Immunoinformatics Driven Prediction of Multiepitopic Vaccine Against Klebsiella pneumoniae and Mycobacterium tuberculosis Coinfection and Its Validation via In Silico Expression

International Journal of Peptide Research and Therapeutics - Klebsiella pneumoniae and Mycobacterium tuberculosis coinfection is one of the most lethal combinations that has been becoming frequent...     

Prenatal Exposure to Fenugreek Impairs Sensorimotor Development and the Operation of Spinal Cord Networks in Mice

Fenugreek is a medicinal plant whose seeds are widely used in traditional medicine, mainly for its laxative, galactagogue and antidiabetic effects. However, consumption of fenugreek seeds during pregnancy has been associated with a range of congenital malformations, including hydrocephalus, anencephaly and spina bifida in humans. The present study was conducted to evaluate the effects of prenatal treatment of fenugreek seeds on the development of sensorimotor functions from birth to young adults. Pregnant mice were treated by gavage with 1g/kg/day of lyophilized fenugreek seeds aqueous extract (FSAE) or distilled water during the gestational period. Behavioral tests revealed in prenatally treated mice a significant delay in righting, cliff avoidance, negative geotaxis responses and the swimming development. In addition, extracellular recording of motor output in spinal cord isolated from neonatal mice showed that the frequency of spontaneous activity and fictive locomotion was reduced in FSAE-exposed mice. On the other hand, the cross-correlation coefficient in control mice was significantly more negative than in treated animals indicating that alternating patterns are deteriorated in FSAE-treated animals. At advanced age, prenatally treated mice displayed altered locomotor coordination in the rotarod test and also changes in static and dynamic parameters assessed by the CatWalk automated gait analysis system. We conclude that FSAE impairs sensorimotor and coordination functions not only in neonates but also in adult mice. Moreover, spinal neuronal networks are less excitable in prenatally FSAE-exposed mice suggesting that modifications within the central nervous system are responsible, at least in part, for the motor impairments.     

Early lesion of the reticular thalamic nucleus disrupts the structure and function of the mediodorsal thalamus and prefrontal cortex

The thalamic reticular nucleus (TRN), part of the thalamus, is a thin GABAergic cell layer adjacent to the relay nuclei of the dorsal thalamus. It receives input from the cortex and other thalamic nuclei and provides major inhibitory input to each thalamic nucleus, particularly the mediodorsal nucleus (MD). As the MD is important for supporting optimal cortico–thalamo–cortical interactions during brain maturation, we hypothesized that that early damage to the TRN will cause major disturbances to the development and the functioning of the prefrontal cortex (PFC) and the MD. Rat pups at P4 were randomized in three groups: electrolytic lesion of TRN, TRN‐sham‐lesion group, and the classical control group. Seven weeks later, all rats were tested with several behavioral and cognitive paradigms, and then perfused for histological and immunohistochemical studies. Results showed that TRN lesion rats exhibited reduced spontaneous activity, high level of anxiety, learning and recognition memory impairments. Besides the behavioral effects observed after early TRN lesions, our study showed significant cytoarchitectural and functional changes in the cingulate cortex, the dorsolateral and prelimbic subdivisions of the PFC, as well as in the MD. The assessment of the basal levels of neuronal activity revealed a significant reduction of the basal expression of C‐Fos levels in the PFC. These experiments, which are the first to highlight the effects of early TRN lesions, provided evidence that early damage of the anterior part of the TRN leads to alterations that may control the development of the thalamocortical–corticothalamic pathways.     

Status of Selected Heavy Metal Distribution in Scalp Hair of Traffic Control Personnel Exposed to Vehicular Emissions

Scalp hair samples of traffic control personnel (n = 71, ages between 25 to 45 years) were analyzed for 11 selected metals by using an Inductively Coupled Argon Plasma Atomic Emission (ICP-AE) technique using nitric acid–perchloric acid based wet digestion method. The observed order of mean concentrations (μ g/g, dry weight) of the metals, in washed hair samples, was: Ca > Mg > Zn > Fe > Pb > Cu > Ni > Mn > Cr > Cd > Co, with corresponding metal levels at 1042.2, 182.4, 169.7, 13.6, 12.4, 11.1, 2.7, 1.9, 1.9, 0.8, and 0.7 μ g/g, respectively. Most of the unwashed samples exhibited 10–15% higher metal levels compared with those in washed samples. A positive metal-to-metal correlation (p < 0.01) was observed for the metal pairs: Ca-Mg (r = 0.737), Pb-Cr (r = 0.441), and Cu-Zn (0.385). Principal Component Analysis (PCA) extracted 6 factors as metals origin using varimax normalized rotation commutatively representing more than 76% of the total variance. Cluster Analysis (CA) showed five strong clusters of selected metals in the hair of the subjects: Age-Exposure, Ca-Mg, Cd-Fe-Mn, Pb-Cr, and Cu-Zn. The results of the current study were compared with those for two other occupationally exposed groups, metal arc welders and autodrivers, using published literature values. The traffic controllers in our study generally, with the exception of Cu, exhibited lower levels of metals in hair samples than did autodrivers and arc welders.     

