Department of Biotechnology and Bioinformatics

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    2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid (Activator-3) is a potent activator of AMPK
    ( 2018-12-01) Bung, Navneet ; Surepalli, Sobhitha ; Seshadri, Sriram ; Patel, Sweta ; Peddasomayajula, Saranya ; Kummari, Lalith Kumar ; Kumar, Sireesh T. ; Babu, Phanithi Prakash ; Parsa, Kishore V.L. ; Poondra, Rajamohan Reddy ; Bulusu, Gopalakrishnan ; Misra, Parimal
    AMPK is considered as a potential high value target for metabolic disorders. Here, we present the molecular modeling, in vitro and in vivo characterization of Activator-3, 2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid, an AMP mimetic and a potent pan-AMPK activator. Activator-3 and AMP likely share common activation mode for AMPK activation. Activator-3 enhanced AMPK phosphorylation by upstream kinase LKB1 and protected AMPK complex against dephosphorylation by PP2C. Molecular modeling analyses followed by in vitro mutant AMPK enzyme assays demonstrate that Activator-3 interacts with R70 and R152 of the CBS1 domain on AMPK γ subunit near AMP binding site. Activator-3 and C2, a recently described AMPK mimetic, bind differently in the γ subunit of AMPK. Activator-3 unlike C2 does not show cooperativity of AMPK activity in the presence of physiological concentration of ATP (2 mM). Activator-3 displays good pharmacokinetic profile in rat blood plasma with minimal brain penetration property. Oral treatment of High Sucrose Diet (HSD) fed diabetic rats with 10 mg/kg dose of Activator-3 once in a day for 30 days significantly enhanced glucose utilization, improved lipid profiles and reduced body weight, demonstrating that Activator-3 is a potent AMPK activator that can alleviate the negative metabolic impact of high sucrose diet in rat model.
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    23 years of the discovery of Helicobacter pylori: Is the debate over?
    ( 2005-10-31) Ahmed, Niyaz
    The Gram negative curved bacillus H. pylori has become the prize bug of all times. Barry Marshall and Robin Warren the two discoverers of this organism have been awarded with this year's Nobel Prize. The Nobel committee at the Karolinska Institute of Sweden has selected this paradigm shift discovery of 1982 as the most impacting in medical sciences. This award has surprised many as the Nobel assembly has selected this 'Robert Koch styled medical detective work' for the prize as compared to many outstanding basic research stories on the waitlist. This editorial briefly touches the significant impact of H. pylori on gastroduodenal management and the path forward as the bug has become quite controversial in recent times. © 2005 Ahmed, licensee BioMed Central Ltd.
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    3D modeling of dengue virus NS4B and Chikungunya virus nsP4: Identification of a common drug target and designing a single antiviral inhibitor
    ( 2014-01-01) Satheesh, Garisekurthi ; Prabhu, Nagu P. ; Venkataramana, Musturi
    Dengue and chikungunya virus infections are one of the major causes of morbidity and mortality in tropical and sub-tropical regions of the world. These two viruses belong to two different families with many similarities and dissimilarities. Both are enveloped viruses and the mode of transmission is also by the same mosquito species. Especially in case of symptom expression, there is confusion between these two viruses. Reports indicate the overlapping endemic areas and co-infections of both viruses in a single patient. The above factors indicate that there is a need for developing a single drug/vaccine for both the viruses. As a first report in this direction, we have used the bioinformatics tools to identify a common target in both the viruses for a single inhibitor molecule. Phylogenetic and distance based analyses using the nucleotide sequences of arthropod and non-arthropod borne viruses indicated a common origin of evolutionary point for mosquito borne viruses, irrespective of their families. Similarly, the amino acid sequences of non-structural protein-4B (NS4B) of dengue virus and non-structural protein-P4 (nsP4) of chikungunya virus showed a common evolutionary origin. Modeled and superimposed 3D-structures of above two proteins showed a common alpha helix. Virtual screening of selected molecules was done to identify the molecules which can bind to the identified common helix and found that N-(p-tolylmethyl)-3-[(3-pyridylmethylamino)methyl]benzamide (TPB) has significant binding characteristics to the common helix. Molecular simulations indicated that both the protein-TPB complexes were stable. Therefore, we propose that TPB or its analogues could act as antiviral agents against both the viruses.
