A high-resolution method for amino acid analysis of physiological fluids containing mixed disulfides

In this paper we report a high-resolution method and special buffers for the analysis for 45 common amino acids and related compounds and seven less-common components occasionally found in physiological fluids. Careful optimization of the chromatographic protocol resolves cysteine-penicillamine mixed disulfide, penicillamine disulfide, and dihydroxyphenylalanine from cystine and methionine; cysteine-homocysteine mixed disulfide is eluted between tyrosine and phenylalanine. Argininosuccinic acid is eluted before ethanolamine; glycerophosphoethanolamine is separated from taurine; glucosaminic acid is eluted between urea and aspartic acid. A Beckman Instruments System 6300 Amino Acid Analyzer was used. System performance is examined in terms of coefficient of variation (CV) over the concentration range of 1.0-5.0 nmol/50 microL; average CV for all components is better than 1.5%. Method performance is assessed through the analysis of normal and abnormal urine and plasma specimens.     

A microplate assay for <Emphasis Type="Italic">Leishmania amazonensis</Emphasis> promastigotes expressing multimeric green fluorescent protein

Convenient and economical assays capable of screening many compounds are vital to advance the development of drug therapy. This is particularly important for many of the infections that occur mainly in the Third World. The development of such a spectrofluorometric assay for the protozoan parasite Leishmania is presented here. Using multimeric (four monomers) green fluorescent protein (GFP), Leishmania amazonensis promastigotes were generated with brightness measurable in 96-well microtiter plates. The promastigotes maintained the parental characteristics, were infective to murine macrophages and to mice, and the level of GFP fluorescence corresponded to the number of inoculated cells. The feasibility of using this assay for testing drugs kinetically and in a concentration-dependent manner, under microplate culture condition, was demonstrated with amphotericin B and the herbicide oryzalin, respectively. This assay is the first to allow a real-time analysis of antileishmanial agents with live promastigotes. The method of expressing multimeric GFP for in vitro drug screening is likely to be extendable to many species of parasitic protozoa.     

Mechanisms of trafficking in axons and dendrites: implications for development and neurodegeneration

In the area of routing and sorting of dendritic traffic, the current phenomenological data beg questions about the cellular mechanisms utilized not only to transport material but also to modulate activity in a process, even apoptosis. To aid in formulating testable hypotheses, many plausible models are developed here and linked with some of the preliminary data that supports them. We first assume that in long dendrites the sorting of membranous proteins into transport vesicles also involves the linkage of motor proteins to the vesicles. Second, we assume that the cytoskeleton in dendrites is altered from the cytoskeleton in axons and the cell body. Viral glycoproteins, MAP2 and specific mRNA sorting into dendrites provide the simplest models for analyzing vesicular, cytoskeletal and RNA sorting. In the case of viral glycoproteins, initial sorting appears to occur at the Golgi but additional routing steps involve further complexities that could best be served by an additional sorting step at the junction of the cell body and the process. Transport of the specialized cytoskeletal proteins and specific mRNAs as well as vesicular material could be controlled by a similar gatekeeper at the mouth of a process. Studies of the microtubule-organelle motor complex, regulation of microtubule-based motility by microtubule-associated proteins, and slow axonal transport all provide insights into important aspects of the routing and sorting. These processes are in turn controlled by extracellular signals such as those generated by matrix molecules or their hydrolysis products in the case of amyloid precursor protein (APP). Routing and sorting mechanisms may be central to the development of Alzheimer's disease in view of evidence that APP processing is affected, transport is disturbed, and intracellular vesicles (early endosomes) hypertrophied. Further it is possible that routing mechanisms play a role in cell–cell interactions as, for example, the possibility that pathogenic/cellular stress signals may be passed along circuits transsynaptically.     

MDR1 P-glycoprotein is expressed by endothelial cells of newly formed capillaries in human gliomas but is not expressed in the neovasculature of other primary tumors.

