Comparative evaluation of different therapeutic protocols for contagious caprine pleuropneumonia in Himalayan Pashmina goats.

Therapeutic management of contagious caprine pleuroneumonia (CCPP) involves mostly the use of oxytetracycline followed by enrofloxacin and rarely tylosin. In many parts of the world including India, the former antibiotics are commonly available than the latter. Therefore, prolonged use of the same leads to the development of antibiotic resistance and decreased efficacy of drug. Besides, inflammatory and allergic pathogenesis of CCPP envisages combination therapy. In this study, we evaluated the effectiveness of the combination therapy using different antibiotics (oxytetracycyline @ 10: group I, enrofloxacin @ 5 group II, and tylosin: group III, @ 10 mg/kg body weight), along with anti-inflammatory (meloxicam @ 0.5 mg/kg) and anti-allergic (pheneramine maleate @ 1.0 mg/kg) drugs. These drugs were given intramuscularly at the interval of 48 h for four times in three test groups (n = 10) of Pashmina goats, viz. groups I, II, and III, respectively, affected with CCPP. Group IV (n = 10) was kept as healthy control when group V (n = 10) treated with oxytetracycline @ 10 mg/kg alone was used as positive control. Clinical signs, clinical parameters, pro-inflammatory cytokine (tumor necrosis factor alpha (TNF-α)), and oxidative stress indices (total oxidant status (TOS), total antioxidant status (TAS)) were evaluated at hours 0, 48, 96, and 144 of experimental trial. Tylosin-based combination therapy resulted in a rapid and favorable recovery resulting in restoration of normal body temperature (102.46 ± 0.31 °F), respiration rate (16.30 ± 0.79 per minute), and heart rate (89.50 ± 2.63 per minute) compared to the oxytetracycline (102.95 ± 0.13, 21.30 ± 1.12, 86.00 ± 2.33, respectively) and enrofloxacin (102.97 ± 0.19, 21.00 ± 1.25, 90.00 ± 2.58, respectively) treated groups. By hour 144, all the groups showed restoration of clinical parameters of normal health and diminishing signs of CCPP, viz. fever, dyspnea, coughing, nasal discharge, weakness, and pleurodynia. Significant (P ≤ 0.05) decrease in levels of TNF-α and non-significant (P > 0.05) decrease in levels of TOS and an increase in levels of TAS were noted from hour 0 to 144 in all the test groups. Within the groups, no significant (P > 0.05) change was noted in TNF-α, TOS, and TAS levels; however, TNF-α levels were comparatively lower in group III. Hematological parameters did not differ significantly (P > 0.05). From these findings, it can be inferred that tylosin-based combination therapy is relatively better for early, rapid, and safe recovery besides minimizing inflammatory and oxidative cascade in CCPP affected Pashmina goats compared to oxytetracycline- and enrofloxacin-based therapies.

Extrahepatic portal vein obstruction and portal vein thrombosis in special situations: Need for a new classification.

Extrahepatic portal vein obstruction is a vascular disorder of liver, which results in obstruction and cavernomatous transformation of portal vein with or without the involvement of intrahepatic portal vein, splenic vein, or superior mesenteric vein. Portal vein obstruction due to chronic liver disease, neoplasm, or postsurgery is a separate entity and is not the same as extrahepatic portal vein obstruction. Patients with extrahepatic portal vein obstruction are generally young and belong mostly to Asian countries. It is therefore very important to define portal vein thrombosis as acute or chronic from management point of view. Portal vein thrombosis in certain situations such as liver transplant and postsurgical/liver transplant period is an evolving area and needs extensive research. There is a need for a new classification, which includes all areas of the entity. In the current review, the most recent literature of extrahepatic portal vein obstruction is reviewed and summarized.

Clinical course, laboratory parameters and outcome of TTP pediatric patients presenting with posterior reversible encephalopathy syndrome.

