Differential susceptibility of macrophage cell lines to Bacillus anthracis-Vollum 1B.

Bacillus anthracis (BA) is a spore forming bacterium and the causative agent of anthrax disease. Macrophages (Mphis) play a central role in anthrax disease. An important step in disease progression is the ability of BA to secrete lethal toxin (LeTx) that kills Mphis. LeTx is a heterodimer composed of protective antigen (PA) and lethal factor (LF). Researchers have shown that Mphi cell lines demonstrate differential susceptibility to purified LeTx; for example RAW264.7 and J774A.1 Mphis are sensitive to LeTx whereas IC-21 Mphis are resistant. Research has also suggested that exogenous factors, including other BA proteins, can influence the activity of LeTx. For this reason, the objective of the current work was to examine if RAW264.7, J774A.1, and IC-21 Mphis demonstrated differential susceptibility when cultured with a LeTx-producing strain of BA. Here, we co-cultured Mphis with LeTx+ Vollum 1B (V1B) spores for >15 h and assayed for Mphi cell death by morphology, trypan blue (TB) staining, neutral red (NR) activity, and lactate dehydrogenase (LDH) activity in the culture media. Following the addition of V1B spores, necrosis (approximately 50% mortality) was observed in RAW264.7 and J774A.1 Mphis at 7.5 and 10 h, respectively. By 15 h, both RAW264.7 and J774A.1 Mphis demonstrated 100% mortality. In contrast, IC-21 Mphis, under identical culture conditions, remained viable (98%) and activated throughout the course of the experiment (>24 h). The mechanism of RAW264.7 cell death appeared to involve LeTx because the V1B-induced cytotoxicity was dose-dependently reversed by the addition of anti-PA antibody to the culture media. These observations suggest there is differential susceptibility of Mphi cell lines to the LeTx+ V1B strain of BA. Further development of this in vitro model may be useful to further characterize the interactions between Mphis and BA spores.     

Dynamic Interactions of Post Cleaved NS2B Cofactor and NS3 Protease Identified by Integrative Structural Approaches

Diseases caused by flaviviruses such as dengue virus (DENV) and West Nile Virus (WNV), are a serious threat to public health. The flavivirus single-stranded RNA genome is translated into a polyprotein which is cleaved into three structural proteins and seven non-structural proteins by the viral and cellular proteases. Non-structural (NS) protein 3 is a multifunctional protein that has N-terminal protease and C-terminal helicase domains. The NS3 protease requires co-factor NS2B for enzymatic activity and folding. Due to its essential role in viral replication, NS2B-NS3 protease is an attractive target for antiviral drugs. Despite the availability of crystal structures, dynamic interactions of the N- and C-termini of NS2B co-factor have been elusive due to their flexible fold. In this study, we employ integrative structural approaches combined with biochemical assays to elucidate the dynamic interactions of the flexible DENV4 NS2B and NS3 N- and C-termini. We captured the crystal structure of self-cleaved DENV4 NS2B₄₇NS3 protease in post cleavage state. The intermediate conformation adopted in the reported structure can be targeted by allosteric inhibitors. Comparison of our new findings from DENV4 against previously studied ZIKV NS2B-NS3 proteins reveals differences in NS2B-NS3 function between the two viruses. No inhibition of protease activity was observed for unlinked DENV NS2B-NS3 in presence of the cleavage site while ZIKV NS2B-NS3 cleavage inhibits protease activity. Another difference is that binding of the NS2B C-terminus to DENV4 eNS2B₄₇NS3Pro active site is mediated via interactions with P4-P6 residues while for ZIKV, the binding of NS2B C-terminus to active site is mediated by P1-P3 residues. The mapping of NS2B N- and C-termini with NS3 indicates that these intermolecular interactions occur mainly on the beta-barrel 2 of the NS3 protease domain. Our integrative approach enables a comprehensive understanding of the folding and dynamic interactions of DENV NS3 protease and its cofactor NS2B.     

Nusinersen treatment significantly improves hand grip strength,hand motor function and MRC sum scores in adult patients with spinal muscular atrophy types 3 and 4

Journal of Neurology - Nusinersen recently became available as the first treatment for Spinal Muscular Atrophy (SMA) and data on its effectiveness and safety in adult SMA patients are still scarce....     