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    3D modeling of dengue virus NS4B and Chikungunya virus nsP4: Identification of a common drug target and designing a single antiviral inhibitor
    ( 2014-01-01) Satheesh, Garisekurthi ; Prabhu, Nagu P. ; Venkataramana, Musturi
    Dengue and chikungunya virus infections are one of the major causes of morbidity and mortality in tropical and sub-tropical regions of the world. These two viruses belong to two different families with many similarities and dissimilarities. Both are enveloped viruses and the mode of transmission is also by the same mosquito species. Especially in case of symptom expression, there is confusion between these two viruses. Reports indicate the overlapping endemic areas and co-infections of both viruses in a single patient. The above factors indicate that there is a need for developing a single drug/vaccine for both the viruses. As a first report in this direction, we have used the bioinformatics tools to identify a common target in both the viruses for a single inhibitor molecule. Phylogenetic and distance based analyses using the nucleotide sequences of arthropod and non-arthropod borne viruses indicated a common origin of evolutionary point for mosquito borne viruses, irrespective of their families. Similarly, the amino acid sequences of non-structural protein-4B (NS4B) of dengue virus and non-structural protein-P4 (nsP4) of chikungunya virus showed a common evolutionary origin. Modeled and superimposed 3D-structures of above two proteins showed a common alpha helix. Virtual screening of selected molecules was done to identify the molecules which can bind to the identified common helix and found that N-(p-tolylmethyl)-3-[(3-pyridylmethylamino)methyl]benzamide (TPB) has significant binding characteristics to the common helix. Molecular simulations indicated that both the protein-TPB complexes were stable. Therefore, we propose that TPB or its analogues could act as antiviral agents against both the viruses.
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    A biochemical analysis of topoisomerase II α and β kinase activity found in HIV-1 infected cells and virus
    ( 2005-09-01) Kondapi, Anand K. ; Padmaja, Gade ; Satyanarayana, N. ; Mukhopadyaya, Robin ; Reitz, Marvin S.
    Human topoisomerase II plays a crucial role in DNA replication and repair. It exists in two isoforms: topoisomerase II alpha (α) and topoisomerase II beta (β). The α isoform is localized predominantly in the nucleus, while the β isoform exhibits a reticular pattern of distribution both in the cytosol and in the nucleus. We show that both isoforms of topoisomerase II are phosphorylated in HIV infected cells and also by purified viral lysate. An analysis of the phosphorylation of topoisomerase II isoforms showed that extracts of HIV infected cells at 8 and 32 h. post-infection (p.i.) contain maximal phosphorylated topoisomerase II α, whereas infected cell extracts at 4 and 64 h p.i. contain maximum levels of phosphorylated topoisomerase II β. In concurrent to phosphorylated topoisomerase II isoforms, we have also observed increased topoisomerase II α kinase activity after 8 h p.i and topoisomerase β kinase activity at 4 and 64 h p.i. These findings suggest that both topoisomerase II α and β kinase activities play an important role in early as well as late stages of HIV-1 replication. Further analysis of purified virus showed that HIV-1 virion contained topoisomerase II isoform-specific kinase activities, which were partially isolated. One of the kinase activities of higher hydrophobicity can phosphorylate both topoisomerase II α and β, while lower hydrophobic kinase could predominantly phosphorylate topoisomerase II α. The phosphorylation status was correlated with catalytic activity of the enzyme. Western blot analysis using phosphoamino-specific antibodies shows that both the kinase activities catalyze the phosphorylation at serine residues of topoisomerase II α and β. The catalytic inhibitions by serine kinase inhibitors further suggest that the α and β kinase activities associated with virus are distinctly different. © 2005 Elsevier Inc. All rights reserved.
