (P=.01) and Hispanic participants (P=.03). Hispanic participants also evidenced significant improvement in their confidence to self-manage pain (P=.003) and reported fewer arthritis-related symptoms (P=.02). All three race/ethnicity groups reported substantial increases in the number of days spent doing stretching, endurance and relaxation exercises (P< or =.01). CONCLUSION: Positive results were noted for all three race/ethnicity groups, particularly in the areas of pain reduction and uptake of stretching, endurance and relaxation exercises. Our findings support efforts to disseminate broadly the ASHP in community settings that serve older African American, Hispanic and non-Hispanic white adults.

A role for p11 in the antidepressant action of brain-derived neurotrophic factor.

BACKGROUND: The protein p11 (also called S100A10) is downregulated in human and rodent depressive-like states. Considerable experimental evidence also implicates p11 in the mechanism of action of antidepressant drugs and electroconvulsive seizures, in part due to its interaction with specific serotonin receptors. Brain-derived neurotrophic factor (BDNF) has been linked to the therapeutic activity of antidepressants in rodent models and humans. In the current study, we investigated whether BDNF regulates p11 in vitro and in vivo. METHODS: We utilized primary neuronal cultures, in vivo analyses of transgenic mice, and behavioral techniques to assess the effects of BDNF on p11. RESULTS: Results indicate that BDNF stimulates p11 expression through tropomyosin-related kinase B (trkB) receptors and via the mitogen-activated protein kinase signaling pathway. Brain-derived neurotrophic factor-induced changes in p11 in vivo correlate with changes in ligand binding to the 5-hydroxytryptamine receptor 1B, the subcellular localization of which is known to be regulated by p11. Behavioral studies demonstrate that p11 knockout mice are insensitive to the antidepressant actions of BDNF. CONCLUSIONS: Taken together, our data demonstrate that p11 levels are regulated by BDNF in vitro and in vivo and that the antidepressant-like effect of BDNF in two well-established behavioral models requires p11. These data support a role for p11 in the antidepressant activity of neurotrophins.