In silico design of a promiscuous chimeric multi-epitope vaccine against Mycobacterium tuberculosis.

Tuberculosis (TB) is a global health threat, killing approximately 1.5 million people each year. The eradication of Mycobacterium tuberculosis, the main causative agent of TB, is increasingly challenging due to the emergence of extensive drug-resistant strains. Vaccination is considered an effective way to protect the host from pathogens, but the only clinically approved TB vaccine, Bacillus Calmette-Guérin (BCG), has limited protection in adults. Multi-epitope vaccines have been found to enhance immunity to diseases by selectively combining epitopes from several candidate proteins. This study aimed to design a multi-epitope vaccine against TB using an immuno-informatics approach. Through functional enrichment, we identified eight proteins secreted by M. tuberculosis that are either required for pathogenesis, secreted into extracellular space, or both. We then analyzed the epitopes of these proteins and selected 16 helper T lymphocyte epitopes with interferon-γ inducing activity, 15 cytotoxic T lymphocyte epitopes, and 10 linear B-cell epitopes, and conjugated them with adjuvant and Pan HLA DR-binding epitope (PADRE) using appropriate linkers. Moreover, we predicted the tertiary structure of this vaccine, its potential interaction with Toll-Like Receptor-4 (TLR4), and the immune response it might elicit. The results showed that this vaccine had a strong affinity for TLR4, which could significantly stimulate CD4+ and CD8+ cells to secrete immune factors and B lymphocytes to secrete immunoglobulins, so as to obtain good humoral and cellular immunity. Overall, this multi-epitope protein was predicted to be stable, safe, highly antigenic, and highly immunogenic, which has the potential to serve as a global vaccine against TB.

Clp protease and antisense RNA jointly regulate the global regulator CarD to mediate mycobacterial starvation response.

Under starvation conditions, bacteria tend to slow down their translation rate by reducing rRNA synthesis, but the way they accomplish that may vary in different bacteria. In Mycobacterium species, transcription of rRNA is activated by the RNA polymerase (RNAP) accessory transcription factor CarD, which interacts directly with RNAP to stabilize the RNAP-promoter open complex formed on rRNA genes. The functions of CarD have been extensively studied, but the mechanisms that control its expression remain obscure. Here, we report that the level of CarD was tightly regulated when mycobacterial cells switched from nutrient-rich to nutrient-deprived conditions. At the translational level, an antisense RNA of carD (AscarD) was induced in a SigF-dependent manner to bind with carD mRNA and inhibit CarD translation, while at the post-translational level, the residual intracellular CarD was quickly degraded by the Clp protease. AscarD thus worked synergistically with Clp protease to decrease the CarD level to help mycobacterial cells cope with the nutritional stress. Altogether, our work elucidates the regulation mode of CarD and delineates a new mechanism for the mycobacterial starvation response, which is important for the adaptation and persistence of mycobacterial pathogens in the host environment.

Phytochemical analysis, antioxidant and anti-inflammatory potential of FERETIA APODANTHERA root bark extracts.

BACKGROUND: Inflammation has been implicated in many disorders, including cancer and available therapies elicit adverse effects. Plants of the family Rubiaceae have shown potency against inflammation. The anti-inflammatory and anti-oxidant potential of Feretia apodanthera was investigated in this study to evaluate its effectiveness. METHODS: The phytochemical, antioxidant and anti-inflammatory potential of root bark (n-Hexane, diethyl ether, ethanol and aqueous) extracts of Feretia apodanthera was investigated in this study. The extracts were subjected to various chemical tests for phytochemical constituents; their antioxidant activity was determined using in-vitro DPPH radical scavenging activity assay and their anti-inflammatory activity was determined using carrageenan induced paw oedema model. FTIR and GCMS analysis was done to determine the compounds present. RESULTS: Phytochemical screening of extracts revealed the presence of unsaturated steroids, triterpenes, cardiac glycosides, tannins, saponin and alkaloids. Vitamin C had a median inhibitory concentration (IC50) of 0.038 mg/ml which was lower than IC50 of all the extracts. Of all the extracts, ethanol extract had the lowest IC50 (0.044 mg/ml) which is comparable to vitamin C. Anti-inflammatory studies showed that the inflammation inhibition potential of 400 mg/kg body weight of all the extracts was significantly lower (p < 0.05) than the standard ketoprofen (50 mg/kg) at the first three hours but significantly higher (p < 0.05) at the fourth hour. At the fifth hour, the inflammation inhibition potential of diethyl ether, ethanol and aqueous extracts were significantly higher (p < 0.05) than that of the standard. FTIR analysis showed the presence of ketones, amines, alkenes and carboxylic groups. GCMS analysis revealed compounds that are potential anti-inflammatory agents. CONCLUSION: This study revealed that extracts of Feretia apodanthera possess anti-inflammatory effects against right hind paw oedema of albino rats and can act as an effective antioxidant.

