Vps18 contributes to phagosome membrane integrity in Mycobacterium tuberculosis-infected macrophages.

Mycobacterium tuberculosis (Mtb) has evolved to be exquisitely adapted to survive within host macrophages. The capacity to damage the phagosomal membrane has emerged as central to Mtb virulence. While Mtb factors driving membrane damage have been described, host factors that repair that damage to contain the pathogen remain largely unknown. We used a genome-wide CRISPR screen to identify novel host factors required to repair Mtb-damaged phagosomal membranes. Vacuolar protein sorting-associated protein 18 (Vps18), a member of the HOPS and CORVET trafficking complexes, was among the top hits. Vps18 colocalized with Mtb in macrophages beginning shortly after infection, and Vps18-knockout macrophages demonstrated increased damage of Mtb-containing phagosomes without impaired autophagy. Mtb grew more robustly in Vps18-knockout cells, and the first-line anti-tuberculosis antibiotic pyrazinamide was less effective. Our results identify Vps18 as required for phagosomal membrane integrity in Mtb-infected cells and suggest that modulating phagosome integrity may hold promise for improving the efficacy of antibiotic treatment for TB.

The response to stressors in adulthood depends on the interaction between prenatal exposure to glucocorticoids and environmental context.

Maternal stress during reproduction can influence how offspring respond to stress later in life. Greater lifetime exposure to glucocorticoid hormones released during stress is linked to greater risks of behavioral disorders, disease susceptibility, and mortality. The immense variation in individual's stress responses is explained, in part, by prenatal glucocorticoid exposure. To explore the long-term effects of embryonic glucocorticoid exposure, we injected Japanese quail (Coturnix japonica) eggs with corticosterone. We characterized the endocrine stress response in offspring and measured experienced aggression at three different ages. We found that prenatal glucocorticoid exposure affected (1) the speed at which the stress response was terminated suggesting dysregulated negative feedback, (2) baseline corticosterone levels in a manner dependent on current environmental conditions with higher levels of experienced aggression associated with higher levels of baseline corticosterone, (3) the magnitude of an acute stress response based on baseline concentrations. We finish by proposing a framework that can be used to test these findings in future work. Overall, our findings suggest that the potential adaptive nature of prenatal glucocorticoid exposure is likely dependent on environmental context and may also be tempered by the negative effects of longer exposure to glucocorticoids each time an animal faces a stressor.

Methylation of genes and regulation of inflammatory processes on emotional response in young adults with alcoholic parents.

Many Americans are adult children of an alcoholic parent (ACoA), which can confer an increased risk of trauma and hazardous alcohol use, as well as heritable and environmental genetic influence. Psychological health and related neural activity can be influenced by inflammation responses, but it is not clear how these factors interact regarding risk or resilience to hazardous alcohol use. The goals of this study were to better understand the relationships between current alcohol use and inflammation, how these are modified by single nucleotide polymorphisms (SNPs) and/or epigenetic modifications of inflammation-associated genes; and how these alter neural reactivity to emotionally-salient stimuli. To do so, ACoA participants were dichotomized as resilient (not engaged in hazardous alcohol use) or vulnerable (currently engaged in hazardous alcohol use). Measures of blood-oxygen-level-dependent (BOLD) activity within regions of interest (ROIs), SNPs and DNA methylation of specific inflammation regulatory genes, and biological markers of inflammation were compared between these groups. Vulnerable ACoAs exhibited higher plasma C-reactive protein (CRP) and greater BOLD activity in the right hippocampus and ventral anterior cingulate cortex in response to emotional cues as well as reduced methylation of CRP and glucocorticoid-related genes. Path analysis revealed significant relationships between alcohol use, SNPs, DNA methylation of inflammatory-related genes, CRP levels, and BOLD activity to emotional stimuli. Taken together, these findings suggest a complex association related to hazardous alcohol use in ACoAs that may predict current inflammation and neural reactivity to emotional stimuli. A better understanding of these associations could direct the future of individual treatment options.

Obesity and exercise training alter inflammatory pathway skeletal muscle small extracellular vesicle microRNAs.

