Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1

Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations. T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide. This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT).     

R620W functional polymorphism of protein tyrosine phosphatase non-receptor type 22 is not associated with pulmonary tuberculosis in Zahedan, southeast Iran

The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene, which encodes an intracellular lymphoid-specific phosphatase, is considered an important regulator of T-cell activation. We investigated a possible association between the PTPN22 C1858T (R620W) polymorphism and pulmonary tuberculosis in an Iranian population. Single nucleotide polymorphisms of PTPN22 C1858T (rs2476601) were genotyped in 172 pulmonary tuberculosis cases and 204 normal subjects from Zaheden, Iran. Frequencies of genotypes CC, CT and TT of the PTPN22 C1858T polymorphism were 98.3, 1.7 and 0% in the pulmonary tuberculosis patients, and 96.1, 3.9 and 0% in the control group, respectively (P = 0.239). The frequency of the minor (T) allele was 0.8% in pulmonary tuberculosis patients and 2.0% in controls. Significant differences were not observed in genotype or allele frequencies of PTPN22 C1858T in the comparison between pulmonary tuberculosis patients and healthy subjects in our Iranian population sample.     

A joint experimental and theoretical investigation of kinetics and mechanistic study in a synthesis reaction between triphenylphosphine and dialkyl acetylenedicarboxylates in the presence of benzhydrazide

Stable crystalline phosphorus ylides were obtained in excellent yields from the 1:1:1 addition reaction between triphenylphosphine (TPP) and dialkyl acetylenedicarboxylates, in the presence of NH-acids, such as benzhydrazide. To determine the kinetic parameters of the reactions, they were monitored by UV spectrophotometery. The second order fits were automatically drawn and the values of the second order rate constant (k₂) were calculated using standard equations within the program. At the temperature range studied the dependence of the second order rate constant (Ln k₂) on reciprocal temperature was compatible with Arrhenius equation. This provided the relevant plots to calculate the activation energy of all reactions. Furthermore, useful information were obtained from studies of the effect of solvent, structure of reactants (different alkyl groups within the dialkyl acetylenedicarboxylates) and also concentration of reactants on the rate of reactions. On the basis of experimental data the proposed mechanism was confirmed according to the obtained results and a steady state approximation and the first step (k₂) and third (k₃) steps of the reactions were recognized as the rate determining steps, respectively. In addition, three speculative proposed mechanisms were theoretically investigated using quantum mechanical calculation. The results, arising from the second and third speculative mechanisms, were far from the experimental data. Nevertheless, there was a good agreement between the theoretical kinetic data, emerge from the first speculative mechanism, and experimental kinetic data of proposed mechanism.

Defective pulmonary innervation and autonomic imbalance in congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is associated with significant mortality due to lung hypoplasia and pulmonary hypertension. The role of embryonic pulmonary innervation in normal lung development and lung maldevelopment in CDH has not been defined. We hypothesize that developmental defects of intrapulmonary innervation, in particular autonomic innervation, occur in CDH. This abnormal embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH. To define patterns of pulmonary innervation in CDH, human CDH and control lung autopsy specimens were stained with the pan-neural marker S-100. To further characterize patterns of overall and autonomic pulmonary innervation during lung development in CDH, the murine nitrofen model of CDH was utilized. Immunostaining for protein gene product 9.5 (a pan-neuronal marker), tyrosine hydroxylase (a sympathetic marker), vesicular acetylcholine transporter (a parasympathetic marker), or VIP (a parasympathetic marker) was performed on lung whole mounts and analyzed via confocal microscopy and three-dimensional reconstruction. Peribronchial and perivascular neuronal staining pattern is less complex in human CDH than control lung. In mice, protein gene product 9.5 staining reveals less complex neuronal branching and decreased neural tissue in nitrofen-treated lungs from embryonic day 12.5 to 16.5 compared with controls. Furthermore, nitrofen-treated embryonic lungs exhibited altered autonomic innervation, with a relative increase in sympathetic nerve staining and a decrease in parasympathetic nerve staining compared with controls. These results suggest a primary defect in pulmonary neural developmental in CDH, resulting in less complex neural innervation and autonomic imbalance. Defective embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH.     

Phenotyping of circulating CD8⁺ T cell subsets in human cutaneous leishmaniasis

Recovery from CL is usually accompanied with long-lasting protection and induction of strong immune response. The phenotypes, generation and maintenance of central (=T_CM) and effector (=T_EM) memory T cell subsets in human leishmaniasis are not well known. Profile of T cell subsets were analyzed on peripheral CD8⁺ T cells from volunteers with history of cutaneous leishmaniasis (HCL).In HCL and control groups, mean frequencies of CCR7⁺CD45RA⁺CD8⁺ naïve and CCR7^?CD45RA^?CD8⁺ T_EM cells were higher than other subsets before culture, but after stimulation with soluble Leishmania antigen, the frequency of naïve T cells was significantly decreased and the frequency of T_EM cells was significantly increased. T_EM phenotype composed the highest portion of proliferating Carboxy Fluorescein diacetate Succinimidyl Ester (CFSE)-dim population which was significantly higher in HCL volunteers than in control group. Stimulation of isolated CD8⁺ memory T cells, but not naïve T cells, from HCL volunteers induced a significantly higher IFN-γ production compared with that of healthy controls. Intracellular IFN-γ staining provided the same result.Memory population is shown to be responsible for Leishmania-induced IFN-γ production. Leishmania-reactive proliferating T_EM cells were identified as the most frequent subset which may play a role in recall immune response and protection against Leishmania infection.     