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    A comparative whole genome analysis of Helicobacter pylori from a human dense South Asian setting
    ( 2021-02-01) Qumar, Shamsul ; Nguyen, Trang Hoa ; Nahar, Shamsun ; Sarker, Nishat ; Baker, Stephen ; Bulach, Dieter ; Ahmed, Niyaz ; Rahman, Motiur
    Helicobacter pylori, a Gram-negative bacterium, is associated with a wide range of gastric diseases such as gastritis, duodenal ulcer, and gastric cancer. The prevalence of H pylori and risk of disease vary in different parts of the world based on the prevailing bacterial lineage. Here, we present a contextual and comparative genomics analysis of 20 clinical isolates of H pylori from patients in Bangladesh. Despite a uniform host ethnicity (Bengali), isolates were classified as being part of the HpAsia2 (50%) or HpEurope (50%) population. Out of twenty isolates, eighteen isolates were cagA positive, with two HpEurope isolates being cagA negative, three EPIYA motif patterns (AB, AB-C, and ABC-C) were observed among the cagA-positive isolates. Three vacA genotypes were observed with the s1m1i1dic1 genotype observed in 75% of isolates; the s1m2i1d1c2 and s2m2i2d2c2 genotypes were found to be 15% and 10% of isolates, respectively. The non-virulent genotypes s2m2i2d2c2 was only observed in HpEurope population isolates. Genotypic analysis of oipA gene, present in all isolates, revealed five different patterns of the CT repeat; all HpAsia2 isolates were in “ON” while 20% of HpEurope isolates were genotypically “OFF.” The three blood group antigen binding adhesins encoded genes (bab genes) examined and we observed that the most common genotype was (babA/babB/-) found in eight isolates, notably six were HpAsia2 isolates. The babA gene was found in all HpAsia2 isolates but present in only half of the HpEurope isolates. In silico antibiotic susceptibility analysis revealed that 40% of the strains were multi-drug resistant. Mutations associated with resistance to metronidazole, fluoroquinolone, and clarithromycin were detected 90%, 45%, and 5%, respectively, in H pylori strain. In conclusion, it is evident that two populations of H pylori with similar antibiotic profiles are predominant in Bangladesh, and it appears that genotypically the HpAisa2 isolates are potentially more virulent than the HpEurope isolates.
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    A conserved molecular action of native and recombinant Epap-1 in inhibition of HIV-1 gp120 mediated viral entry
    ( 2006-12-01) Roda Rani, K. P. ; Pelluru, Dheeraj ; Kondapi, Anand K.
    The early expression of Epap-1 (early pregnancy associated protein), a 90 kDa anti-HIV-1 active glycoprotein, in the first trimester placental tissue suggests that it is one of the innate immune factors/proteins protecting the fetus from HIV infections. In the present investigation, we have cloned and expressed Epap-1 in bacterial and baculovirus expression systems. The recombinant Epap-1 as well as native Epap-1 shows a conserved molecular mode of action. These proteins exhibit significant antiviral activity and inhibit the cell fusion reaction between gp120 expressing HeLa (HL2/3) cells and T cell line (SupT1). Further, the rhodamine labeled Epap-1 specifically bound to gp120 expressed on the surface of HL2/3 cells during fusion reaction thereby inhibiting viral entry. Analysis of the interacting gp120 epitopes revealed that Epap-1 binds specifically to epitopes of gp120, recognizing constant-5 (C5) region and the variable-3 (V3) epitope of gp120 expressed on HL2/3 cells; It exhibits specific interaction with C5 region of cell-free virus in four HIV-1 isolates suggesting that the molecular interaction of Epap-1 is specific and is highly conserved in binding to gp120 leading to inhibition of viral entry. Epap-1 can thus be a very efficient natural protection mechanism against cell-free and cell-associated viral infections during early pregnancy. © 2006 Elsevier Inc. All rights reserved.
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    A core and pan gene map of Leptospira genus and its interactions with human host
    ( 2022-01-01) Lata, Kumari Snehkant ; Kumar, Swapnil ; Vindal, Vaibhav ; Patel, Saumya ; Das, Jayashankar
    Leptospira species are the etiological agent of an emerging zoonotic disease known as “Leptospirosis” that substantially affects both human health and economy across the globe. Despite the global importance of the disease, pathogenetic features, host-adaptation and proper diagnosis of this bacteria remains lacking. To accomplish these gaps, pan-genome of Leptospira genus was explored in the present study. The pan-genome of Leptospira genus was comprised of core (692) and accessory parts (softcore:1804, shell:6432, cloud:16,600). The functional analysis revealed the abundancy of “Translation, ribosomal structure and biogenesis” COG class in core-genes; whereas in accessory parts, genes involved in signal transduction was the most abundant. Furthermore, pathogen-host interaction (PHI) analysis of core and accessory proteins with human proteins showed the presence of a total of 599 and 510 interactions, respectively. There were eight hubs in core PHI network and five hubs in PHI network of accessory proteins. The human's proteins involved in these interactions were found functionally enriched in metabolic processes, responses to stimulus and immune system processes. Further, pan-genome based phylogeny separated the Leptospira genus in three major clades (belonging to P1, P2 and S) which relates with their pathogenicity level. Additionally, pathogenic and saprophytic clade specific genes of Leptospira have also been identified and functionally annotated for COG, KEGG and virulence factors. The results revealed the presence of 102 pathogenic and 215 saprophytic group specific gene clusters. The COG functional annotation of pathogen specific genes showed that defence mechanism followed by signal transduction mechanisms category were most significantly enriched COG categories; whereas in saprophytic group, signal transduction mechanisms was the most abundant COG, suggesting their role in adaptation and hence important for microbe's evolution and survival. In conclusion, this study provides a new insight of genomic features of Leptospira genus which may further be implemented for development of better control actions of the disease.