NEW FINDINGS: What is the central question of this study? Is 1 week of exercise training sufficient to reduce local and systemic inflammation? Do obesity and short-term concurrent aerobic and resistance exercise training alter skeletal muscle extracellular vesicle (EV) contents? What is the main finding and its importance? Obesity alters skeletal muscle small EV microRNAs targeting inflammatory and growth pathways. Exercise training alters skeletal muscle small EV microRNAs targeting inflammatory pathways, indicative of reduced inflammation. Our findings provide support for the hypotheses that EVs play a vital role in intercellular communication during health and disease and that EVs mediate many of the beneficial effects of exercise. ABSTRACT: Obesity is associated with chronic inflammation characterized by increased levels of inflammatory cytokines, whereas exercise training reduces inflammation. Small extracellular vesicles (EVs; 30-150 nm) participate in cell-to-cell communication in part through microRNA (miRNA) post-transcriptional regulation of mRNA. We examined whether obesity and concurrent aerobic and resistance exercise training alter skeletal muscle EV miRNA content and inflammatory signalling. Vastus lateralis biopsies were obtained from sedentary individuals with (OB) and without obesity (LN). Before and after 7 days of concurrent aerobic and resistance training, muscle-derived small EV miRNAs and whole-muscle mRNAs were measured. Pathway analysis revealed that obesity alters small EV miRNAs that target inflammatory (SERPINF1, death receptor and Gαi ) and growth pathways (Wnt/ß-catenin, PTEN, PI3K/AKT and IGF-1). In addition, exercise training alters small EV miRNAs in an anti-inflammatory manner, targeting the IL-10, IL-8, Toll-like receptor and nuclear factor-κB signalling pathways. In whole muscle, IL-8 mRNA was reduced by 50% and Jun mRNA by 25% after exercise training, consistent with the anti-inflammatory effects of exercise on skeletal muscle. Obesity and 7 days of concurrent exercise training differentially alter skeletal muscle-derived small EV miRNA contents targeting inflammatory and anabolic pathways.

BDNF rs6265 Genotype Influences Outcomes of Pharmacotherapy and Subthalamic Nucleus Deep Brain Stimulation in Early-Stage Parkinson's Disease.

INTRODUCTION: The efficacy of pharmacotherapy and deep brain stimulation of the subthalamic nucleus in treating Parkinson's disease motor symptoms is highly variable and may be influenced by patient genotype. The relatively common (prevalence about one in three) and protein-altering rs6265 single nucleotide polymorphism (C > T) in the gene BDNF has been associated with different clinical outcomes with levodopa. OBJECTIVE: We sought to replicate this reported association in early-stage Parkinson's disease subjects and to examine whether a difference in clinical outcomes was present with subthalamic nucleus deep brain stimulation. MATERIALS AND METHODS: Fifteen deep brain stimulation and 13 medical therapy subjects were followed for 24 months as part of the Vanderbilt DBS in Early Stage PD clinical trial (NCT00282152, FDA IDE #G050016). Primary outcome measures were the Unified Parkinson's Disease Rating Scale (UPDRS) and Parkinson's Disease Questionnaire-39. RESULTS: Outcomes with drug therapy in subjects carrying the rs6265 T allele were significantly worse following 12 months of treatment compared to C/C subjects (UPDRS: +20 points, p = 0.019; PDQ-39: +16 points, p = 0.018). In contrast, rs6265 genotype had no effect on overall motor response to subthalamic nucleus deep brain stimulation at any time point; further, rs6265 C/C subjects treated with stimulation were associated with worse UPDRS part II scores at 24 months compared to medical therapy. CONCLUSIONS: Genotyping for the rs6265 polymorphism may be useful for predicting long-term response to drug therapy and counseling Parkinson's disease patients regarding whether to consider earlier subthalamic nucleus deep brain stimulation. Validation in a larger cohort of early-stage Parkinson's disease subjects is warranted.

Research led by people who use drugs: centering the expertise of lived experience.

