Insight into trichomonas vaginalis genome evolution through metabolic pathways comparison

Trichomonas vaginalis causes the trichomoniasis, in women and urethritis and prostate cancer in men. Its genome draft published by TIGR in 2007 presents many unusual genomic and biochemical features like, exceptionally large genome size, the presence of hydrogenosome, gene duplication, lateral gene transfer mechanism and the presence of miRNA. To understand some of genomic features we have performed a comparative analysis of metabolic pathways of the T. vaginalis with other 22 significant common organisms. Enzymes from the biochemical pathways of T. vaginalis and other selected organisms were retrieved from the KEGG metabolic pathway database. The metabolic pathways of T. vaginalis common in other selected organisms were identified. Total 101 enzymes present in different metabolic pathways of T. vaginalis were found to be orthologous by using BLASTP program against the selected organisms. Except two enzymes all identified orthologous enzymes were also identified as paralogous enzymes. Seventy-five of identified enzymes were also identified as essential for the survival of T. vaginalis, while 26 as non-essential. The identified essential enzymes also represent as good candidate for novel drug targets. Interestingly, some of the identified orthologous and paralogous enzymes were found playing significant role in the key metabolic activities while others were found playing active role in the process of pathogenesis. The N-acetylneuraminate lyase was analyzed as the candidate of lateral genes transfer. These findings clearly suggest the active participation of lateral gene transfer and gene duplication during evolution of T. vaginalis from the enteric to the pathogenic urogenital environment

Labor analgesia with intradermal sterile water block in a patient with dilated cardiomyopathy

Epidural analgesia is the gold standard for providing labor analgesia, but an obstetric anesthesiologist should be well versed with many other non-pharmacological modalities of pain management. The present case highlights the importance of non-pharmacological methods of labor analgesia that might be the only options available in certain subset of patients to provide adequate labor analgesia

Use of Comparison Films for Breast Cancer Screening and Diagnosis Among Florida Radiologists

Presentation of results of a study finding links between mammogram comparison ad the rates of failure in breast cancer diagnosis

Importance of Amino Acids, Gln-119 and Tyr-376, in the S1 Pocket of Escherichia coli Peptidase N in Determining Substrate Specificity

Peptidase N (PepN) is a broad specific metallo-peptidase and the sole member of the M1 class encoded by Escherichia coli. Comparative analysis of residues present in the S1 subsite of E. coli PepN with other family members revealed that Tyr-381 is conserved whereas Glu-121, Gln-119 and Tyr-376 are partially conserved. The functional importance of these amino acids was investigated by protein engineering studies. The change in Glu-121 to Gln and Tyr-381 to Phe led to catalytically inactive PepN. At the same time, the change in Gln-119 to His (Q119H) and Tyr-376 to Phe (Y376F) led to alterations in substrate specificity. Kinetic studies revealed that purified PepN variants, Q119H and Y376F, cleaved some substrates (e.g. Arg) similar to wild type PepN. However, these variants displayed lower efficacy with other substrates (e.g. Tyr, AAF and Suc-AAF). Q119H or Y376F, cleave a natural peptide (insulin B chain) and a loosely folded protein (casein) with greatly reduced efficacy. The double mutant, i.e. harboring both Q119H and Y376F, displays greatly reduced catalytic activity with respect to all substrates studied. The in vivo significance was addressed by expressing these variants in Delta pepN during nutritional downshift and high temperature (NDHT) stress. Compared to wild type PepN, the Y376F and Q119H variants display lower intracellular amounts of free N-terminal amino acids and reduction in growth during NDHT stress. Finally, structural modeling, using the crystal structure of E. coli PepN bound to substrates, Arg or Tyr, shed insights into the roles of Q119H and Y376F in determining substrate preferences

Interferon-Gamma and Nitric Oxide Synthase 2 Mediate the Aggregation of Resident Adherent Peritoneal Exudate Cells: Implications for the Host Response to Pathogens

<div><p>Interferon-gamma (Ifnγ), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifnγ to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifnγ, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to <i>Cd11b</i>. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/nitric oxide adduct (DETA/NO), and <i>Nos2</i>^-/- mice identified Nitric oxide (NO) induced by Ifnγ as a key regulator of aggregation of APECs. Further studies with <i>Nos2</i>^-/- APECs revealed that some Ifnγ responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifnγ and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with <i>Salmonella</i> Typhimurium (<i>S</i>. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon <i>ex vivo</i> culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifnγ contain fewer intracellular <i>S</i>. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifnγ and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or “group behavior” of APECs are discussed in the context of host resistance to infectious organisms.</p></div

