Prevalence of mutations at codon 463 of katG gene in MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and application of the method in rapid diagnosis

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. In respect to high GC content of Mycobacterium tuberculosis, nonsynonymous mutations are dominant in this group. In this study a collection of 145 M. tuberculosis isolates was used to evaluate the conferring mutations in nucleotide 1388 of katG gene (KatG463) in resistance to isoniazid. A PCR-RFLP method was applied in comparison with DNA sequencing and anti-mycobacterial susceptibility testing. From all studied patients, 98 (67.6%) were men, 47 (32.4%) were women, 3% were <15 and 9% were >65 years old; male to female ratio was 1:2.4. PCR result of katG for a 620-bp amplicon was successful for all purified M. tuberculosis isolates and there was no positive M. tuberculosis culture with PCR negative results (100% specificity). Subsequent PCR RFLP of the katG identified mutation at KatG463 in 33.3%, 57.8% and 59.2% of our clinically susceptible, multidrug resistant TB (MDR) and extensively drug resistant (XDR) isolates, respectively. Strains of H37Rv and Academic had no any mutations in this codon. M. bovis was used as a positive control for mutation in KatG463. Automated DNA sequencing of the katG amplicon from randomly selected INH-susceptible and resistant isolates verified 100% sequence accuracy of the point mutations detected by PCR-RFLP. We concluded that codon 463 was a polymorphic site that is associated to INH resistance (a missense or "quiet" mutation). RFLP results of katG amplicons were identical to those of sequence method. Our PCR-RFLP method has a potential application for rapid diagnosis of M. tuberculosis with a high specificity.     

Enhanced methane production from wool textile residues by thermal and enzymatic pretreatment

Methane production from two types of wool textile wastes (TW1 and TW2) was investigated. To improve the digestibility of these textiles, different pretreatments were applied, and comprised thermal treatment (at 120 °C for 10 min), enzymatic hydrolysis (using an alkaline endopeptidase at different levels of enzymatic loading, at 55 °C for 0, 2, and 8 h), and a combination of these two treatments. Soluble protein concentration and sCOD (soluble chemical oxygen demand) were measured to evaluate the effectivity of the different pretreatment conditions to degrade wool keratin. The sCOD as well as the soluble protein content had increased in both textile samples in comparison to untreated samples, as a response to the different pretreatments indicating breakdown of the wool keratin structure.The combined treatments and the thermal treatments were further evaluated by anaerobic batch digestion assays at 55 °C. Combined thermal and enzymatic treatment of TW1 and TW2 resulted in methane productions of 0.43 N m³/kg VS and 0.27 N m³/kg VS, i.e., 20 and 10 times higher yields, respectively, than that gained from untreated samples. The application of thermal treatment by itself was less effective and resulted in increasing the methane production by 10-fold for TW1 and showing no significant improvement for TW2.     

Evidence for crossing the blood barrier of adult rat brain by human adipose-derived mesenchymal stromal cells during a 6-month period of post-transplantation

Background aimsTherapeutic promises of adult stem cells have been overshadowed by an elicited immune response, low maintenance of implanted cells or concerns regarding their migration to non-target sites. These problems might be lessened by the use of immune privilege cells and tissues for implantation.MethodsIn this study, human adipose-derived mesenchymal stromal cells (hADMSCs) were stably transfected with a vector containing Turbo green fluorescent protein (GFP) and JRed, which allows tracing the cells after transplantation. Labeled hADMSCs were transplanted into the adult rat brain followed by assessment of their survival and migration during 6 months after transplantation.ResultsResults indicate that there were no postsurgical complications, and the animals thrived after transplantation. The lesions of the surgical process were remarkable at the first weeks, and a high number of transplanted cells were accumulated around them. Cell populations declined over time as they partly migrated away from the injection sites; nonetheless, they were detectable at each examination time point. Although the cells could survive and remain at the injection site for up to 6 months, some of them drifted to spleen, which is an indication of their ability to cross the blood-brain barrier.ConclusionsDespite the high survival rate of hADMSCs in the xenogenic condition, which is an ideal criterion in cell therapy, irregular migration tendency must be handled with caution.     

