First report of molecular confirmation and phylogenetic analysis of ocular seteriasis in buffalo in India using 12S rRNA.

An adult Indian buffalo (Bubalus bubalis) presented with corneal opacity, irritation, and excessive lacrimation from the left eye in the Referral Veterinary Polyclinic-Teaching Veterinary Clinical Complex (RVC-TVCC), Indian Veterinary Research Institute, Izatnagar. Clinical examination revealed a whitish thread-like worm in the left eye's anterior chamber. The worm was surgically removed from the eye with supportive nerve blocks. Light microscopy was used for parasite morphological identification, which provided insight into the worm as female Setaria sp. Genomic DNA was isolated, and polymerase chain reaction amplification of 12S rRNA was conducted for molecular confirmation of the parasite. The amplicon was sequenced and analysed by bioinformatics software. Sequence data showed an amplicon size of 243 bp. Phylogenetic analysis with reference data from the NCBI Genbank database revealed the worm was S. digitata, with a similarity of 99.17%. The common predilection site of S. digitata is in the peritoneal cavity of natural hosts like cattle and buffalo and is mostly non-pathogenic. The aberrant migration of the parasite larva to the brain and eye commonly occurs in goats, sheep, and horses, causing clinical conditions like cerebrospinal nematodiasis (lumbar paralysis) and ocular setariasis, respectively. Nevertheless, until now, there have been no reports of ocular setariasis in buffalo. This report is the first unusual occurrence of ocular setariasis in buffalo and its molecular confirmation and phylogenetic analysis using 12S rRNA.

Oriental theileriosis associated with a new genotype of Theileria orientalis in buffalo (Bubalus bubalis) calves in Uttar Pradesh, India.

Theileria orientalis is known to cause a benign infection in cattle and buffalo (Bubalus bubalis). However, the Ikeda and Chitose genotypes of the parasite cause lethal disease in beef and dairy cattle. Recently an outbreak of clinical oriental theileriosis occurred in buffalo calves in a Government Animal Husbandry and Agricultural Farm located in Uttar Pradesh, India. Examination of Giemsa stained thin blood smears revealed typical rod-shaped T. orientalis piroplasms in the erythrocytes. The clinical signs included pyrexia, nasal discharge, lacrimation, lethargy, inappetence and anaemia with varying degrees of paleness of the visible mucous membranes. Vascular congestion in internal organs, pulmonary emphysema and consolidation of lungs, focal areas of necrosis in the heart with mononuclear cell infiltration, focal mononuclear cell aggregation in the cortex and tubular degeneration of the kidney were significant necropsy findings. The T. orientalis major piroplasm surface protein (MPSP) gene was amplified by polymerase chain reaction (PCR) using specific primers. The nucleotide sequence analysis of the PCR product revealed 84.8% identity between the T. orientalis Uttar Pradesh isolate and other reference genotypes available in the public domain. Furthermore, the phylogenetic analysis of the MPSP gene sequence ratified that this is a new genotype of T. orientalis. This is the first report of a clinical outbreak of oriental theileriosis in Indian buffalo calves caused by a novel genotype of T. orientalis.

Role of α- and ß-adrenergic signaling in phenotypic targeting: significance in benign and malignant urologic disease.

The urinary tract is highly innervated by autonomic nerves which are essential in urinary tract development, the production of growth factors, and the control of homeostasis. These neural signals may become dysregulated in several genitourinary (GU) disease states, both benign and malignant. Accordingly, the autonomic nervous system is a therapeutic target for several genitourinary pathologies including cancer, voiding dysfunction, and obstructing nephrolithiasis. Adrenergic receptors (adrenoceptors) are G-Protein coupled-receptors that are distributed throughout the body. The major function of α1-adrenoceptors is signaling smooth muscle contractions through GPCR and intracellular calcium influx. Pharmacologic intervention of α-and ß-adrenoceptors is routinely and successfully implemented in the treatment of benign urologic illnesses, through the use of α-adrenoceptor antagonists. Furthermore, cell-based evidence recently established the antitumor effect of α1-adrenoceptor antagonists in prostate, bladder and renal tumors by reducing neovascularity and impairing growth within the tumor microenvironment via regulation of the phenotypic epithelial-mesenchymal transition (EMT). There has been a significant focus on repurposing the routinely used, Food and Drug Administration-approved α1-adrenoceptor antagonists to inhibit GU tumor growth and angiogenesis in patients with advanced prostate, bladder, and renal cancer. In this review we discuss the current evidence on (a) the signaling events of the autonomic nervous system mediated by its cognate α- and ß-adrenoceptors in regulating the phenotypic landscape (EMT) of genitourinary organs; and (b) the therapeutic significance of targeting this signaling pathway in benign and malignant urologic disease. Video abstract.