Molecular profiling of sepsis in mice using Fourier Transform Infrared Microspectroscopy

Sepsis is a life threatening condition resulting from a high burden of infection. It is a major health care problem and associated with inflammation, organ dysfunction and significant mortality. However, proper understanding and delineating the changes that occur during this complex condition remains a challenge. A comparative study involving intra-peritoneal injection of BALB/c mice with Salmonella Typhimurium (infection), lipopolysaccharide (endotoxic shock) or thioglycollate (sterile peritonitis) was performed. The changes in organs and sera were profiled using immunological assays and Fourier Transform Infrared (FTIR) micro-spectroscopy. There is a rapid rise in inflammatory cytokines accompanied with lowering of temperature, respiratory rate and glucose amounts in mice injected with S. Typhimurium or lipopolysaccharide. FTIR identifies distinct changes in liver and sera: decrease in glycogen and protein/lipid ratio and increase in DNA and cholesteryl esters. These changes were distinct from the pattern observed in mice treated with thioglycollate and the differences in the data obtained between the three models are discussed. The combination of FTIR spectroscopy and other biomarkers will be valuable in monitoring molecular changes during sepsis. GRAPHICS] Intra-peritoneal infection with high dose of Salmonella Typhimurium leads to rapid increase in inflammatory cytokines, e.g. Tnf alpha (A). FTIR analysis of liver (B) and sera (C) identifies several metabolic changes: glycogen, protein/lipid, cholesteryl esters and DNA

The cortical stability of cytoskeleton elements, Actin and Tubulin, is regulated in a Nos2 dependent manner upon Ifnγ treatment.

<p>Fluorescence microscopic images, with the scale bar of 10 μm, of F-Actin (green) and α-Tubulin (red) in APECs from C57BL/6 mice and <i>Nos2</i>^<i>-/-</i> mice treated without or with 25 U/ml of Ifnγ for 36 h. In addition, <i>Nos2</i>^<i>-/-</i> APECs were treated with 100 μM of SNAP in the absence or presence of Ifnγ. The white arrows indicate cortical arrangement of F-Actin and α-Tubulin.</p

CD11b is required for aggregation of APECs in response to Ifnγ.

<p>The relative amounts of E-Selectin (A, E) and CD11b (B, F) on APECs upon treatment with Ifnγ (25 U/ml) in the absence or presence of indicated amounts of Reopro (A-D) or siRNA to <i>Cd11b</i> (E-H) was measured after 36 h. The amount of nitrite (C,G) and cell aggregates (D,H) upon 25 U/ml of Ifnγ treatment in the absence or presence of indicated doses of Reopro or 200 nM siRNA to <i>Cd11b</i> is shown. The data is represented as mean ± S.E from three independent experiments and control refers to untreated cells alone. The significance with respect to untreated controls and Ifnγ treated C57BL/6 APECs controls are represented as * and θ respectively.</p

Nos2 derived NO mediates the Ifnγ induced aggregation of APECs.

<p>Kinetic analysis of amounts of ROS (A), nitrite (D) of APECs treated with 25 U/ml of Ifnγ. The amounts of ROS (B) and the number of cell aggregates (C) in APECs treated with Ifnγ in the presence or absence of indicated doses of PEG-Catalase (PC) for 36 h. The amount of nitrite (E) and the number of cell aggregates (F) of APECs treated with Ifnγ in the presence or absence of indicated doses of LNMA for 36 h. Kinetic analysis of the number of viable APECs from C57BL/6 and <i>Nos2</i>^<i>-/-</i> mice left untreated or upon treatment with 25 U/ml of Ifnγ (G). The amount of nitrite (H) and the number of cell aggregates (I) from Ifnγ treated <i>Nos2</i>^<i>-/-</i> APECs in the absence or presence of indicated doses of SNAP for 36 h in comparison to C57BL/6 APECs treated with 25 U/ml Ifnγ for 36 h. The data is represented as mean ± S.E from three independent experiments. The significance with respect to untreated controls, Ifnγ treated C57BL/6 APECs controls and untreated <i>Nos2</i>^<i>-/-</i> APECs controls are represented as *, θ and Δ respectively.</p