BACKGROUND: Research collaborations between people who use drugs (PWUD) and researchers are largely underutilized, despite the long history of successful, community-led harm reduction interventions and growing health disparities experienced by PWUD. PWUD play a critical role in identifying emerging issues in the drug market, as well as associated health behaviors and outcomes. As such, PWUD are well positioned to meaningfully participate in all aspects of the research process, including population of research questions, conceptualization of study design, and contextualization of findings. MAIN BODY: We argue PWUD embody unparalleled and current insight to drug use behaviors, including understanding of novel synthetic drug bodies and the dynamics at play in the drug market; they also hold intimate and trusting relationships with other PWUD. This perfectly situates PWUD to collaborate with researchers in investigation of drug use behaviors and development of harm reduction interventions. While PWUD have a history of mistrust with the medical community, community-led harm reduction organizations have earned their trust and are uniquely poised to facilitate research projects. We offer the North Carolina Survivors Union as one such example, having successfully conducted a number of projects with reputable research institutions. We also detail the fallacy of meaningful engagement posed by traditional mechanisms of capturing community voice. As a counter, we detail the framework developed and implemented by the union in hopes it may serve as guidance for other community-led organizations. We also situate research as a mechanism to diversify the job opportunities available to PWUD and offer a real-time example of the integration of these principles into public policy and direct service provision. CONCLUSION: In order to effectively mitigate the risks posed by the fluid and volatile drug market, research collaborations must empower PWUD to play meaningful roles in the entirety of the research process. Historically, the most effective harm reduction interventions have been born of the innovation and heart possessed by PWUD; during the current overdose crisis, there is no reason to believe they will not continue to be.

Treatment experiences for skin and soft tissue infections among participants of syringe service programs in North Carolina.

INTRODUCTION: Bacterial and fungal infections, such as skin and soft tissue infections (SSTIs) and infective endocarditis (IE), are increasing among people who use drugs in the United States. Traditional healthcare settings can be inaccessible and unwelcoming to people who use drugs, leading to delays in getting necessary care. The objective of this study was to examine SSTI treatment experiences among people utilizing services from syringe services programs. This study was initiated by people with lived experience of drug use to improve quality of care. METHODS: We conducted a cross-sectional survey among participants of five syringe services programs in North Carolina from July through September 2020. Surveys collected information on each participant's history of SSTIs and IE, drug use and healthcare access characteristics, and SSTI treatment experiences. We examined participant characteristics using counts and percentages. We also examined associations between participant characteristics and SSTI history using binomial linear regression models. RESULTS: Overall, 46% of participants reported an SSTI in the previous 12 months and 10% reported having IE in the previous 12 months. Those with a doctor they trusted with drug use-related concerns had 27 fewer (95% confidence interval = - 51.8, - 2.1) SSTIs per every 100 participants compared to those without a trusted doctor. Most participants with a SSTI history reported delaying (98%) or not seeking treatment (72%) for their infections. Concerns surrounding judgment or mistreatment by medical staff and self-treating the infection were common reasons for delaying or not seeking care. 13% of participants used antibiotics obtained from sources other than a medical provider to treat their most recent SSTI. Many participants suggested increased access to free antibiotics and on-site clinical care based at syringe service programs to improve treatment for SSTIs. CONCLUSIONS: Many participants had delayed or not received care for SSTIs due to poor healthcare experiences. However, having a trusted doctor was associated with fewer people with SSTIs. Improved access to non-judgmental healthcare for people who use drugs with SSTIs is needed. Expansion of syringe services program-based SSTI prevention and treatment programs is likely a necessary approach to improve outcomes among those with SSTI and IE.

Tenofovir Alafenamide for HIV Prevention: Review of the Proceedings from the Gates Foundation Long-Acting TAF Product Development Meeting.