Transcriptome Landscape of Mycobacterium smegmatis.

The non-pathogenic bacterium Mycobacterium smegmatis mc2155 has been widely used as a model organism in mycobacterial research, yet a detailed study about its transcription landscape remains to be established. Here we report the transcriptome, expression profiles and transcriptional structures through growth-phase-dependent RNA sequencing (RNA-seq) as well as other related experiments. We found: (1) 2,139 transcriptional start sites (TSSs) in the genome-wide scale, of which eight samples were randomly selected and further verified by 5'-RACE; (2) 2,233 independent monocistronic or polycistronic mRNAs in the transcriptome within the operon/sub-operon structures which are classified into five groups; (3) 47.50% (1016/2139) genes were transcribed into leaderless mRNAs, with the TSSs of 41.3% (883/2139) mRNAs overlapping with the first base of the annotated start codon. Initial amino acids of MSMEG_4921 and MSMEG_6422 proteins were identified by Edman degradation, indicating the presence of distinctive widespread leaderless features in M. smegmatis mc2155. (4) 150 genes with potentially wrong structural annotation, of which 124 proposed genes have been corrected; (5) eight highly active promoters, with their activities further determined by ß-galactosidase assays. These data integrated the transcriptional landscape to genome information of model organism mc2155 and lay a solid foundation for further works in Mycobacterium.

HISTOLOGICAL FEATURES OF PSEUDOTUMOR AFTER SMALL HEAD DIAMETER METAL-ON-METAL TOTAL HIP ARTHROPLASTY.

The pathologic aspects of periprosthetic tissues in failed second-generation metal-on-metal (MoM) resurfacing hip arthroplasties have been widely described in terms of necrosis and inflammation. To our knowledge little data are reported on the association of this lesion with the use of small head diameter (less than 32 mm). In this study we present a small series of pseudo-tumor in small head metal-on-metal total hip arthroplasty focusing our attention on the histologic aspects of the harvested pathologic tissue. The histological examination of our cases showed a presence of lymphocytic infiltrate suggesting a delayed hypersensitivity reaction to metal of type IV (ALVAL) but different to each other in terms of the prevalence of the cellular component. If macrophages are predominant, the pathogenetic mechanism seems to be the reaction against metallic particle. On the other hand, if granulocytes are predominant, it is possible to consider a hypersensitivity reaction. Our observation suggests that the evidence of Pseudotumor in case of small-head metal-on-metal arthroplasty should be considered with the same properties of big-head and therefore these patients should be followed scrupulously.

The use of a preformed spacer in two-stage revision of infected hip arthroplasties.

Two-stage revision with the use of an antibiotic-loaded cement spacer has spread widely as a successful treatment for THA infection. Between 1999 and 2008, 28 patients with infected THA were treated with two-stage implant revision using a preformed spacer. The spacer was left in situ for 5.5 months (range 1-13 months), and the patients were allowed to walk with partial weight bearing. At a mean follow-up of 53 months (range 18-106 months), recurrence of infection was observed in only one patient. Complications were observed in five patients: three spacer dislocations, one distal femoral fracture occurred during stem removal, and one femoral artery pseudo-aneurysm. The mean HHS increased from 43 points (range 13-77) to 82 points (range 35-96). Though small prospective studies are reported in literature, good eradication rate and good functional outcomes encourage for the use of an antibiotic-loaded cement spacer. The industrial production ensures procedure standardization, well-defined physical and chemical properties to the device and eliminates time necessary to intraoperatory manufacturing.

Etiology of portal vein thrombosis in adults. A prospective evaluation of primary myeloproliferative disorders.

In a prospective study of 33 adults with portal vein thrombosis unrelated to a liver tumor, we have assessed the prevalence of primary myeloproliferative disorders using conventional criteria and cultures of bone marrow progenitor cells. A primary myeloproliferative disorder was documented in 14 patients investigated at the time of recognition of portal vein thrombosis. Among these 14 patients, the main clue to the presence of the myeloproliferative disorder was (a) the observation of suggestive abnormalities of peripheral blood cell counts in 4 patients; (b) characteristic findings at bone marrow biopsy or determination of total red cell volume in 3 patients; and (c) formation of "spontaneous" erythroid colonies in cultures of bone marrow progenitor cells in erythropoietin-poor medium in 7 patients. In 2 other patients, agnogenic myeloid metaplasia with myelosclerosis of apparently recent onset developed 5 yr after recognition of portal vein thrombosis. In conclusion, primary myeloproliferative disorders--in a full-blown or latent form, or at an early stage--are a major cause of portal vein thrombosis.