AIM: The clinical course and outcome of children with thrombotic thrombocytopenic purpura and posterior reversible encephalopathy has not been observed and studied till date. The aim of the present study was to know the clinical course and outcome of children with thrombotic thrombocytopenic purpura and posterior reversible encephalopathy. Results from our observation invite potential insight for further research on this subject. METHODS: From January 2005 to February 2013, seven children diagnosed with thrombotic thrombocytopenic purpura and posterior reversible encephalopathy syndromes were admitted in a tertiary care hospital in Srinagar, Kashmir. The demographic parameters, clinical characteristics and laboratory data were noted. The outcome was defined in the form of complete recovery or death. Thrombotic thrombocytopenic purpura was diagnosed on clinical grounds, laboratory parameters and renal biopsy. The diagnosis was established after an expert opinion from a hematologist. Posterior reversible encephalopathy syndrome was defined on neuroimaging. RESULTS: The common clinical parameters which were shared by all the patients were hypertension and altered sensorium. Four (57.1%) patients showed clinical deterioration and died within one week of admission even after intensive management. Three (42.8%) patients improved clinically and recovered fully and were discharged in stable clinical condition. Repeat imaging on discharge was normal. CONCLUSION: This series of seven pediatric patients is the first series on this subject. The presence of posterior reversible encephalopathy syndrome in pediatric patients with thrombotic thrombocytopenic purpura complicates the clinical course and worsens the prognosis.

Polycythemia, increased erythropoietin levels in a patient with renal lymphoma.

A young male presented to our clinic with 3 months history of shortness of breathness and progressive distension of abdomen. On investigations, patient had renal failure, polycythemia and nephromegaly. A diagnosis of non-Hodgkin's lymphoma was made on renal and lymph node biopsy. Serum erythropoietin concentrations were physiologically inappropriate. - Erythropoietin immunohistochemistry on renal tissue samples demonstrated positive staining for tumor cells. This patient was managed as a case of infiltrative lymphoproliferative disorder with kidney involvement having polycythemia owing to paraneoplastic Erythropoietin production and possibly local hypoxia produced by tumor cells. With maximum efforts, we could not find such an association in the literature.

Renal allograft transplant recipient with ruptured hydatid native kidney.

Echinococcosis of the kidneys in a renal transplant recipient is extremely rare and its occurrence being related to immunosuppression is a possibility which needs further characterisation. Ruptured renal hydatid in a renal transplant recipient is not reported so far to our best knowledge. We present a 42-year-old renal allograft receipient who presented one year after transplant with left flank pain, palpable left lumbar mass and gross hydatiduria. Investigations revealed a ruptured native hydatid kidney. Patient was managed with a combination of chemotherapy and left native nephrectomy and discharged in a satisfactory condition.

Combination of novel green fluorescent protein mutant TSapphire and DsRed variant mOrange to set up a versatile in planta FRET-FLIM assay.

Förster resonance energy transfer (FRET) measurements based on fluorescence lifetime imaging microscopy (FLIM) are increasingly being used to assess molecular conformations and associations in living systems. Reduction in the excited-state lifetime of the donor fluorophore in the presence of an appropriately positioned acceptor is taken as strong evidence of FRET. Traditionally, cyan fluorescent protein has been widely used as a donor fluorophore in FRET experiments. However, given its photolabile nature, low quantum yield, and multiexponential lifetime, cyan fluorescent protein is far from an ideal donor in FRET imaging. Here, we report the application and use of the TSapphire mutant of green fluorescent protein as an efficient donor to mOrange in FLIM-based FRET imaging in intact plant cells. Using time-correlated single photon counting-FLIM, we show that TSapphire expressed in living plant cells decays with lifetime of 2.93 +/- 0.09 ns. Chimerically linked TSapphire and mOrange (with 16-amino acid linker in between) exhibit substantial energy transfer based on the reduction in the lifetime of TSapphire in the presence of the acceptor mOrange. Experiments performed with various genetically and/or biochemically known interacting plant proteins demonstrate the versatility of the FRET-FLIM system presented here in different subcellular compartments tested (cytosol, nucleus, and at plasma membrane). The better spectral overlap with red monomers, higher photostability, and monoexponential lifetime of TSapphire makes it an ideal FRET-FLIM donor to study protein-protein interactions in diverse eukaryotic systems overcoming, in particular, many technical challenges encountered (like autofluorescence of cell walls and fluorescence of pigments associated with photosynthetic apparatus) while studying plant protein dynamics and interactions.

Interaction between SGT1 and cytosolic/nuclear HSC70 chaperones regulates Arabidopsis immune responses.