The ability to successfully develop a safe and effective vaccine for the prevention of HIV infection has proven challenging. Consequently, alternative approaches to HIV infection prevention have been pursued, and there have been a number of successes with differing levels of efficacy. At present, only two oral preexposure prophylaxis (PrEP) products are available, Truvada and Descovy. Descovy is a newer product not yet indicated in individuals at risk of HIV-1 infection from receptive vaginal sex, because it still needs to be evaluated in this population. A topical dapivirine vaginal ring is currently under regulatory review, and a long-acting (LA) injectable cabotegravir product shows strong promise. Although demonstrably effective, daily oral PrEP presents adherence challenges for many users, particularly adolescent girls and young women, key target populations. This limitation has triggered development efforts in LA HIV prevention options. This article reviews efforts supported by the Bill & Melinda Gates Foundation, as well as similar work by other groups, to identify and develop optimal LA HIV prevention products. Specifically, this article is a summary review of a meeting convened by the foundation in early 2020 that focused on the development of LA products designed for extended delivery of tenofovir alafenamide (TAF) for HIV prevention. The review broadly serves as technical guidance for preclinical development of LA HIV prevention products. The meeting examined the technical feasibility of multiple delivery technologies, in vivo pharmacokinetics, and safety of subcutaneous (SC) delivery of TAF in animal models. Ultimately, the foundation concluded that there are technologies available for long-term delivery of TAF. However, because of potentially limited efficacy and possible toxicity issues with SC delivery, the foundation will not continue investing in the development of LA, SC delivery of TAF products for HIV prevention.

Consumer discernment of fentanyl in illicit opioids confirmed by fentanyl test strips: Lessons from a syringe services program in North Carolina.

BACKGROUND: The United States (U.S.) continues to witness an unprecedented increase in opioid overdose deaths driven by precipitous growth in the supply and use of illicitly-manufactured fentanyls (IMF). Fentanyl's growing market share of the illicit opioid supply in the U.S. has led to seismic shifts in the composition of the country's heroin supply. The growth in fentanyl supply has transformed illicit opioid markets once offering heroin with fairly consistent purity and potency to a supply overpopulated with fentanyl(s) of inconsistent and unpredictable potency. In response, people who inject drugs (PWID) have developed a number of sensory strategies to detect fentanyl in illicit opioids. The current study examined the accuracy of sensory discernment strategies by measuring study participants' descriptions of the last opioid injected and checked with a fentanyl test strip (FTS) by that test's positive/negative result. The primary objective was to determine associations between FTS results and descriptions of the illicit opioid's physical appearance and physiological effects. METHODS: Between September-October 2017, a total of 129 PWID were recruited from a syringe services program in Greensboro, North Carolina and completed an online survey about their most recent use of FTS. Participants were instructed to describe the appearance and effects associated with the most recent opioid they injected and tested with FTS. We conducted bivariate and multivariate analyses to determine differences in positive vs negative FTS results and the physical characteristics and physiological experiences reported. An exploratory analysis was also conducted to describe the types and bodily locations of unusual sensations experienced by PWID reporting positive FTS results. RESULTS: For physical characteristics, 32% reported that the drug was white before adding water and 38% reported the solution was clear after adding water. For physiological effects compared to heroin, 42% reported a stronger rush, 30% a shorter high, 30% a shorter time to the onset of withdrawal symptoms, and 42% experienced unusual sensations. In the multivariable model adjusting for demographics and polydrug correlates, white color of drug before adding water, stronger rush, shorter time to withdrawal, and unusual sensations were significantly associated with a positive FTS result. The most common unusual sensations were pins and needles (51%), warming of the head and face (35%), and lightheadedness (30%), and the most common locations where sensations occurred were face and neck (61%), arms/legs (54%), and chest (37%). CONCLUSION: We found positive FTS results were significantly associated with the physical characteristics and physiological effects described by PWID. Descriptions concerning physical appearance were consistent with law enforcement profiles of illicitly-manufactured fentanyl and physiological effects were concomitant with scientific and clinical medical literature on iatrogenic fentanyl use. Taken together, these findings suggest sensory strategies for detecting fentanyl in illicit opioids may be an effective risk reduction tool to help consumers navigate unpredictable markets more safely.

High-Throughput Rapid Yeast Chronological Lifespan Assay.

