Comparative genomic and proteomic analyses of PE/PPE multigene family of Mycobacterium tuberculosis H₃₇Rv and H₃₇Ra reveal novel and interesting differences with implications in virulence

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease taking one human life every 15 s globally. The two well-characterized strains H37Rv and H37Ra, derived from the same parental strain M. tuberculosis H37, show dramatically different pathogenic phenotypes. PE/PPE gene family, comprising of 176 open reading frames and present exclusively in genus Mycobacterium, accounts for ∼10% of the M. tuberculosis genome. Our comprehensive in silico analyses of PE/PPE family of H37Ra and virulent H37Rv strains revealed genetic differences between these strains in terms of several single nucleotide variations and InDels and these manifested in changes in physico-chemical properties, phosphorylation sites, and protein: protein interacting domains of the corresponding proteomes. Similar comparisons using the 13 sigma factor genes, 36 members of the mammalian cell entry family, 13 mycobacterial membrane protein large family members and 11 two-component signal transduction systems along with 5 orphaned response regulators and 2 orphaned sensor kinases failed to reveal very significant difference between H37Rv and H37Ra, reinforcing the importance of PE/PPE genes. Many of these changes between H37Rv and H37Ra can be correlated to differences in pathogenesis and virulence of the two strains

Efficacy of sub-Tenon's block using an equal volume of local anaesthetic administered either as a single or as divided doses. A randomised clinical trial

<p>Abstract</p> <p>Background</p> <p>Sub-Tenon's anaesthetic is effective and reliable in producing both akinesia and anaesthesia for cataract surgery. Our clinical experience indicates that it is sometimes necessary when absolute akinesia is required during surgery to augment the block with 1–2 ml of local anaesthetic. Hypothesis was that after first injection some of the volume injected may spill out and before second injection the effect of hyaluronidase has taken place and second volume injectate will have desired effect.</p> <p>Methods</p> <p>A prospective, randomised, control trial in which patients were randomly allocated to one of two groups. In group 1, single injection of 5 ml of local anaesthetic was injected. In group 2, 3 ml of the same anaesthetic solution was injected followed by application of gentle orbital pressure for 2 minutes. A further 2 ml of the same anaesthetic solution was injected through the same conjunctival incision. Measurement of movement in four quadrants of eye was done by the surgeon at 3 and 6 minutes. Intraocular pressure, chemosis, and subconjuctival haemorrhage were also measured.</p> <p>Results</p> <p>Significant differences at 3 minutes between groups for overall movement, medial, superior, and lateral quadrants occurred. At 6 minutes no significant group differences emerged for the overall movement or for any of four quadrants.</p> <p>Conclusion</p> <p>Single injection of local anaesthesia for sub-Tenon's block with mixture of lignocaine with adrenaline, bupivacaine and hyaluronidase was found to be superior to provide akinesia of ocular muscles compared to divided dose given by two injections. No difference in groups in terms of haemorrhage, chemosis, patient's satisfaction and intraocular pressure was found.</p> <p>Trial registration</p> <p>Trial registration no-ISRCTN73431052</p

Physiologically relevant screening of polyphenol-rich commercial preparations for bioactivity in vascular endothelial cells and application to healthy volunteers: A viable workflow and a cautionary tale

YesThis study describes the screening of 13 commercially-available plant extracts for pharmacological activity modulating vascular function using an endothelial cell model. A French maritime pine bark extract (FMPBE) was found to have the greatest effect upon nitric oxide availability in control (181% ± 36% of untreated cells) and dysfunctional cells (132% ± 8% of untreated control cells). In healthy volunteers, the FMPBE increased plasma nitrite concentrations 8 h post-consumption compared to baseline (baseline corrected median 1.71 ± 0.38 (25% IQR) and 4.76 (75% IQR) µM, p 0.05), however HPLC-UV fingerprinting showed that the new batch had a 5-15% in major constituents (including procyanidins A2, B1 and B2) compared to the original batch. This research describes a robust mechanism for screening bioactive extracts for vascular effects. It also highlights batch variability as a significant limitation when using complex extracts for pharmacological activity, and suggests the use of in vitro systems as a tool to identify this problem in future studies

Concurrent Proinflammatory and Apoptotic Activity of a Helicobacter pylori Protein (HP986) Points to Its Role in Chronic Persistence