Urine Extracellular Vesicle GATA2 mRNA Discriminates Biopsy Result in Men with Suspicion of Prostate Cancer.

PURPOSE: Prostate specific antigen has limited performance in detecting prostate cancer. The transcription factor GATA2 is expressed in aggressive prostate cancer. We analyzed the predictive value of urine extracellular vesicle GATA2 mRNA alone and in combination with a multigene panel to improve detection of prostate cancer and high risk disease. MATERIALS AND METHODS: GATA2 mRNA was analyzed in matched extracellular vesicles isolated from urines before and after prostatectomy (16) and paired urine and tissue prostatectomy samples (19). Extracellular vesicle GATA2 mRNA performance to distinguish prostate cancer and high grade disease was tested in training (52) and validation (165) cohorts. The predictive value of a multigene score including GATA2, PCA3 and TMPRSS2-ERG (GAPT-E) was tested in both cohorts. RESULTS: Confirming its prostate origin, urine extracellular vesicle GATA2 mRNA levels decreased significantly after prostatectomy and correlated with prostate cancer tissue GATA2 mRNA levels. In the training and validation cohort GATA2 discriminated prostate cancer (AUC 0.74 and 0.66) and high grade disease (AUC 0.78 and 0.65), respectively. Notably, the GAPT-E score improved discrimination of prostate cancer (AUC 0.84 and 0.72) and high grade cancer (AUC 0.85 and 0.71) in both cohorts when compared with each biomarker alone and PT-E (PCA3 and TMPRSS2-ERG). A GAPT-E score for high grade prostate cancer would avoid 92.1% of unnecessary prostate biopsies, compared to 61.9% when a PT-E score is used. CONCLUSIONS: Urine extracellular vesicle GATA2 mRNA analysis improves the detection of high risk prostate cancer and may reduce the number of unnecessary biopsies.

BjuWRR1, a CC-NB-LRR gene identified in Brassica juncea, confers resistance to white rust caused by Albugo candida.

KEY MESSAGE: BjuWRR1, a CNL-type R gene, was identified from an east European gene pool line of Brassica juncea and validated for conferring resistance to white rust by genetic transformation. White rust caused by the oomycete pathogen Albugo candida is a significant disease of crucifer crops including Brassica juncea (mustard), a major oilseed crop of the Indian subcontinent. Earlier, a resistance-conferring locus named AcB1-A5.1 was mapped in an east European gene pool line of B. juncea-Donskaja-IV. This line was tested along with some other lines of B. juncea (AABB), B. rapa (AA) and B. nigra (BB) for resistance to six isolates of A. candida collected from different mustard growing regions of India. Donskaja-IV was found to be completely resistant to all the tested isolates. Sequencing of a BAC spanning the locus AcB1-A5.1 showed the presence of a single CC-NB-LRR protein encoding R gene. The genomic sequence of the putative R gene with its native promoter and terminator was used for the genetic transformation of a susceptible Indian gene pool line Varuna and was found to confer complete resistance to all the isolates. This is the first white rust resistance-conferring gene described from Brassica species and has been named BjuWRR1. Allelic variants of the gene in B. juncea germplasm and orthologues in the Brassicaceae genomes were studied to understand the evolutionary dynamics of the BjuWRR1 gene.

Detection of Antibodies Against Toxoplasma gondii in Indian Cattle by Recombinant SAG2 Enzyme-Linked Immunosorbent Assay.