The budding yeast is a valuable model system for discovering molecular mechanisms underlying cellular aging. This is due to the ease of performing genetic manipulations in yeast and the vast number of evolutionarily conserved genes that have been found to regulate cellular health and lifespan from yeast to humans. Lifespan assays are an essential tool for examining the effects of these genes on longevity. There are two ways lifespan is measured in yeast: replicative lifespan (RLS) and chronological lifespan (CLS). RLS is a measure of how many divisions an individual mother cell will undergo. CLS measures the length of time nondividing cells survive. Previously described CLS assays involved diluting and plating cells of a culture and counting the colonies that arose. While effective, this method is both time and labor intensive. Here, we describe a method for a high-throughput rapid CLS assay that is both time- and cost-efficient.

Performance and Stability of Tenofovir Alafenamide Formulations within Subcutaneous Biodegradable Implants for HIV Pre-Exposure Prophylaxis (PrEP).

A critical need exists to develop diverse biomedical strategies for the widespread use of HIV Pre-Exposure Prophylaxis (HIV PrEP). This manuscript describes a subcutaneous reservoir-style implant for long-acting delivery of tenofovir alafenamide (TAF) for HIV PrEP. We detail key parameters of the TAF formulation that affect implant performance, including TAF ionization form, the selection of excipient and the exposure to aqueous conditions. Both in-vitro studies and shelf stability tests demonstrate enhanced performance for TAF freebase (TAFFB) in this long-acting implant platform, as TAFFB maintains higher chemical stability than the TAF hemifumarate salt (TAFHF). We also examined the hydrolytic degradation profiles of various formulations of TAF and identified inflection points for the onset of the accelerated drug hydrolysis within the implant using a two-line model. The compositions of unstable formulations are characterized by liquid chromatography-mass spectrometry (LC-MS) and are correlated to predominant products of the TAF hydrolytic pathways. The hydrolysis rate of TAF is affected by pH and water content in the implant microenvironment. We further demonstrate the ability to substantially delay the degradation of TAF by reducing the rates of drug release and thus lowering the water ingress rate. Using this approach, we achieved sustained release of TAFFB formulations over 240 days and maintained > 93% TAF purity under simulated physiological conditions. The opportunities for optimization of TAF formulations in this biodegradable implant supports further advancement of strategies to address long-acting HIV PrEP.

Enhancing myocardial repair with CardioClusters.

Cellular therapy to treat heart failure is an ongoing focus of intense research, but progress toward structural and functional recovery remains modest. Engineered augmentation of established cellular effectors overcomes impediments to enhance reparative activity. Such 'next generation' implementation includes delivery of combinatorial cell populations exerting synergistic effects. Concurrent isolation and expansion of three distinct cardiac-derived interstitial cell types from human heart tissue, previously reported by our group, prompted design of a 3D structure that maximizes cellular interaction, allows for defined cell ratios, controls size, enables injectability, and minimizes cell loss. Herein, mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and c-Kit+ cardiac interstitial cells (cCICs) when cultured together spontaneously form scaffold-free 3D microenvironments termed CardioClusters. scRNA-Seq profiling reveals CardioCluster expression of stem cell-relevant factors, adhesion/extracellular-matrix molecules, and cytokines, while maintaining a more native transcriptome similar to endogenous cardiac cells. CardioCluster intramyocardial delivery improves cell retention and capillary density with preservation of cardiomyocyte size and long-term cardiac function in a murine infarction model followed 20 weeks. CardioCluster utilization in this preclinical setting establish fundamental insights, laying the framework for optimization in cell-based therapeutics intended to mitigate cardiomyopathic damage.

Predicting Pharmacokinetics of a Tenofovir Alafenamide Subcutaneous Implant Using Physiologically Based Pharmacokinetic Modelling.