Helicobacter pylori induces cytokine mediated changes in gastroduodenal pathophysiology, wherein, the activated macrophages at the sub-mucosal space play a central role in mounting innate immune response against the antigens. The bacterium gains niche through persistent inflammation and local immune-suppression causing peptic ulcer disease or chronic gastritis; the latter being a significant risk factor for the development of gastric adenocarcinoma. What favors persistence of H. pylori in the gastric niches is not clearly understood. We report detailed characterization of a functionally unknown gene (HP986), which was detected in patient isolates associated with peptic ulcer and gastric carcinoma. Expression and purification of recombinant HP986 (rHP986) revealed a novel, ∼29 kDa protein in biologically active form which associates with significant levels of humoral immune responses in diseased individuals (p<0.001). Also, it induced significant levels of TNF-α and Interleukin-8 in cultured human macrophages concurrent to the translocation of nuclear transcription factor-κB (NF-κB). Further, the rHP986 induced apoptosis of cultured macrophages through a Fas mediated pathway. Dissection of the underlying signaling mechanism revealed that rHP986 induces both TNFR1 and Fas expression to lead to apoptosis. We further demonstrated interaction of HP986 with TNFR1 through computational and experimental approaches. Independent proinflammatory and apoptotic responses triggered by rHP986 as shown in this study point to its role, possibly as a survival strategy to gain niche through inflammation and to counter the activated macrophages to avoid clearance

Mechanistic Insights into a Novel Exporter-Importer System of Mycobacterium tuberculosis Unravel Its Role in Trafficking of Iron

Elucidation of the basic mechanistic and biochemical principles underlying siderophore mediated iron uptake in mycobacteria is crucial for targeting this principal survival strategy vis-à-vis virulence determinants of the pathogen. Although, an understanding of siderophore biosynthesis is known, the mechanism of their secretion and uptake still remains elusive.Here, we demonstrate an interplay among three iron regulated Mycobacterium tuberculosis (M.tb) proteins, namely, Rv1348 (IrtA), Rv1349 (IrtB) and Rv2895c in export and import of M.tb siderophores across the membrane and the consequent iron uptake. IrtA, interestingly, has a fused N-terminal substrate binding domain (SBD), representing an atypical subset of ABC transporters, unlike IrtB that harbors only the permease and ATPase domain. SBD selectively binds to non-ferrated siderophores whereas Rv2895c exhibits relatively higher affinity towards ferrated siderophores. An interaction between the permease domain of IrtB and Rv2895c is evident from GST pull-down assay. In vitro liposome reconstitution experiments further demonstrate that IrtA is indeed a siderophore exporter and the two-component IrtB-Rv2895c system is an importer of ferrated siderophores. Knockout of msmeg_6554, the irtA homologue in Mycobacterium smegmatis, resulted in an impaired M.tb siderophore export that is restored upon complementation with M.tb irtA.Our data suggest the interplay of three proteins, namely IrtA, IrtB and Rv2895c in synergizing the balance of siderophores and thus iron inside the mycobacterial cell

Combination of adenoviral virotherapy and temozolomide chemotherapy eradicates malignant glioma through autophagic and apoptotic cell death in vivo

Conditionally replicative adenoviruses (CRAds) represent a novel treatment strategy for malignant glioma. Recent studies suggest that the cytopathic effect elicited by these vectors is mediated through autophagy, a form of programmed cell death. Likewise, temozolomide (TMZ), a chemotherapeutic agent used for the treatment of malignant gliomas, also triggers autophagic cell death. In this study, we examined the potential to combine the two treatments in the setting of experimental glioma. In vitro, pretreatment with TMZ followed by CRAd-Surivin-pk7 enhanced cytotoxicity against a panel of glioma cell lines. Western blot analysis showed increased expression of BAX and p53, decreased expression of BCL2 and elevated level of APG5. Treatment with TMZ followed by CRAd-Survivin-pk7 (CRAd-S-pk7) led to a significant over-expression of autophagy markers, acidic vesicular organelles and light-chain 3 (LC3). These results were further evaluated in vivo, in which 90% of the mice with intracranial tumours were long-term survivors (>100 days) after treatment with TMZ and CRAd-S-pk7 (P<0.01). Analysis of tumours ex vivo showed expression of both LC3 and cleaved Caspase-3, proving that both autophagy and apoptosis are responsible for cell death in vivo. These results suggest that combination of chemovirotherapy offers a powerful tool against malignant glioma and should be further explored in the clinical setting