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    A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany
    ( 2016-11-01) Sanchini, Andrea ; Semmler, Torsten ; Mao, Lei ; Kumar, Narender ; Dematheis, Flavia ; Tandon, Kshitij ; Peddireddy, Vidyullatha ; Ahmed, Niyaz ; Lewin, Astrid
    Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n = 41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A–H (sized 6.2–73.3 kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.
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    A knowledge driven supervised learning approach to identify gene network of differentially up-regulated genes during neuronal senescence in Rattus norvegicus
    ( 2015-09-01) Dholaniya, Pankaj Singh ; Ghosh, Soumitra ; Surampudi, Bapi Raju ; Kondapi, Anand K.
    Various approaches have been described to infer the gene interaction network from expression data. Several models based on computational and mathematical methods are available. The fundamental thing in the identification of the gene interaction is their biological relevance. Two genes belonging to the same pathway are more likely to affect the expression of each other than the genes of two different pathways. In the present study, interaction network of genes is described based on upregulated genes during neuronal senescence in the Cerebellar granule neurons of rat. We have adopted a supervised learning method and used it in combination with biological pathway information of the genes to develop a gene interaction network. Further modular analysis of the network has been done to identify senescence-related marker genes. Currently there is no adequate information available about the genes implicated in neuronal senescence. Thus identifying multipath genes belonging to the pathway affected by senescence might be very useful in studying the senescence process.
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    A knowledge driven supervised learning approach to identify gene network of differentially up-regulated genes during neuronal senescence in Rattus norvegicus
    ( 2015-09-01) Dholaniya, Pankaj Singh ; Ghosh, Soumitra ; Surampudi, Bapi Raju ; Kondapi, Anand K.
    Various approaches have been described to infer the gene interaction network from expression data. Several models based on computational and mathematical methods are available. The fundamental thing in the identification of the gene interaction is their biological relevance. Two genes belonging to the same pathway are more likely to affect the expression of each other than the genes of two different pathways. In the present study, interaction network of genes is described based on upregulated genes during neuronal senescence in the Cerebellar granule neurons of rat. We have adopted a supervised learning method and used it in combination with biological pathway information of the genes to develop a gene interaction network. Further modular analysis of the network has been done to identify senescence-related marker genes. Currently there is no adequate information available about the genes implicated in neuronal senescence. Thus identifying multipath genes belonging to the pathway affected by senescence might be very useful in studying the senescence process.
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    A membrane-bound cAMP receptor protein, SyCRP1 mediates inorganic carbon response in Synechocystis sp. PCC 6803
    ( 2022-04-01) Bantu, Lingaswamy ; Chauhan, Suraj ; Srikumar, Afshan ; Hirakawa, Yoshihisa ; Suzuki, Iwane ; Hagemann, Martin ; Prakash, Jogadhenu S.S.
    The availability of inorganic carbon (Ci) as the source for photosynthesis is fluctuating in aquatic environments. Despite the involvement of transcriptional regulators CmpR and NdhR in regulating genes encoding Ci transporters at limiting CO2, the Ci-sensing mechanism is largely unknown among cyanobacteria. Here we report that a cAMP-dependent transcription factor SyCRP1 mediates Ci response in Synechocystis. The mutant ∆sycrp1 exhibited a slow-growth phenotype and reduced maximum rate of bicarbonate-dependent photosynthetic electron transport (Vmax) compared to wild-type at the scarcity of CO2. The number of carboxysomes was decreased significantly in the ∆sycrp1 at low CO2 consistent with its reduced Vmax. The DNA microarray analysis revealed the upregulation of genes encoding Ci transporters in ∆sycrp1. The membrane-localized SyCRP1 was released into the cytosol in wild-type cells shifted from low to high CO2 or upon cAMP treatment. Soluble His-tagged SyCRP1 was shown to target DNA-binding sites upstream of the Ci-regulated genes sbtA and ccmK3. In addition, cAMP enhanced the binding of SyCRP1 to its target sites. Our data collectively suggest that the Ci is sensed through the second messenger cAMP releasing membrane-bound SyCRP1 into cytoplasm under sufficient CO2 conditions. Hence, SyCRP1 is a possible regulator of carbon concentrating mechanism, and such a regulation might be mediated via sensing Ci levels through cAMP in Synechocystis.