Undifferentiated sinonasal carcinoma in a patient with nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome is an autosomal dominant multisystem disorder characterized by developmental anomalies and occurrence of multiple basal cell carcinomas and other tumors in early childhood. In this article, the authors report a case of a 19-year-old African American male with nevoid basal cell carcinoma syndrome and a history of medulloblastoma at age 2, meningioma at age 14, thyroid follicular adenomas with papillary carcinoma at age 15, and 2 basal cell carcinomas at ages 16 and 18. Recently, he developed sinonasal undifferentiated carcinoma (SNUC). The radiology and pathology of the sinonasal carcinoma are presented in this report. Review of the literature reveals that this is the first case of SNUC occurring in a patient with nevoid basal cell carcinoma syndrome.     

Alcohol and HIV risk taking among intravenous drug users

PURPOSE: To determine if drug risk days are also alcohol use days for active injection drug users (IDUs). METHODS: Cross-sectional interview of 187 AUDIT-positive (> or = 8) active IDUs recruited between 2/98 and 10/99 from a needle exchange program (NEP) in Providence, RI. A drug risk day is defined as "using needles, cotton, or cookers after someone else had used it," measured using a 30-day Timeline Follow-Back procedure. RESULTS: The sample was 64% male, 87% white, with 85% meeting DSM-IV criteria for alcohol abuse/dependence. Of the total days analyzed (n = 5610), 25% were drug risk days; on 40% of these days, drinking also occurred. Using a generalized estimating equation (GEE) model to cluster by subject, alcohol use was associated with drug risk days (OR 1.53; 95% CI 1.2-1.9; P < .001), controlling for gender, age, race, cocaine use, number of daily injections, methadone treatment, and partner drug use. CONCLUSIONS: Using a data analytic strategy that allows examination of self-reports of behaviors on a day-to-day basis, we found that alcohol use is associated with drug risk taking behavior among IDUs. Whether alcohol use precedes or is subsequent to risky HIV behaviors remains to be determined.     

Absence of drug abuse in prisoners in a major correctional institution

Laboratory evaluation of illicit drug abuse was conducted in a major correctional institution. Fifty-eight prison inmates were screened for common drugs of abuse in conjunction with a triazolo-benzodiazipine compound tolerance protocol. Urine specimens were collected on days -1, 1, 15, 29, and 49 and assayed for amphetamines, opiates, barbiturates, and alcohol. Quinine was detected in two individuals and was probably derived from cold remedy preparations rather than the illegal use of heroin. While occasional illicit drugs may find their way into a prison population, this study showed no evidences of drug abuse in one correctional institution whose prisoners participated in Phase I drug evaluations.     

Correlations of somatophysiological, biochemical, psychosocial and behavioral risk factors of cardiovascular diseases in a sample of employees of Prague enterprises and institutions

The study is aimed at the detection and intervention of risk factors for cardiovascular diseases (CVD) and at the part played by stress in their development. The methods applied are designed to eliminate the risks and, simultaneously, to identify the function of the two main parts of stress reaction, i.e., in terms of the extent, level and type of the stressors experienced, and in terms of the level of mental resilience and related psychosocial factors which affect the experiencing of and coping with stress. The purpose of medical examination based on the patient's history, subjective complaints and objective tests, including a study of dietary habits and psychological methods was to establish the status praesens and identify the main risk factors and symptoms leading to ischemic heart disease (IHD). Intervention was performed in subjects with increased cardiovalscular risk factors.     

Disturbance of smooth muscle regulatory function by Eisenia foetida toxin lysenin: Insight into the mechanism of smooth muscle contraction

Lysenin, a toxin present in the coelomic fluid of the earthworm Eisenia foetida, is known to cause a long-lasting contraction of rat aorta smooth muscle strips. We addressed the mechanisms underlying its action on smooth muscle cells and present the first report demonstrating a completely new property of lysenin unrelated to its basic sphingomyelin-binding ability. Here we report lysenin enhancement effect on smooth muscle actomyosin ATPase activity and the ability of networking the actin filaments. The maximum enhancement of the ATPase activity of actomyosin at 120 mM KCl was observed at a molar ratio of lysenin to actin of about 1:10⁵, while at 70 mM KCl at the ratio of about 1:10⁶. The effect of lysenin became most pronounced only when both smooth muscle regulatory proteins, tropomyosin and caldesmon, were present. Co-sedimentation experiments indicated that lysenin did not displace neither tropomyosin nor caldesmon from the thin filament. Thus, the lysenin-dependent abolishment of the inhibitory effect of caldesmon on the ATPase activity was related rather to the modification of the filament structure. The ability of the toxin to exert its stimulatory effect at extremely low concentrations (as low as one molecule of lysenin per 10⁶ actin molecules) may result from the long-range cooperative transitions in the entire thin filament with an involvement of smooth muscle tropomyosin, while the role of caldesmon may be limited exclusively to the inhibition of ATPase activity.