Biochemical characterization of S&#402;9 Sp-family-like protein factors reveals interesting features

We earlier documented the involvement of novel Sp-family-like protein factors in transcription from the Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin (polh) gene promoter [Ramachandran et al. (2001) J. Biol. Chem. 276: 23440-23449]. These zinc-dependent Sp-like factors bind to two putative Sp-factor-binding motifs, present within the AcSp sequence upstream of the polh promoter, with very high affinity (Kd = 2.1 &#215; 10-12M). Like other polh-promoter-associated host transcription factors, these Sp-like factors display tolerance to high ion concentrations up to even 3M NaCl. An electrophoretic mobility shift assay demonstrated a probable cross-talk between the Spodoptera frugiperda (S&#402;9) Sp-family-like proteins and the TFIID complex. In complementary experiments, specific replacements of the Sp-factor-binding motifs with TATA-like elements resulted in expression of a luciferase reporter gene to almost the same level as that obtained with a wild-type native construct. Our results point to the possibility of the involvement of TFIID and S&#402;9 Sp protein interaction in transcription from the baculovirus polyhedrin promoter

A multicopy DNA sequence from Meconopsis simplicifolia discriminates between the different species of this endangered Himalayan poppy

Clones harboring multicopy DNA sequences were isolated on the basis of reverse genome hybridization to Meconopsis paniculata (Himalayan yellow poppy) DNA from a Sau3A partial genomic plasmid library of M. simplicifolia (Himalayan blue poppy). Restriction-endonuclease-dependent genetic polymorphism between five species of Meconopsis, M. aculeata, M. paniculata, M. simplicifolia, M. sinuata and M. villosa, belonging to geographically isolated populations, was evident in genomic DNA filter hybridizations when probed with a clone (pIMS10) isolated from this library. Pooled DNA from seedlings originating from plants of individual populations of M. paniculata, M. simplicifolia and M. villosa gave similar band patterns, with respect to a given enzyme, suggesting intra-population genetic homogeneity

Molecular cloning of human satellite DNA sequences and their use in detecting DNA polymorphism

A approximately 400 bp HaeIII human genomic satellite DNA band was cloned into pUC18 to construct a partial library. A fragment of bacteriophage M13 containing a sequence homologous to the human minisatellite core was cloned in pUC18 and was used as a probe to isolate a approximately 350 bp human satellite clone (pTRF5.6) from the partial library. Other clones from this library showed a wide variation in terms of size and hybridization to the pTRF5.6 clone. Human DNA from different individuals was digested with restriction enzymes, Southern transferred and probed with TRF5.6. Individual-specific complex pattern of DNA bands was produced. TRF5.6, therefore, could be useful as a probe for detecting genetic polymorphism

β-Subunit of human chorionic gonadotropin hormone and firefly luciferase simultaneously synthesized in insect cells using a recombinant baculovirus are differentially expressed and transported

A recombinant baculovirus vAcβhCG-luc was constructed that carried the cDNAs encoding firefly luciferase (luc) and β-subunit of human chorionic gonadotropin (βhCG) placed under the transcriptional control of individual copies of the baculovirus polyhedrin gene promoter. The simple, rapid, and sensitive detection of LUC expression was used for selecting recombinant viruses that simultaneously expressed βhCG, which was identical in all respects to that synthesized using a recombinant baculovirus carrying the βhCG gene alone. Immunofluorescence staining of virus-infected cells using anti-LUC antibodies revealed that LUC, a nonglycosylated, intracellular protein was retained within the cells whereas βhCG, an extensively glycosylated, secretory protein, was processed and secreted into the culture medium. LUC and βhCG were both immunoreactive on Western blot. βhCG was bioactive, as evident from its ability to associate with ahCG and bind with the receptor and produce testosterone in an in vitro mouse Leydig cell assay system. Comparison of recombinant LUC and βhCG synthesized by the virus-infected insect cells surprisingly revealed that the level of the former was quantitatively higher by at least 10-fold than the latter. A blot of total RNA isolated from vAcβhCG-luc-infected insect cells, when probed with probes corresponding to the 3' region of the βhCG or lue genes, indicated differential transcription of the two genes. Computer-aided sequence analysis indicated extensive secondary structure and stem-loop complex-forming potential of the βhCG gene, which could be responsible for the transcriptional difference observed