Long-acting (LA) administration using a subcutaneous (s.c.) implant presents opportunities to simplify administration of antiretroviral drugs, improve pharmacological profiles, and overcome suboptimal adherence associated with daily oral formulations. Tenofovir alafenamide (TAF) is a highly potent nucleoside reverse transcriptase inhibitor (NRTI) and an attractive agent for LA delivery, with a high potency and long intracellular half-life. The aim of this study was to predict minimum TAF doses required to achieve concentrations effective for HIV preexposure prophylaxis (PrEP). Daily drug release requirements were then ascertained by averaging across the dosing interval. A TAF physiologically based pharmacokinetic (PBPK) model was developed and partially qualified against available oral single- and multiple-dose pharmacokinetics. The models were assumed to be qualified when simulated values were within 2-fold of the observed mean. TAF s.c. implants were simulated in five hundred individuals, reporting predicted TAF plasma and tenofovir (TFV) plasma concentrations for various release rates. Intracellular TFV diphosphate (TFV-DP) concentrations were also simulated in peripheral blood cells and cervical and rectal tissues. The minimum dose predicted to achieve intracellular TFV-DP levels above a target concentration of 48 fmol/106 cells for a month was identified. TAF, TFV, and TFV-DP concentrations for release rates between 1.0 and 1.6 mg/day were simulated. The PBPK model indicated that a minimum release of 1.4 mg/day TAF is necessary to achieve TFV-DP concentrations above the identified target in peripheral blood mononuclear cells (PBMCs). TFV-DP cervical and rectal tissue concentrations were predicted to be between 1.5 and 2.0 fmol/106 cells and 0.9 and 1.1 fmol/106 cells, respectively, for release rates between 1.3 and 1.6 mg/day. These simulations provide target minimum doses for LA TAF PrEP in humans. Based on the generated results, multiple implants delivering a total of 1.4 mg/day of TAF subcutaneously could provide protection levels for approximately 6 months to 1 year. This modeling may inform future design of s.c. implants to mitigate adherence issues for effective PrEP applications.

Characterization of a Reservoir-Style Implant for Sustained Release of Tenofovir Alafenamide (TAF) for HIV Pre-Exposure Prophylaxis (PrEP).

Long-acting (LA) HIV pre-exposure prophylaxis (PrEP) offers the potential to improve adherence by lowering the burden of daily or on-demand regimens of antiretroviral (ARV) drugs. This paper details the fabrication and in vitro performance of a subcutaneous and trocar-compatible implant for the LA delivery of tenofovir alafenamide (TAF). The reservoir-style implant comprises an extruded tube of a biodegradable polymer, poly(ε-caprolactone) (PCL), filled with a formulation of TAF and castor oil excipient. Parameters that affect the daily release rates of TAF are described, including the surface area of the implant, the thickness of the PCL tube walls (between 45 and 200 µm), and the properties of the PCL (e.g., crystallinity). In vitro studies show a linear relationship between daily release rates and surface area, demonstrating a membrane-controlled release mechanism from extruded PCL tubes. Release rates of TAF from the implant are inversely proportional to the wall thickness, with release rates between approximately 0.91 and 0.15 mg/day for 45 and 200 µm, respectively. The sustained release of TAF at 0.28 ± 0.06 mg/day over the course of 180 days in vitro was achieved. Progress in the development of this implant platform addresses the need for new biomedical approaches to the LA delivery of ARV drugs.

Bovine leukemia virus discovered in human blood.

BACKGROUND: Bovine leukemia virus (BLV) infection is widespread in cattle globally and is present in marketed beef and dairy products. Human infection with BLV has been reported in breast and lung cancer tissues and was significantly associated with breast cancer in 3 case-control studies. The purpose of this current research was to determine if BLV is present in human blood cells and if antibodies to BLV are related to blood cell infection. METHODS: Standard liquid PCR and Sanger DNA sequencing were used to test for BLV in buffy coat cells (leukocytes and platelets) of blood specimens from 95 self-selected female subjects. Enzyme-linked immunosorbent assay (ELISA) for IgG, IgM, and IgA was used to detect antibodies to BLV in the plasma of the corresponding blood samples. RESULTS: BLV DNA was detected in the buffy coat cells of blood in 33/95 (38%) of the subjects by PCR and DNA sequencing. IgG antibodies were detected in 30/95(32%), IgM in 55/95(58%), and IgA in 30/95(32%) of the subjects. There was no significant correlation between presence of the antibodies and presence of BLV DNA. CONCLUSIONS: This first report of BLV in human blood raises the question of whether infection of leukocytes could conceivably lead to leukemia as it does in infected cattle. Also, system wide circulation of infected blood cells could facilitate BLV transit to various internal tissues/organs with potential for their infection and subsequent development of cancer. The most likely route of BLV transmission to humans would be zoonotic, as a foodborne infection. Although eradicated from cattle in some countries, BLV still has a high rate of infection in the Americas, the Middle East, and parts of Europe and Asia. This report of BLV in the blood layer containing human leukocytes/platelets adds important information which could be useful to elucidate possible routes of transmission of BLV to humans and to prevent further human infection.