BACKGROUND: A large number of recombinant proteins are tested over a period of time for diagnosis of toxoplasmosis throughout the world. However, such literature is very much limited from an Indian perspective. PURPOSE: The aim of the study was to assess the sero prevalence of Toxoplasma gondii in adult cattle from Kerala, India, using recombinant surface antigen 2 (recSAG2) protein. METHODS: An antibody detection recombinant ELISA specific for T gondii was laboratory-standardized using recSAG2 protein. The optimum antigen concentration, serum concentration, and conjugate dilutions were determined by the initial checkerboard titrations. Subsequently, the diagnostic potential of the recombinant protein was assessed with 258 field sera samples cattle and compared with indirect fluorescent antibody test (IFAT). RESULTS: Among the cattle sera tested, 61.5% showed sero positivity of T. gondii-speciic IgG. When compared to IFAT, the sensitivity of the recSAG2 ELISA was found to be 80.00% with 88.57% specificity at 95% confidence interval with substantial agreement between the tests. CONCLUSION: The results of present study have revealed the presence of high seroprevalence of the parasite and, hence, immense public health significance.

Randomized trial to assess safety and clinical efficacy of intensive blood pressure reduction in acute spontaneous intracerebral haemorrhage.

BACKGROUND: Haematoma expansion due to raised blood pressure in spontaneous intracerebral haemorrhage may determine outcome. The aim of this study was to determine safety and efficacy of lowering blood pressure in acute spontaneous intracerebral haemorrhage. METHODS: This open label, multicentric trial randomized patients ≥18 years with spontaneous intracerebral haemorrhage with no secondary cause within 72 h of onset to tight BP control arm where treatment was initiated if mean arterial pressure (MAP) was ≥115 mm of Hg and conventional BP control arm where treatment was initiated if MAP was ≥130 mm of Hg. The MAP was maintained in the respective arm for another 72 h after which both arms had MAP below 115 mm of Hg. Primary outcome was modified Rankin Scale at 90 days. RESULTS: 118 patients, 59 in each arm were included. Follow up was available for all. Baseline characteristics were similar. At 90 days there was no significant difference between median mRS between the two arms. Odds Ratio for "poor outcome" (mRS 3-6) in the tight control arm (safety of the intervention) against "good outcome" (mRS 0-2) was not significant (OR 0.70 [95% CI 0.34-1.47] p = 0.35). Efficacy of the intervention in the form of Odds Ratio for "good outcome" in the tight control arm was not significant (OR 1.43 [95% CI 0.68-2.99], p = 0.35). CONCLUSION: In patients with spontaneous intracerebral haemorrhage who present within 72 h of the onset of symptoms, MAP can be safely lowered if it crosses 115 mm of Hg but it does not improve clinical outcome.

Pathobiology of human RH strain induced experimental toxoplasmosis in murine model.

Of late, toxoplasmosis has gained immense importance as an opportunist parasite in immunocompromised patients. In immunocompromised subjects, the disease is supposed to occur in acute form and causes acute toxoplasmic encephalitis. However, the exact pathogenesis of other vital organs, particularly in acute form of infection, is still a matter of debate. Therefore, an attempt was made to study the pathogenesis of acute form of toxoplasmosis using cryopreserved human RH strain of the parasite in murine models. For this, 100 tachyzoites were given to individual mice and upon the setup of acute form of infection, the mice were euthanized and the organs were processed for histopathology. Histopathology revealed tachyzoites in liver only while severe necrosis due to multiplication of tachyzoites were visible in liver, spleen, lungs and brain. Kidneys and heart appeared more or less normal. Finally, the pathology of disease in these organs is described in detail. The present research has generated some vital information regarding necrotic changes in tissues due to acute toxoplasmosis and will defiantly help the researchers in the better understanding of disease particularly in humans and putting up of suitable treatment regime for human subjects infected with acute toxoplasmosis.

Prostate biopsies: Available technique, approaches and diagnostic accuracy.

Despite advances in the diagnosis of prostate cancer over the past century, it remains a leading cause of cancer related death. A recent recommendation against screening has further complicated the diagnosis and management of this condition. It remains to be demonstrated if newer diagnostic modalities will have an impact on mortality rates. Most certainly, not all prostate cancers need to be diagnosed, and methods of accurately diagnosing those cancers that lead to death needs more work. In this review article, we describe the different techniques, approaches and diagnostic accuracies of the currently used biopsy methods.

An overview on kinetoplastid paraflagellar rod.