Optimization of non-detergent treatment for enveloped virus inactivation using the Taguchi design of experimental methodology (DOE)

Abstract

In mammalian cell culture technology, viral contamination is one of the main challenges; and, so far, various strategies have been taken to remove or inactivate viruses in the cell-line production process. The suitability and feasibility of each method are determined by different factors including effectiveness in target virus inactivation, maintaining recombinant protein stability, easiness—in terms of the process condition, cost-effectiveness, and eco-friendliness. In this research, Taguchi design-of-experiments (DOE) methodology was used to optimize a non-detergent viral inactivation method via considering four factors of temperature, time, pH, and alcohol concentration in an unbiased (orthogonal) fashion with low influence of nuisance factors. Herpes Simplex Virus-1 (HSV1) and Vero cell-line were used as models for enveloped viruses and cell-line, respectively. Examining the cytopathic effects (CPE) in different dilutions showed that pH (4), alcohol (15%), time (120?min), and temperature (25?°C) were the optimal points for viral inactivation. Evaluating the significance of each parameter in the HSV-1 inactivation using Taguchi and ANOVA analyses, the contributions of pH, alcohol, temperature and time were 56.5%, 19.2%, 12%, and 12%, respectively. Examining the impact of the optimal viral treatment condition on the stability of model recombinant protein-recombinant human erythropoietin, no destabilization was detected.     

Acute and chronic exposure of chick embryo to ethanol alters brain neurosteroid levels

Neurosteroids are modulators of neuronal function that may play important role in brain maturation. The aim of the present investigation was to study the effect of prenatal exposure to acute and chronic ethanol on brain progesterone, estradiol, and testosterone concentration on 10th and 15th days following egg incubation. Eggs were exposed to ethanol at 10 % in chronic treatment and 70 % in acute treatment. Progesterone, estradiol, and testosterone were assayed by radioimmunoassay method. It was shown that brain progesterone level was significantly decreased (P?<?0.05) in chronic ethanol group on embryonic day 10, but it was significantly decreased (P?<?0.05) in acute and chronic groups on embryonic day 15. Brain estradiol level was significantly increased (P?<?0.05) in chronic ethanol group on embryonic day 10, and it was decreased (P?<?0.05) in acute and chronic groups of ethanol on embryonic day 15. Brain testosterone was significantly increased (P?<?0.05) in acute and chronic ethanol-exposed groups on embryonic days 10 and 15. Our observations suggest that ethanol may modulate neurosteroid synthesis in the brain.     

Improved exercise performance and increased aerobic capacity after endurance training of patients with stable polymyositis and dermatomyositis

Introduction

This randomized, controlled study on patients with polymyositis or dermatomyositis was based on three hypotheses: patients display impaired endurance due to reduced aerobic capacity and muscle weakness, endurance training improves their exercise performance by increasing the aerobic capacity, and endurance training has general beneficial effects on their health status.

Methods

In the first part of this study, we compared 23 patients with polymyositis or dermatomyositis with 12 age- and gender-matched healthy controls. A subgroup of patients were randomized to perform a 12-week endurance training program (exercise group, n = 9) or to a non-exercising control group (n = 6). We measured maximal oxygen uptake (VO₂ max) and the associated power output during a progressive cycling test. Endurance was assessed as the cycling time to exhaustion at 65% of VO₂ max. Lactate levels in the vastus lateralis muscle were measured with microdialysis. Mitochondrial function was assessed by measuring citrate synthase (CS) and β-hydroxyacyl-CoA dehydrogenase (β-HAD) activities in muscle biopsies. Clinical improvement was assessed according to the International Myositis Assessment and Clinical Studies Group (IMACS) improvement criteria. All assessors were blinded to the type of intervention (that is, training or control).

Results

Exercise performance and aerobic capacity were lower in patients than in healthy controls, whereas lactate levels at exhaustion were similar. Patients in the exercise group increased their cycling time, aerobic capacity and CS and β-HAD activities, whereas lactate levels at exhaustion decreased. Six of nine patients in the exercise group met the IMACS improvement criteria. Patients in the control group did not show any consistent changes during the 12-week study.

Conclusions

Polymyositis and dermatomyositis patients have impaired endurance, which could be improved by 12 weeks of endurance training. The clinical improvement corresponds to increases in aerobic capacity and muscle mitochondrial enzyme activities. The results emphasize the importance of endurance exercise in addition to immunosuppressive treatment of patients with polymyositis or dermatomyositis.

