Decreased myoblast differentiation in chronic binge alcohol-administered simian immunodeficiency virus-infected male macaques: role of decreased miR-206.

Skeletal muscle stem cells play a critical role in regeneration of myofibers. We previously demonstrated that chronic binge alcohol (CBA) markedly attenuates myoblast differentiation potential and myogenic gene expression. Muscle-specific microRNAs (miRs) are implicated in regulation of myogenic genes. The aim of this study was to determine whether myoblasts isolated from asymptomatic CBA-administered simian immunodeficiency virus (SIV)-infected macaques treated with antiretroviral therapy (ART) showed similar impairments and, if so, to elucidate potential underlying mechanisms. Myoblasts were isolated from muscle at 11 mo after SIV infection from CBA/SIV macaques and from time-matched sucrose (SUC)-treated SIV-infected (SUC/SIV) animals and age-matched controls. Myoblast differentiation and myogenic gene expression were significantly decreased in myoblasts from SUC/SIV and CBA/SIV animals compared with controls. SIV and CBA decreased muscle-specific miR-206 in plasma and muscle and SIV decreased miR-206 expression in myoblasts, with no statistically significant changes in other muscle-specific miRs. These findings were associated with a significant increase in histone deacetylase 4 (HDAC4) and decrease in myogenic enhancer factor 2C (MEF2C) expression in CBA/SIV muscle. Transfection with miR-206 inhibitor decreased myotube differentiation, increased expression of HDAC4, and decreased MEF2C, suggesting a critical role of miR-206 in myogenesis. Moreover, HDAC4 was confirmed to be a direct miR-206 target. These results support a mechanistic role for decreased miR-206 in suppression of myoblast differentiation resulting from chronic alcohol and SIV infection. The parallel changes in skeletal muscle and circulating levels of miR-206 warrant studies to establish the possible use of plasma miR-206 as an indicator of impaired muscle function.

2H stable isotope analysis of human tooth enamel: a new tool for forensic human provenancing?

Stable isotope analysis of biogenic tissues such as tooth enamel and bone mineral has become a well-recognised and increasingly important method for determining the provenance of human remains, and it has been used successfully in bio-archaeological studies as well as forensic investigations. In particular, (18)O and (2)H stable isotope signatures of bone and hair, respectively, are well-established proxies of climate (temperature) and source water and are therefore considered as indicators of geographic life trajectories of animals and humans. While the methodology for (2)H analysis of human hair, fingernails, and bone collagen is currently used to determine human provenance, i.e. geographic origin and identify possible migration patterns, studies involving the analysis of (2)H in tooth enamel appear to be nonexistent in the scientific literature. Ground tooth enamel was analysed by continuous-flow isotope ratio mass spectrometry (IRMS) coupled on-line to a high-temperature conversion elemental analyser (TC/EA). An array of tooth enamel samples from archaeological and modern teeth has been analysed under different experimental conditions, and the results of this proof-of-concept study are presented. While no significant differences in (2)H abundance were noted as a result of H exchange studies or different sample preparation protocols, no significant differences or trends in measured δ(2)H-values were observed either with regard to known differences in geographical provenance. We concluded that the δ(2)H-values obtained from tooth enamel could not be used as proxy for a person's geographical origin during adolescence.

A review of optical pacing with infrared light.

Optical pacing (OP) uses pulsed infrared light to initiate heartbeats in electrically excitable cardiac tissues without employing exogenous agents. OP is an alternative approach to electrical pacing that may overcome some its disadvantages for some applications. In this review, we discuss the initial demonstrations, mechanisms, safety, advantages and applications of OP.

Localization and imaging with radioiodine-labeled monoclonal antibodies in a xenogeneic tumor model for human B-cell lymphoma.

Two MoAbs directed towards human B-cell malignancies have been studied in a preclinical animal model to evaluate their potential for in vivo imaging and therapy of B-cell lymphomas. Anti-B1 reacts with virtually all immunoglobulin-bearing malignancies and non-T acute lymphoblastic leukemia. Anti-J5 reacts with the common acute lymphoblastic leukemia antigen found on non-T acute lymphoblastic leukemia and follicular lymphomas. Anti-T1 which recognizes the CD5 antigen on most T-cell leukemias and lymphomas was used as a control antibody. These monoclonal antibodies were radiolabeled with 125I or 131I by the ICl method. Namalwa (B-cell) and MOLT-4 (T-cell) tumors were grown s.c. in irradiated nude mice. The highest tissue concentration of 125I-labeled anti-J5 in Namalwa-bearing mice was in blood and tumor. The tumor/blood ratio ranged from 0.7-1.2, with the highest ratio 4 days after injection. Pharmacokinetic analysis indicated that the t1/2 beta of anti-J5 from blood and other tissues ranged from 40-50 h, while the t1/2 beta for tumor averaged 65 h. The area under the curve of tumor was 2- to 5-fold higher than the area under the curve of liver, kidney, skin, and muscle. The peak tissue levels of 125I-labeled anti-B1 in Namalwa-bearing mice were again in blood and tumor and 6 days following injection more than 5-fold greater activity was found in tumor compared to normal tissues other than blood. The tumor/blood ratio was 1.2 and 0.7 at 4 and 6 days after injection. 125I-labeled anti-B1 showed minimal uptake in antigen-negative MOLT-4 tumors and 125I-labeled anti-T1 showed little uptake in Namalwa tumors. Scintigraphic images were obtained following the injection of 131I-labeled anti-J5 and anti-B1 in nude mice bearing Namalwa tumors. These results indicate that radiolabeled anti-J5 and anti-B1 show promise as diagnostic and possibly therapeutic agents for human B-cell lymphoma, although there may be a limitation to clinical utility due to cross-reactivity with some normal cells.