Altered serum pro-inflammatory cytokines in children with Down's syndrome

There are reports showing that pro-inflammatory cytokines are dysregulated in patients with Down's syndrome (DS). However, most of these reports concern adults. We analyzed cytokine levels in serum samples from children with DS, and compared them with samples from intellectually disabled (ID), and healthy, control children. Blood samples were collected from 24 DS, 24 age-/sex-matched ID, and 24 age-/sex-matched healthy, control children. Serum levels of the cytokines IL-5, IL-10, IL-13, IFN-γ, and TNF-α were measured using a sandwich ELISA method, . The age range of the children was 1-15 years, with a mean ± SD of 5.75 ± 4.36 years. TNF-α levels were significantly higher in the DS and ID groups compared with those found in healthy, control children (P<0.05). The DS and ID groups had significantly higher IFN-γ levels compared with healthy, control children (P = 0.0002 and P<0.01, respectively), with significant higher levels in the DS than the ID group (P<0.05). Serum from the ID group showed significantly higher IL-10 levels compared with those from the DS group (P<0.05), but not the healthy, control group. Significant correlations were found between the differences in TNF-α and IFN-γ levels, in both ID (rs = 0.558; P = 0.005) and DS children (rs = 0.405; P<0.05). There were no significant differences found in serum levels of IL-13 between the groups, and IL-5 was not detectable in any of the serum samples. Levels of TNF-α and IFN-γ were increased, and IL-10 decreased in serum from children with DS. It may be that these differences contribute to the clinical symptoms seen in DS: consequently, these pro-inflammatory cytokines might be useful as early biomarkers of the disorders associated with DS.     

Adenovirus-mediated expression of the HO-1 protein within MSCs decreased cytotoxicity and inhibited apoptosis induced by oxidative stresses

The capacity of mesenchymal stem cells (MSCs) to survive and engraft in the target tissue may lead to promising therapeutic effects. However, the fact that the majority of MSCs die during the first few days following transplantation complicates cell therapy. Hence, it is necessary to strengthen the stem cells to withstand the rigors of the microenvironment to improve the efficacy of cell therapy. In this study, we manipulated MSCs to express a cytoprotective factor, heme oxygenase-1 (HO-1), to address this issue. Full-length cDNA of human HO-1 was isolated and cloned into TOPO vector by TOPO cloning reaction. Then, the construct was ligated to gateway adapted adenovirus expression vector by LR recombination reaction. Afterwards, the recombinant virus expressing HO-1 was produced in appropriate mammalian cell line and used to infect MSCs. The HO-1 engineered MSCs were exposed to hypoxic and oxidative stress conditions followed by evaluation of the cells’ viability and apoptosis. Transient expression of HO-1 was detected within MSCs. It was observed that HO-1 expression could protect MSCs against cell death and the apoptosis triggered by hypoxic and oxidative stress conditions. The MSCs-HO-1 retained their ability to differentiate into adipogenic, chondrogenic, or osteogenic lineages. These findings could be applied as a strategy for prevention of graft cell death in MSCs-based cell therapy and is a good demonstration of how an understanding of cellular stress responses can be used for practical applications.     

Antiproliferative activity of novel thiopyran analogs on MCF-7 breast and HCT-15 colon cancer cells: synthesis, cytotoxicity, cell cycle analysis, and DNA-binding

A series of 6H-thiopyran-2,3-dicarboxylate derivatives 4a-d were synthesized and evaluated for their cytotoxic effect against HCT-15 colon and MCF-7 breast cancer cell lines using Sulforhodamine B (SRB) assay. The results showed that these compounds could exhibit a good cytotoxicity to both cell lines. In addition, these compounds were found to exhibit significant DNA-binding affinity. Ultraviolet-visible light (UV-Vis) spectroscopy was conducted to determine the ability of the ligand under analysis. The effect of ligand complexation on DNA structure led to overall affinity constants of K(4a)=3.5×10(4) M(-1), K(4b)=6.4×10(4) M(-1), K(4c)=3.2×10(4) M(-1), and K(4d)=2.4×10(4) M(-1). Our findings could provide new evidence showing the relationship between the chemical structure and biological activity and may be useful for the discovery of new anti-cancer drugs.     

(-)-Epicatechin maintains endurance training adaptation in mice after 14 days of detraining

The purpose of this study was to determine whether (-)-epicatechin (mainly found in cocoa) could attenuate detraining effects in the hindlimb muscles of mice. Thirty-two male mice were randomized into 4 groups: control, trained, trained with 14 d of detraining and vehicle (DT-14-W), and trained with 14 d of detraining and (-)-epicatechin [DT-14-(-)-Epi]. DT-14-(-)-Epi received (-)-epicatechin (1.0 mg/kg 2 ×/d), whereas water was given to the DT-14-W group. The latter 3 groups performed 5 wk of endurance training 5 ×/wk. Hindlimb muscles were harvested, and Western blots, as well as enzyme analyses, were performed. Training significantly increased capillary-to-fiber ratio (≈ 78.8%), cytochrome-c oxidase (≈ 35%), and activity (≈ 144%) compared to controls. These adaptations returned to control levels for the DT-14-W group, whereas the DT-14-(-)-Epi group was able to maintain capillary-to-fiber ratio (≈ 44%), CcO protein expression (≈ 45%), and activity (≈ 108%) above control levels. In addition, the increase in capillarity was related to decreased protein expression of thrombospondin-1, an antiangiogenic regulator. Furthermore, there were no significant differences in endurance capacity between the trained and DT-14-(-)-Epi groups. Our data suggest that (-)-epicatechin may be a suitable compound to maintain exercise-induced improved capillarity and mitochondrial capacity, even when exercise regimens are discontinued.