Transduction of multiple cell types using improved conditions for gene delivery and expression of SV40 pseudovirions packaged in vitro

This comprehensive study demonstrates highly efficient transduction of a wide variety of human, murine, and monkey cell lines, using a procedure for in vitro packaging of plasmid DNA in recombinant simian virus 40 (SV40) capsid proteins to form pseudovirions. The pseudovirions are encapsidated by the VP1 major capsid protein, with no SV40 sequence requirement, and are able to carry up to 17.7 kb of supercoiled plasmid DNA. We developed a procedure to scale-up production of SV40 pseudovirions, as well as an efficient protocol to concentrate the virions with no loss of activity. We also developed a method that allows transduction of 10 times more cells than the original protocol. This protocol was tested using supercoiled in vitro-packaged plasmid carrying the human multidrug-resistance gene (MDR1 encoding P-glycoprotein; P-gp), or the enhanced green fluorescent protein reporter gene (EGFP) in .45 human lymphoblastoid cells and in K562 human erythroleukemia cells. Multiple transductions at 24-h intervals were shown to increase expression using the EGFP reporter gene. The protocols developed in this study establish in vitro-packaged SV40 pseudovirions as one of the most efficient gene delivery systems.     

A New Broad-Spectrum Peptide Antibiotic Produced by <Emphasis Type="Italic">Bacillus brevis</Emphasis> Strain MH9 Isolated from Margalla Hills of Islamabad,Pakistan

The antimicrobial peptide from a bacterial strain is isolated from soil sample of Margalla Hills of Islamabad, Pakistan. The peptide is found to significantly inhibit the growth of both Gram-positive (Staphylococcus aureus ATCC 6538 and Micrococcus luteus ATCC 10240) and Gram-negative (Escherichia coli ATCC 25922 and Salmonella typhi ATCC 14028) bacteria as compared to gramicidin as standard. The bacterium is identified as Bacillus brevis strain MH9 based on phenotype and phylogenetic analysis. The antibacterial polypeptide was produced optimally at 35 °C after 48 h of growth, precipitated by 50 % ammonium sulphate, and further purified using HPLC. The sequential steps of purification decrease the peptide contents with prominent antibacterial activity. The peptide composed of 11 amino acid was further characterized by FT-IR and NMR. Results suggested that the peptide molecule is a novel antibacterial agent that is effective against both Gram-positive and Gram-negative bacteria. This study may have important implications for new peptide antibiotic that could be a new addition to treat infections.     

Impact of interleukin-6 on hypoxia-induced pulmonary hypertension and lung inflammation in mice

Background

Inflammation may contribute to the pathogenesis of various forms of pulmonary hypertension (PH). Recent studies in patients with idiopathic PH or PH associated with underlying diseases suggest a role for interleukin-6 (IL-6).

Methods

To determine whether endogenous IL-6 contributes to mediate hypoxic PH and lung inflammation, we studied IL-6-deficient (IL-6^-/-) and wild-type (IL-6^+/+) mice exposed to hypoxia for 2 weeks.

Results

Right ventricular systolic pressure, right ventricle hypertrophy, and the number and media thickness of muscular pulmonary vessels were decreased in IL-6^-/- mice compared to wild-type controls after 2 weeks'' hypoxia, although the pressure response to acute hypoxia was similar in IL-6^+/+ and IL-6^-/- mice. Hypoxia exposure of IL-6^+/+ mice led to marked increases in IL-6 mRNA and protein levels within the first week, with positive IL-6 immunostaining in the pulmonary vessel walls. Lung IL-6 receptor and gp 130 (the IL-6 signal transducer) mRNA levels increased after 1 and 2 weeks'' hypoxia. In vitro studies of cultured human pulmonary-artery smooth-muscle-cells (PA-SMCs) and microvascular endothelial cells revealed prominent synthesis of IL-6 by PA-SMCs, with further stimulation by hypoxia. IL-6 also markedly stimulated PA-SMC migration without affecting proliferation. Hypoxic IL-6^-/- mice showed less inflammatory cell recruitment in the lungs, compared to hypoxic wild-type mice, as assessed by lung protein levels and immunostaining for the specific macrophage marker F4/80, with no difference in lung expression of adhesion molecules or cytokines.

Conclusion

These data suggest that IL-6 may be actively involved in hypoxia-induced lung inflammation and pulmonary vascular remodeling in mice.     

Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma

Background

Complex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes have been limited. Deciphering the molecular mechanism that differentiate between normal and disease state may lead to identification of biomarkers for carcinoma.

Results

Many proteins displayed differential expression when nuclear membrane proteome of hepatocellular carcinoma (HCC), fibrotic liver, and HepG2 cell line were assessed using 2-DE and ESI-Q-TOF MS/MS. From the down regulated set in HCC, we have identified for the first time a 15 KDa cytochrome b5A (CYB5A), ATP synthase subunit delta (ATPD) and Hemoglobin subunit beta (HBB) with 11, 5 and 22 peptide matches respectively. Furthermore, nitrosylation studies with S-nitrosocysteine followed by immunoblotting with anti SNO-cysteine demonstrated a novel and biologically relevant post translational modification of thiols of CYB5A in HCC specimens only. Immunofluorescence images demonstrated increased protein S-nitrosylation signals in the tumor cells and fibrotic region of HCC tissues. The two other nuclear membrane proteins which were only found to be nitrosylated in case of HCC were up regulated ATP synthase subunit beta (ATPB) and down regulated HBB. The decrease in expression of CYB5A in HCC suggests their possible role in disease progression. Further insight of the functional association of the identified proteins was obtained through KEGG/ REACTOME pathway analysis databases. String 8.3 interaction network shows strong interactions with proteins at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor of liver pathology.

Conclusion

These findings may have broader implications for understanding the mechanism of development of carcinoma. However, large scale studies will be required for further verification of their critical role in development and progression of HCC.