Long-term treatment of social phobia with clonazepam.

Twenty-six socially phobic outpatients were treated with clonazepam for the relief of symptoms. At evaluation, which took place after an average of 11.3 months of continuous treatment, 22 (84.6%) patients showed good improvement and 4 (14.4%) showed no improvement or were not recovered. The dose declined over time, from a peak mean of 2.1 mg/day to a mean of 0.94 mg/day at follow-up. Side effects are described, along with individual case descriptions that illustrate important aspects of the use of benzodiazepines for the treatment of social phobia.

Magnetic resonance spectroscopy in social phobia: preliminary findings.

Proton localized magnetic resonance spectroscopy was studied in 20 social phobics and 20 age- and sex-matched controls. Stimulated Echo Acquisition Mode volume element localization was used with chemical shift imaging. Choline and creatine signal-to-noise ratios (SNRs) were significantly lower in social phobia than in controls in subcortical, thalamic, and caudate areas. In the social phobic group, N-acetylaspartate (NAA) SNR was significantly lower in cortical and subcortical regions, and ratios of NAA to other metabolites were lower in social phobia. Choline, creatine, and NAA SNRs were inversely correlated to total social phobia and fear symptoms, as measured by the Brief Social Phobia Scale, in the thalamic and noncortical gray areas. In a small number of patients who received clonazepam, posttreatment SNRs generally increased relative to baseline. Our results suggest a promising place for magnetic resonance spectroscopy in social phobia and also indicate potential pharmacodynamic uses of this technique.

The Brief Social Phobia Scale.

An observer measure of social phobic symptoms, referred to as the Brief Social Phobia Scale, consists of 11 items, 7 evaluating commonly feared or avoided situations and 4 additional items measuring autonomic distress. Symptoms represented by the scale items are found frequently in social phobia, and the instrument demonstrates acceptable interrater and test-retest reliability, internal consistency, concurrent validity against other measures of social phobia, and the ability of patients to change as a result of treatment. It can also detect differences between active treatment and placebo treatment. A comparison of the sensitivity of this scale with other scales in detection of size of effect also is presented.

Transient vasopressin-resistant diabetes insipidus of pregnancy.

A case of transient vasopressin-resistant diabetes insipidus is reported, which developed during the seventh month of pregnancy and remitted postpartum. The patient was resistant to large doses of deamino-D-arginine vasopressin as well as to aqueous pitressin. She was treated with hydrochlorothiazide. Evidence that prostaglandins may play a role in this syndrome is discussed.

Transient vasopressin-resistant diabetes insipidus of pregnancy.

A case of transient vasopressin-resistant diabetes insipidus is reported which developed during the seventh gestational month. Polyuria reached 4-6 L daily and urine osmolality remained dilute despite 21 hours of water deprivation followed by 5 U intramuscularly of aqueous pitressin, as well as four days of treatment with intranasal DDAVP (0.1-0.5 mL every 12 hours). Urinary excretion of prostaglandin E2, 1384 ng/24 hours, was fourfold that in nongravid subjects and a plasma arginine vasopressin level of 12 pg/mL was recorded. Indomethacin had no effect on urine osmolality but decreased urine volume markedly. Hydrochlorothiazide, also, decreased urine volumes, and this drug was used to manage the patient until delivery. The syndrome remitted in the puerperium, the patient concentrating her urine to 938 mOsm/kg when tested several months postpartum.

Cine photography and video recording of anterior segment fluorescein angiography.

A description is given of apparatus and technique for carrying out cine photography and video recording of anterior segment fluorescein angiography. We found cine best for single-frame analysis and video tape recording less expensive.

Iris atrophy in sickle cell disease.

Iris atrophy, of unknown origin and believed to be secondary to the vaso-occlusive process of sickle cell disease, has been observed in 25 eyes of 22 patients (two SS disease, 20 SC disease). The crude prevalence was highest in males with SC disease, in whom 14.7% of patients were affected. Iris atrophy was closely associated with proliferative sickle retinopathy in the same eye. Analysis of haematological indices failed to reveal any significant differences between patients with and without iris atrophy. The characteristics and distribution of iris atrophy are described as well as the histopathology in one 68-year-old male patient with SS disease.

Macular vasculature, visual acuity, and irreversibly sickled cells in homozygous sickle cell disease.