New Food and Drug Administration-Approved and Emerging Novel Treatment Options for Acute Myeloid Leukemia.

Advancements in the treatment of acute myeloid leukemia (AML) have been sparse during the past several decades, and the disease continues to have a poor prognosis. However, in 2017 alone, four new medications approved by the U.S. Food and Drug Administration (FDA) for the treatment of AML reached the market. Midostaurin, liposomal cytarabine and daunorubicin, enasidenib, and gemtuzumab ogozamicin all showed benefit in respective clinical trials to gain approval for the treatment of AML in various patient populations. Additionally, many phase II and III clinical trials are currently ongoing to assess the safety and efficacy of other potential therapies for the treatment of AML. In this review, we summarize the results of the landmark clinical trials associated with the newly approved agents as well as the current ongoing clinical trials for the treatment of AML. A literature search was performed to retrieve data on agents currently being studied for use. Although the overall prognosis for patients with AML remains poor, the addition of the newly FDA-approved medications is a step in the right direction for a disease state that has proved difficult to treat.

Rapid Nuclear Exclusion of Hcm1 in Aging Saccharomyces cerevisiae Leads to Vacuolar Alkalization and Replicative Senescence.

The yeast, Saccharomyces cerevisiae, like other higher eukaryotes, undergo a finite number of cell divisions before exiting the cell cycle due to the effects of aging. Here, we show that yeast aging begins with the nuclear exclusion of Hcm1 in young cells, resulting in loss of acidic vacuoles. Autophagy is required for healthy aging in yeast, with proteins targeted for turnover by autophagy directed to the vacuole. Consistent with this, vacuolar acidity is necessary for vacuolar function and yeast longevity. Using yeast genetics and immunofluorescence microscopy, we confirm that vacuolar acidity plays a critical role in cell health and lifespan, and is potentially maintained by a series of Forkhead Box (Fox) transcription factors. An interconnected transcriptional network involving the Fox proteins (Fkh1, Fkh2 and Hcm1) are required for transcription of v-ATPase subunits and vacuolar acidity. As cells age, Hcm1 is rapidly excluded from the nucleus in young cells, blocking the expression of Hcm1 targets (Fkh1 and Fkh2), leading to loss of v-ATPase gene expression, reduced vacuolar acidification, increased α-syn-GFP vacuolar accumulation, and finally, diminished replicative lifespan (RLS). Loss of vacuolar acidity occurs about the same time as Hcm1 nuclear exclusion and is conserved; we have recently demonstrated that lysosomal alkalization similarly contributes to aging in C. elegans following a transition from progeny producing to post-reproductive life. Our data points to a molecular mechanism regulating vacuolar acidity that signals the end of RLS when acidification is lost.

Synthesis and redox reactions of bis(verdazyl)palladium complexes.