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    A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G
    ( 2005-01-01) Li, Jing Jing ; Venkataramana, Musturi ; Wang, Ai Qing ; Sanyal, Suparna ; Janson, Jan Christer ; Su, Zhi Guo
    Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent. Various other hydrophobic interaction adsorbents were also attempted to refold EF-G. However, ligands with high hydrophobicity tended to misfold EF-G, resulting in irreversible adsorption. Various solvents, detergents, and low temperature as well as 8 M urea were tried to release bound EF-G. Only pulse elution with 8 M urea was efficient. Urea concentrations favorable for efficiently refolding EF-G were investigated. Low urea concentration produced more misfolded structures. © 2005 Elsevier Inc. All rights reserved.
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    A novel anti-NS2BNS3pro antibody-based indirect ELISA test for the diagnosis of dengue virus infections
    ( 2021-06-01) Gandikota, Chaitanya ; Gandhi, Lekha ; Maisnam, Deepti ; Kesavulu, Muppuru Muni ; Billoria, Arcy ; Prasad, Vankayalapati Sri Venkateswara ; Venkataramana, Musturi
    Dengue virus reportedly circulates as four genetically distinct serotypes for which there is no widely accepted vaccine or drug at present. Morbidity and mortality caused by this virus are alarming for the possible increased threat to human health. A suitable diagnostic test is the prerequisite for designing and developing control measures. But, the tests being employed at present possess one or the other drawback for this disease diagnosis. During the dengue virus infections, NS2B is essential for the stability and catalytic activity of the NS3 protease. N-terminal 185 amino acids of NS3 protease domain along with hydrophilic portion of NS2B (NS2BNS3pro) is being used to screen dengue inhibitors but not for diagnosis until now. In the present study, we have used purified NS2BNS3pro as an antigen to trap anti-NS2BNS3pro antibodies of the clinical samples. Antibodies were detected successfully in both Western blot analysis and enzyme-linked immunosorbent assay (ELISA) tests. In ELISA, antibodies were detected in both primary and secondary infections of all serotypes. Interestingly, 17 samples declared as other febrile infections by NS1 and IgM/IgG tests were found to be positive in present test, which were further confirmed by reverse-transcription polymerase chain reaction. In silico studies suggested the absence of conserved epitopes between NS2BNS3pro and the counterpart in JEV, Zika, and CHIKV, indicating less possibility of crossreaction, which was in turn confirmed by using synthetic peptides representing the above epitopes. Statistical analysis with 76% specificity, 87% sensitivity, and 95% concordance also supported the present test as a suitable test for large scale diagnosis of dengue virus infections.
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    A novel immunomodulatory function of PHLPP1: Inhibition of iNOS via attenuation of STAT1 ser < sup > 727 < /sup > phosphorylation in mouse macrophages
    ( 2014-01-01) Alamuru, Neeraja P. ; Behera, Soma ; Butchar, Jonathan P. ; Tridandapani, Susheela ; Kaimal Suraj, Sasidhara ; Prakash Babu, P. ; Hasnain, Seyed E. ; Ehtesham, Nasreen Z. ; Parsa, Kishore V.L.