Trial registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT01184625","term_id":"NCT01184625"}}NCT01184625     

Response surface optimized peroxyoxalate chemiluminescence of octahydro‐Schiff base derivative as new luminophor and study of the quenching effect of some cations,amino acids and cholesterol

We report the first detailed study of the characteristics of an octahydro‐Schiff base derivative as a new luminophor in the peroxyoxalate chemiluminescence (POCL) system. The effect of reagents on this new POCL system was investigated. In addition, the response surface methodology was used for evaluating the relative significance of variables in this POCL system, establishing models and determining optimal conditions. The quenching effect of some cations and compounds such as Cu²⁺, Fe³⁺, Hg²⁺, imidazole, histidine and cholesterol on an optimized POCL reaction were studied. The dynamic ranges were up to approximaterly 100 and 175 × 10^?6 M for Cu²⁺ and cholesterol respectively. The detection limits were 3.3 × 10^?6 m and 2.58 × 10^?6 m for Cu²⁺ and histidine, respectively. In all cases the relative standard deviations were 4–5% (n = 4). Copyright © 2014 John Wiley & Sons, Ltd.     

LprG-Mediated Surface Expression of Lipoarabinomannan Is Essential for Virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis employs various virulence strategies to subvert host immune responses in order to persist and cause disease. Interaction of M. tuberculosis with mannose receptor on macrophages via surface-exposed lipoarabinomannan (LAM) is believed to be critical for cell entry, inhibition of phagosome-lysosome fusion, and intracellular survival, but in vivo evidence is lacking. LprG, a cell envelope lipoprotein that is essential for virulence of M. tuberculosis, has been shown to bind to the acyl groups of lipoglycans but the role of LprG in LAM biosynthesis and localization remains unknown. Using an M. tuberculosis lprG mutant, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. The lprG mutant had a normal quantity of LAM in the cell envelope, but its surface was altered and showed reduced expression of surface-exposed LAM. Functionally, the lprG mutant was defective for macrophage entry and inhibition of phagosome-lysosome fusion, was attenuated in macrophages, and was killed in the mouse lung with the onset of adaptive immunity. This study identifies the role of LprG in surface-exposed LAM expression and provides in vivo evidence for the essential role surface LAM plays in M. tuberculosis virulence. Findings have translational implications for therapy and vaccine development.     

Pre-Transplantation Serum Parathyroid Hormone Influences the Number of Mobilized CD34+ Hematopoietic Stem Cells in Autologous Hematopoietic Stem Cell Transplantation

Background:Parathyroid hormone (PTH) is a calcium homeostasis regulator and can affect bone marrow niche. PTH leads to the bone marrow stem cell niche expansion as well as the induction of stem cell mobilization from the bone marrow into peripheral blood. In this study, we evaluated the association between pre- transplantation serum PTH levels and the number of circulating CD34+ cells along with the platelets/white blood cells (Plt/WBC) engraftment in patients who underwent autologous Hematopoietic Stem Cell Transplantation.Methods:Subjects for the study were 100 patients who received autologous hematopoietic stem cell transplantation (auto-HSCT), retrospectively. Serum levels of PTH, calcium, phosphorus, and alkaline phosphatase were measured before mobilization. Their impacts were measured on the number of mobilized CD34+ hematopoietic stem cells, and Plt/WBC engraftment.Results:High levels of serum PTH (> 63.10 pg/mL) was significantly associated with higher number of CD34+ cells in peripheral blood after granulocyte- colony stimulating factor (G-CSF)-induced mobilization (p= 0.079*). Serum calcium at low levels were associated with higher number of circulating CD34+ cells post mobilization. Pre- transplantation serum levels of phosphorus and alkaline phosphatase on CD34+ numbers were not statistically significant. Serum Plt/WBC engraftment was not improved in presence of high levels of serum PTH.Conclusion:We suggested that serum PTH levels before transplantation could be influential in raising the number of circulating CD34+ hematopoietic stem cell after mobilization.Key Words: Auto-HSCT, CD34+ Cell, Pre- transplant PTH