The synthesis and ligand-centered redox chemistry of palladium complexes bearing two potentially bidentate verdazyl ligands is explored. Reaction of 1,5-diisopropyl-3-pyridin-2-yl-6-oxoverdazyl radical 1 with Pd(NCMe)4·2BF4 gives a complex containing two coordinated verdazyl radicals. The structure of this complex consists of one verdazyl bound to Pd in a bidentate mode and the second verdazyl bound in a monodentate fashion through the pyridine substituent; the fourth coordination site is occupied by a solvent molecule (acetonitrile (3) or dimethyl sulfoxide (4)). Two-electron reduction of this complex with decamethylferrocene affords a bis(verdazyl) palladium complex (5) in which both verdazyls have been reduced to their anionic state and are both bound to Pd in bidentate manner. Complex 5 can be independently synthesized by a redox reaction between 1 and Pd2(dba)3. Reduced complex 5 can be re-oxidized to 3 or 4 with AgBF4; in contrast, oxidation with PhICl2 leads to ligand dissociation, ultimately giving radical 1 and a mono(verdazyl)dichloropalladium complex 2. One-electron oxidation using PhICl2 produces a formally "mixed valent" (in ligand) bis(verdazyl)chloropalladium complex (6) with one bidentate verdazyl anion ligand and one monodentate (pyridine-bound) verdazyl radical. Attempted protonation of the verdazyl ligands in complex 5 leads to complete ligand dissociation and protonation of both the tetrazine and pyridine moieties; deprotonation regenerates 5. Subsequent air oxidation of the tetrazane/pyridinium cation (formed as a tetrachloropalladate salt) leads to re-coordination of the verdazyl ligands to give 6 initially, but ultimately produces a combination of free radical 1 and 2.

Nicotine delivery and pharmacologic response from Verve, an oral nicotine delivery product.

Verve, an oral nicotine delivery product (ONDP), was introduced by Nu Mark (Altria Client Group, Richmond VA) for smokers to use in places where smoking is prohibited. This study assessed the effect of this ONDP on plasma nicotine levels, heart rate, product satisfaction, and ability to suppress smoking urge and cigarette cravings. Thirteen daily cigarette smokers [8 men and 5 women; average age 33.4years] attended two laboratory sessions, one occurred after overnight tobacco abstinence. Plasma samples were collected before and after ONDP use and measured for nicotine. In non-abstinent smokers, mean plasma nicotine levels increased from 18.3 to 21.0ng/mL. In abstinent smokers, average nicotine levels increased from 3.1 to 4.5ng/mL. After overnight tobacco abstinence, ONDP use significantly (p<0.01) increased heart rate from 69beats per minute (bpm) to 75bpm; while urge to smoke decreased significantly (p<0.01) from a score of 8.6 to 4.9. Participants indicated moderate product satisfaction that was not changed by tobacco abstinence. Analysis of unused ONDP revealed total nicotine levels of 1.68±0.09mg/disc. Spent ONDP discs were also analyzed to determine % nicotine liberated during chewing; results were 80% in the non-abstinent and 82% in the abstinent conditions (ns). Our study results indicate that ONDP use can increase plasma nicotine levels and heart rate and reduce cigarette cravings in abstinent smokers.

Bond strength of fibrin glue between layers of porcine small intestine submucosa (SIS).

This study investigated the strength of the bond between layers of small intestine submucosa (SIS, Cook Biotech, Inc., West Lafayette, IN) glued with commercially available fibrin glue (Haemacure Corporation). To determine the conditions leading to the highest bond strength, three parameters were varied: the concentration of the fibrin component, the concentration of the thrombin component, and the type of applicator used to apply the two components. Five glue concentrations and two applicator types, a Paasch Airbrush and one provided with the Haemacure glue kit, were studied. To make the test specimens, two pieces of SIS were each sprayed separately with 1 mL of one of the glue components. The two pieces were then adhered and allowed to cure for two minutes. After the panels were glued, frozen, and lyophilized, they were cut to size according to ASTM Standard D 1876: Peel Resistance of Adhesives (T-Peel Test). The panels were then rehydrated, and tests were performed in an MTS tensile testing machine set to pull at a constant rate of 1 mm/sec over a 100 mm span. The mean force over the duration of the test was computed as specified in the ASTM standard. The airbrush was found to produce a stronger bond than the applicator supplied by Haemacure. Judged qualitatively, the airbrush also produced a much more uniform spray and consistent flow rate than the glue manufacturer's applicator. The data suggest that a decrease in concentration of both glue components yields increased bond strength, although variability in the results also increased with decreased glue component concentration.