The expression of human MDR1 P-glycoprotein (Pgp) in the capillary endothelial cells of the central nervous system has been demonstrated. The brain capillary endothelial cells maintain the structure and function of the blood-brain barrier. Recently, the human MDR1 Pgp (and its mouse homologue MDR1a Pgp) has been shown to function as an important part of this barrier, pumping out xenobiotics from endothelial cells into the lumen of capillaries resulting in the protection of the brain parenchyma. To examine whether the endothelial cells of the newly formed capillaries during neoangiogenesis within malignant human brain tumors express MDR1 Pgp, 35 adult surgical brain tumor specimens (29 gliomas and 6 tumors metastatic to the brain) were obtained from previously untreated patients and studied by a new immunohistochemical sandwich method developed in our laboratory using the JSB-1 monoclonal antibody. JSB-1 is specific for the Pgp product of the human MDR1 (and not MDR3) gene. This sensitive method allows the detection of Pgp in capillary endothelial cells of normal brain in conventional paraffin sections after formalin fixation. The endothelial cells of the newly formed capillaries in 25 of 29 gliomas (86%) and 3 of 6 metastatic tumors, immunostained positive for MDR1 Pgp. The tumor cells in 7 of 35 cases were also positive for Pgp. In the 35 brain tumor cases investigated, the endothelial cells were Pgp positive in the tumor-brain border and in the brain further from the tumor. Capillary endothelial cells of neovasculature in 137 malignant tumors (non-brain) obtained from previously untreated patients showed no MDR1 Pgp expression. These results demonstrated that MDR1 Pgp is expressed not only in the capillaries of normal brain but also in the majority of the newly formed capillaries of brain tumors. Multidrug resistance of brain tumors may result not only from the expression of resistance markers in neoplastic cells but also from the MDR1 Pgp expression in endothelial cells of tumor capillaries. Pgp in this special localization can exclude chemotherapeutic agents from tumor cells that are located around the capillaries. The therapeutic benefit and selectivity of chemotherapeutic agents in combination with a Pgp-reversing agent should be evaluated.     

Immunological Studies on the Envelope Component of Venezuelan Equine Encephalomyelitis Virus

Treatment of purified Venezuelan equine encephalomyelitis virus with the nonionic detergent Triton X-100 permitted spearation of the envelope from the core component. The isolated envelope was a noninfectious immunogen which reacted in hemagglutination, hemagglutination inhibition, complement fixation, and neutralization serological reactions.     

Estimating the impact of changes in weight and BMI on EQ-5D-3L: a longitudinal analysis of a behavioural group-based weight loss intervention

Quality of Life Research - To estimate the association between changes in BMI and changes in Health-Related Quality of Life (EQ-5D-3L). The WRAP trial was a multicentre, randomised controlled trial...     

Sex in the Era of COVID-19 in a U.S. National Cohort of Cisgender Men,Transgender Women,and Transgender Men Who Have Sex with Men: April–May 2020

Since the emergence of the COVID-19 pandemic, there has been an increasing body of research focused on the effects that measures like stay-at-home orders and social distancing are having on other aspects of health, including mental health and sexual health. Currently, there are limited extant data on the effects of the pandemic on sexual and gender minorities. Between April 15, 2020, and May 15, 2020, we invited participants in an ongoing U.S. national cohort study (Together 5000) to complete a cross-sectional online survey about the pandemic, and its effects on mental and sexual health and well-being (n?=?3991). Nearly all (97.7%) were living in an area where they were told they should only leave their homes for essentials. Most (70.1%) reported reducing their number of sex partners as a result of the pandemic. Among the 789 participants prescribed HIV pre-exposure prophylaxis (PrEP), 29.9% said they stopped taking their PrEP entirely, and 14.2% started selectively skipping doses. For those who had been taking PrEP, discontinuing PrEP was associated with having no new sex partners (β?=?0.90, 95% CI 0.40–1.40). Among the 152 HIV-positive participants, 30.9% said they were unable to maintain an HIV-related medical appointment because of the pandemic and 13.8% said they had been unable to retrieve HIV medications. Additionally, 35.3% of participants were experiencing moderate to severe anxiety because of the pandemic and 36.7% reported symptoms of depression. In a multivariable logistic regression, reporting a new sex partner in the prior 30 days was significantly associated with being aged 30 or older (vs. not, AOR?=?1.21), being Black (AOR?=?1.79) or Latinx (AOR?=?1.40, vs. white), and being unsure if they had been in close contact with someone diagnosed with COVID-19 (AOR?=?1.32, vs. no contact). It was unassociated with COVID-19-induced anxiety, depression, or knowing someone hospitalized with COVID-19. The pandemic has caused disruptions in sexual behavior (partner reduction) as well as difficulties navigating PrEP and HIV care continua. Findings will guide more comprehensive public health responses to optimize HIV prevention and treatment in the era of COVID-19.