The conserved eukaryotic protein SGT1 (for Suppressor of G2 allele of skp1) has characteristics of an HSP90 (for heat shock protein 90 kD) cochaperone and in plants regulates hormone responses and Resistance gene-triggered immunity. We affinity-purified SGT1-interacting proteins from Arabidopsis thaliana leaf extracts and identified by mass spectrometry cytosolic heat shock cognate 70 (HSC70) chaperones as the major stable SGT1 interactors. Arabidopsis SGT1a and SGT1b proteins associate with HSC70 in vivo and distribute with HSC70 in the cytosol and nucleus. An intact C-terminal SGT1-specific (SGS) domain that is required for all known SGT1b functions in immunity and development is needed for HSC70 interaction and for the nuclear accumulation of SGT1b. Interaction assays of transiently expressed proteins or their domains in Nicotiana benthamiana point to a role of SGT1 as a HSC70 cofactor. Expression of two HSC70 isoforms is upregulated by pathogen challenge, and while loss of function of individual cytosolic HSC70 genes has no defense phenotype, HSC70-1 overexpression disables resistance to virulent and avirulent pathogens. Moreover, mutations in SGT1b lead to a similar degree of heat shock tolerance as deregulation of HSC70-1. We conclude that an HSC70-SGT1 chaperone complex is important for multiple plant environmental responses and that the evolutionarily conserved SGS domain of SGT1 is a key determinant of the HSC70-SGT1 association.

The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods.

Non-invasive fluorophore-based protein interaction assays like fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC, also referred to as "split YFP") have been proven invaluable tools to study protein-protein interactions in living cells. Both methods are now frequently used in the plant sciences and are likely to develop into standard techniques for the identification, verification and in-depth analysis of polypeptide interactions. In this review, we address the individual strengths and weaknesses of both approaches and provide an outlook about new directions and possible future developments for both techniques.

Lipid rafts in plants.

About two decades ago a provocative hypothesis evolved suggesting that the plasma membrane (PM) of mammalian and probably other eukaryotic cells constitutes a mosaic of patches comprising particular molecular compositions. These scattered lipid bilayer microdomains are supposedly enriched in sterols as well as sphingolipids and depleted in unsaturated phospholipids. In addition, the PM microdomains are proposed to host glycosyl-phosphatidylinositol-anchored polypeptides and a subset of integral and peripheral cell surface proteins while excluding others. Though the actual in vivo existence of such "lipid rafts" remains controversial, a range of fundamental biological functions has been put forward for these PM microenvironments. A variety of recent studies provide preliminary evidence that lipid rafts may also occur in plant cells.

Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein.

We performed a structure-function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop-loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family.

Interaction of maize Opaque-2 and the transcriptional co-activators GCN5 and ADA2, in the modulation of transcriptional activity.

Maize Opaque-2 (ZmO2), a bZip class transcription factor has been shown to activate the transcription of a series of genes expressed in the maturation phase of endosperm development. Activation requires the presence of one or more enhancer binding sites, which confer the propensity for activation by ZmO2 on heterologous promoters and in heterologous plant cell types, such as tobacco mesophyll protoplasts. The region of ZmO2 required for conferring transcriptional activation has been localised to a stretch of acidic residues in the N-terminal portion of the ZmO2 sequence, which is conserved between O2-related bZip factor sequences. Previously we identified the maize homologues of yeast transcriptional co-activators GCN5 and ADA2 that are implicated in nucleosome modification and transcription. In the present study we have shown that transcriptional modulation by ZmO2 involves the intranuclear interaction of ZmO2 with ZmADA2 and ZmGCN5. Förster resonance energy transfer (FRET) based techniques have enabled us to estimate the intracellular site of these intermolecular interactions. As a functional readout of these intranuclear interactions, we used the ZmO2 responsive maize b-32 promoter to drive the beta-glucuronidase (GUS) in the presence and absence of ZmGCN5 and ZmADA2. Our results suggest that the likely recruitment of ZmADA2 and ZmGCN5 modulates the transactivation of b-32 promoter by ZmO2 and that there may be a competition between ZmGCN5 and ZmO2 for binding to the amino-terminal of ZmADA2. The results may be taken as a paradigm for other processes of transcriptional modulation in planta involving acidic activation domains.