    PHLPP1 is a novel tumor suppressor, but its role in the regulation of innate immune responses, which are frequently dysregulated in cancer, is unexplored. Here, we report that LPS attenuated PHLPP1 expression at mRNA and protein levels in immune cells, suggesting its involvement in immune responses. To test this, we overexpressed PHLPP1 in RAW 264.7 macrophages and observed a dramatic reduction in LPS/ IFN-γ-induced iNOS expression. Conversely, silencing of PHLPP1 by siRNA or by shRNA robustly augmented LPS/IFN-γ-induced iNOS expression. qPCR and iNOS promoter reporter experiments showed that PHLPP1 inhibited iNOS transcription. Mechanistic analysis revealed that PHLPP1 suppressed LPS/IFN-γ-induced phosphorylation of ser727 STAT1; however, the underlying mechanisms differed. PHLPP1 reduced IFN-γ- stimulated but not LPS-induced ERK1/2 phosphorylation, and inhibition of ERK1/2 abolished IFN-γ-induced ser727 STAT1 phosphorylation and iNOS expression. In contrast, PHLPP1 knockdown augmented LPS-induced but not IFN-γ-elicited p38 phosphorylation. Blockade of p38 abolished LPS-stimulated phosphorylation of ser727 STAT1 and iNOS expression. Furthermore, PHLPP1 suppressed LPS-induced phosphorylation of tyr701 STAT1 by dampening p38-dependent IFN-β feedback. Collectively, our data demonstrate for the first time that PHLPP1 plays a vital role in restricting innate immune responses of macrophages, and further studies may show it to be a potential therapeutic target within the context of dysregulated macrophage activity. © Society for Leukocyte Biology.
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    A novel meta-cleavage product hydrolase from Flavobacterium sp. ATCC27551
    ( 2006-12-22) Khajamohiddin, Syed ; Babu, Pakala Suresh ; Chakka, Deviprasanna ; Merrick, Mike ; Bhaduri, Anirban ; Sowdhamini, Ramanathan ; Siddavattam, Dayananda
    The organophosphate degrading (opd) gene cluster of plasmid pPDL2 of Flavobacterium sp. ATCC27551 contains a novel open-reading frame, orf243. This was predicted to encode an α/β hydrolase distantly related to the meta-fission product (MFP) hydrolases such as XylF, PhnD, and CumD. By homology modeling Orf243 has most of the structural features of MFP hydrolases including the characteristic active site catalytic triad. The purified protein (designated MfhA) is a homotetramer and shows similar affinity for 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD), 2-hydroxymuconic semialdehyde (HMSA), and 2-hydroxy-5-methylmuconic semialdehyde (HMMSA), the meta-fission products of 3-methyl catechol, catechol, and 4-methyl catechol. The unique catalytic properties of MfhA and the presence near its structural gene of cis-elements required for transposition suggest that mfhA has evolved towards encoding a common hydrolase that can act on meta-fission products containing either aldehyde or ketone groups. © 2006 Elsevier Inc. All rights reserved.
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    A novel method of measuring volume changes of mesophyll cell protoplasts and the effect of mercuric chloride on their osmotically-induced swelling
    ( 1999-01-01) Willmer, C. M. ; Padmasree, K. ; Raghavendra, A. S.
    A quick and accurate method of monitoring changes of volume of mesophyll cell protoplasts (MCP) of pea was devised using the difference in absorbance of the protoplasts at 440 nm and 750 nm (OD 440-750); when protoplasts expanded in response to changing the external medium from 0.4 M sorbitol to 0.3 M sorbitol OD 440-750 values increased and, conversely, when protoplasts were transferred from 0.4 to 0.5 M sorbitol, protoplasts contracted. The kinetics of expansion or contraction of the protoplasts could also be monitored using this method and the half-time for water exchange for expanding protoplasts (about 10 s) was slightly higher than that for contracting protoplasts. A study of the effects of the water channel blocker, mercuric chloride, on swelling protoplasts showed that 500 μM irreversibly damaged protoplasts, 5-10 μM had a negligible inhibitory effect on swelling while 100 μM had a large inhibitory effect often completely inhibiting swelling. A preliminary study indicated that mercapto-ethanol reversed the inhibitory effect of mercuric chloride on protoplast swelling.