Observations of visual acuity and the conjunctival, macular, and perimacular vascularity have been assessed in patients with homozygous sickle cell (SS) disease. There were 17 matched pairs, each consisting of one patient with a high count (greater than or equal to 15%) and one with a low count (less than or equal to 5%) of irreversibly sickled cells (ISCs). The macular vascular bed was assessed by measurements of the foveal avascular zone (FAZ), perimacular avascular zones, and counts of perimacular vascular abnormalities (perimacular counts). Small foveal avascular zones and high perimacular counts were commoner in younger than older patients and there was a significant inverse correlation between size of the FAZ and the perimacular count. These observations were compatible with the hypothesis that perimacular vessel anomalies represent the early vaso-occlusive phase which progresses to ischaemia and the formation and enlargement of avascular areas. Visual acuity was assessed by Snellen's test type and by measuring contrast sensitivity. There was no obvious relationship between acuity measured by the 2 methods and no relationship between acuity and observations of macular vascularity. High ISC counts were significantly related to abnormalities of the conjunctival vasculature, but no relationship was noted with abnormalities of the macular vasculature or with visual acuity.

Choroidal neovascularisation induced by photocoagulation in sickle cell disease.

Choroidal neovascularisation occurred in 35 out of 57 (61%) patients during a trial of xenon are photocoagulation in proliferative sickle retinopathy. Nineteen patients manifested abnormal vessel systems in the plane of the retina which tended to be benign, not enlarging, and without complications (choroidoretinal neovascularisation). In 16 patients the abnormal vessel systems grew into the vitreous, tended to increase in size, and to be associated with vitreous haemorrhage and retinal traction (choroidovitreal neovascularisation). Both forms of neovascularisation were significantly more common at younger ages. Treatment was generally unsatisfactory, frequently resulting in further enlargement of the lesions. Although the natural history of choroidal neovascularisation and its ultimate effect on visual function remains to be recorded, these lesions constitute a common and potentially serious complication of xenon are photocoagulation in proliferative sickle retinopathy as currently performed.

Nanoliter-scale sample preparation methods directly coupled to polymethylmethacrylate-based microchips and gel-filled capillaries for the analysis of oligonucleotides.

We are currently developing miniaturized, chip-based electrophoresis devices fabricated in plastics for the high-speed separation of oligonucleotides. One of the principal advantages associated with these devices is their small sample requirements, typically in the nanoliter to sub-nanoliter range. Unfortunately, most standard sample preparation protocols, especially for oligonucleotides, are done off-chip on a microliter-scale. Our work has focused on the development of capillary nanoreactors coupled to micro-separation platforms, such as micro-electrophoresis chips, for the preparation of sequencing ladders and also polymerase chain reactions (PCRs). These nanoreactors consist of fused-silica capillary tubes (10-20 cm x 20-50 microns I.D.) with fluid pumping accomplished using the electroosmotic flow generated by the tubes. These reactors were situated in fast thermal cyclers to perform cycle sequencing or PCR amplification of the DNAs. The reactors could be interfaced to either a micro-electrophoresis chips via capillary connectors micromachined in polymethylmethacrylate (PMMA) using deep X-ray etching (width 50 microns; depth 50 microns) or conventional capillary gel tubes using zero-dead volume glass unions. For our chips, they also contained an injector, separation channel (length 6 cm; width 30 microns; depth 50 microns) and a dual fiber optic, near-infrared fluorescence detector. The sequencing nanoreactor used surface immobilized templates attached to the wall via a biotin-streptavidin-biotin linkage. Sequencing tracks could be directly injected into gel-filled capillary tubes with minimal degradation in the efficiency of the separation process. The nanoreactor could also be configured to perform PCR reactions by filling the capillary tube with the PCR reagents and template. After thermal cycling, the PCR cocktail could be pooled from multiple reactors and loaded onto a slab gel or injected into a capillary tube or microchip device for fractionation.

Characterization of rabbit primary proximal tubule kidney cell cultures grown on Millicell-HA membrane filters.

With the aim of developing a kidney cell culture system that can be used to assess renal toxicity in vivo, freshly isolated rabbit proximal tubules were plated on Millipore cellulose filters mounted in plastic inserts (Millicell-HA). DNA synthesis peaked on day 6 of culture and cells reached confluency by days 12-14. The integrity of the monolayer was confirmed by exclusion of [(14)C]inulin and cell viability demonstrated by linearity of protein synthesis over a 24-hr period. In confluent cultures, the organic anion, [(14)C]p-aminohippuric acid (PAH) and cation [(14)C]tetraethylammonium bromide (TEA) were shown to be transported from the basolateral to the apical side at a rate 5-6 times greater than that from the apical to basolateral side during the first 60 min of exposure. Probenecid decreased PAH transport by 60% and N-methylnicotinamide and quinine inhibited TEA transport by 40 and 56%, respectively. Uptake of [(14)C]alpha-methylglucopyranoside into the cells was three times greater when label was added to the apical side than when label was added to the basolateral side. Apical uptake of glucose was sodium dependent and inhibited by more than 90% with phloridzin. Thus, kidney proximal tubule cells in the filter insert culture system display functional polarity which appears to mimic function in vivo and may be useful for examining mechanisms of nephrotoxicity.