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    A novel reading scheme for assessing the extent of radiographic abnormalities and its association with disease severity in sputum smear-positive tuberculosis: An observational study in Hyderabad/India
    ( 2015-09-18) Grozdanovic, Zarko ; Berrocal Almanza, Luis C. ; Goyal, Surabhi ; Hussain, Abid ; Klassert, Tilman E. ; Driesch, Dominik ; Tokaryeva, Viktoriya ; Loschmann, Yvonne Yi Na ; Sumanlatha, Gadamm ; Ahmed, Niyaz ; Valluri, Vijayalakshmi ; Schumann, Ralf R. ; Lala, Birgit ; Slevogt, Hortense
    Background Existing reading schemes for chest X-ray (CXR) used to grade the extent of disease severity at diagnosis in patients with pulmonary tuberculosis (PTB) are often based on numerical scores that summate specific radiographic features. However, since PTB is known to exhibit a wide heterogeneity in pathology, certain features might be differentially associated with clinical parameters of disease severity. Objective We aimed to grade disease severity in PTB patients at diagnosis and after completion of DOTS treatment by developing a reading scheme based on five different radiographic manifestations and analyze their association with the clinical parameters of systemic involvement and infectivity. Methods 141 HIV-negative adults with newly diagnosed sputum smear-positive PTB were enrolled in a prospective observational study in Hyderabad, India. The presence and extent on CXRs of five radiographic manifestations, i.e., lung involvement, alveolar infiltration, cavitation, lymphadenopathy and pleural effusion, were classified using the new reading scheme by using a four-quadrant approach. We evaluated the inter-reader reliability of each manifestation, and its association with BMI and sputum smear positivity at diagnosis. The presence and extent of these radiographic manifestations were further compared with CXRs on completion of DOTS treatment. Results At diagnosis, an average lung area of 51.7% +/- 23.3%was affected by radiographic abnormalities. 94% of the patients had alveolar infiltrates, with 89.4%located in the upper quadrants, suggesting post primary PTB and in 34.8% of patients cavities were found. We further showed that the extent of affected lung area was a negative predictor of BMI (β value -0.035, p 0.019). No significant association of BMI with any of the other CXR features was found. The extent of alveolar infiltrates, along with the presence of cavitation, were strongly associated with sputum smear positivity. The microbiological cure rate in our cohort after 6 months of DOTS treatment was 95%. The extent of the affected lung area in these patients decreased from 56.0% +/- 21.5%to 31.0 +/- 20% and a decrease was also observed in the extent of alveolar infiltrates from 98.4%to 25.8% in at least one quadrant, presence of cavities from 34.8% to 1.6%, lymphadenopathy from 46.8% to 16.1%, and pleural effusion from 19.4% to 6.5%. Conclusions We established a new assessment scheme for grading disease severity in PTB by specifically considering five radiographic manifestations which were differently associated with the BMI and sputum smear positivity, changed to a different extent after 6 months of treatment and exhibited an excellent agreement between radiologists. Our results suggest that this reading scheme might contribute to the estimation of disease severity with respect to differences in disease pathology. Further studies are needed to determine a correlation with short and long-term pulmonary function impairment and whether there would be any benefit in lengthening or modulating therapy based on this CXR severity assessment. Copyright:
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    A novel transcriptional regulator, Sll1130, negatively regulates heat-responsive genes in Synechocystis sp. PCC6803
    ( 2013-02-01) Krishna, Pilla Sankara ; Rani, Balaga Radha ; Mohan, M. Karthik ; Suzuki, Iwane ; Shivaji, Sisinthy ; Prakash, Jogadhenu S.S.
    A conserved hypothetical protein, Sll1130, is a novel transcription factor that regulates the expression of major heat-responsive genes in Synechocystis sp. PCC6803. Synechocystis exhibited an increased thermotolerance due to disruption of sll1130. Δsll1130 cells recovered much faster than wild-type cells after they were subjected to heat shock (50°C) for 30 min followed by recovery at 34°C for 48 h. In Δsll1130 cultures, 70% of the cells were viable compared with the wild-type culture in which only 30% of the cells were viable. DNA microarray analysis revealed that in Δsll1130, expression of the heat-responsive genes such as htpG, hspA, isiA, isiB and several hypothetical genes were up-regulated. Sll1130 binds to a conserved inverted-repeat (GGCGATCGCC) located in the upstream region of the above genes. In addition, both the transcript and protein levels of sll1130 were immediately down-regulated upon shift of wild-type cells from 34 to 42°C. Collectively the results of the present study suggest that Sll1130 is a heat-responsive transcriptional regulator that represses the expression of certain heat-inducible genes at optimum growth temperatures. Upon heat shock, a quick drop in the Sll1130 levels leads to de-repression of the heat-shock genes and subsequent thermal acclimation. On the basis of the findings of the present study, we present a model which describes the heat-shock response involving Sll1130. © The Authors Journal compilation © 2013 Biochemical Society.