Kinetoplastids, the evolutionary ancient organisms exhibit a rich and diverse biology which epitomizes many of the fascinating topics of recent interest and study. These organisms possess a multifunctional organelle, the flagellum containing a canonical 9 + 2 axoneme which is involved in vital roles, viz. parasite cell division, morphogenesis, motility and immune evasion. Since Antony Van Leeuwenhoek's innovative explanation of 'little legs' helping the movements of microbes in 1975, this biological nanomachine has captured the thoughts of scientists. The core structure of kinetoplastid flagellum is embroidered with a range of extra-axonemal structures such as paraflagellar rod (PFR), a large lattice like structure which extends alongside the axoneme from the flagellar pocket to the flagellar tip. The coding sequences for significant components of PFR are highly conserved throughout the Kinetoplastida and Euglenida. The high order organization and restricted evolutionary distribution of the PFR components and structure makes the PFR a particularly valuable therapeutic and prophylactic target. This review focuses on the recent developments in identification of ultra structural components of PFR in order to understand the function of this intriguing organelle and devising strategies for therapeutic interventions.

Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model.

OBJECTIVE: To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models. METHODS: A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite. Tissue samples from lung, liver, spleen, brain, heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR, using primers directed to the multicopy of SAG 3 gene, in dublicates. RESULTS: Histopathology revealed presence of tachyzoites only in liver while along with lung, liver, spleen and brain tissue yielded desired positive PCR amplicons. CONCLUSIONS: The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

In vitro inhibition activity of essential oils from some Lamiaceae species against phytopathogenic fungi.

Natural products have been in focus as alternative, effective and safe materials against the phytopathogens. Investigations show Nepeta oils as effective in controlling the food crops decay. The inhibitory effects of essential oils derived from Nepeta leucophylla, Nepeta ciliaris, Nepeta clarkei and Calamintha umbrosa against five phytopthogenic fungi have been determined. In vitro antifungal activity varied with their constituents and target species. More active being the oils containing oxygenated terpenoids. Helminthosporium maydis was sensitive to the all oils, IC50 values have 43.6-109.3 µg mL(-1). The N. leucophylla oil possessing oxygenated iridoids was more effective against H. maydis (IC50 value of 43.6 µg mL(-1)) while N. ciliaris was more active against Fusarium oxysporum (IC50 value of 219.2 µg mL(-1)). The oils were effective against the spore germination of all the tested plant pathogens.

An Insight into the Behavior, Course and Kinetics of Acute Infection of Toxoplasma gondii Human RH Strain in Experimentally Infected Murine Model.

BACKGROUND: Toxoplasma gondii, an apicomplexan parasite, is capable of infecting a broad range of intermediate warm-blooded hosts including humans. The parasite seems to be capable of altering the natural behavior of the host to favor its transmission in the environment. The aim of this study was to evaluate the course, alterations in behavior along with normal kinetics of the abnormally induced experimental acute toxoplasmosis in murine models. METHODS: Ten Swiss albino mice were intraperitoneally inoculated with 100 virulent RH strain tachyzoites and finally, the alterations in behavior were described and compared with other known alterations in humans and animals. RESULTS: The behavior and the other symptoms of the acute toxoplasmosis were recorded. Such mice showed typical symptoms like normal coat, severe ascites with pendulous abdomen and tachypnoea exhibited by resting fore legs either on walls of the cage, or nozzle of water bottle or other resting mice and yielded a creamy colored cloudy natured peritoneal fluid on aspiration. CONCLUSIONS: Finally the alterations in behavior were described and compared with other known alterations in humans and animals. The study has generated some important data related to possible causes of behavioral alterations and generation of suitable strategies for control of these alterations in behavior vis-à-vis better understanding of the effect of acute infection of parasite on normal behavior of infected intermediate host.

Reliability of motor parameters for follow-up after local steroid injection in carpal tunnel syndrome.

BACKGROUND: Local steroid injection is one of the treatment modalities for carpal tunnel syndrome (CTS). Symptomatic and electrophysiological improvement has been previously documented. The electrophysiological parameter, which represents the most consistent change after local steroid injection, is not well-known. OBJECTIVE: The objective of this study was to evaluate the changes in electrophysiological parameters 1 month after local steroid injection and to determine the parameter that is able to depict improvement across the severity spectrum of CTS. MATERIALS AND METHODS: Forty-seven patients (27 with bilateral disease, 74 hands totally) were included. The electrophysiological parameters studied at baseline included sensory onset latency, sensory nerve action potential amplitude, sensory conduction velocity (CV), distal motor latency, compound muscle action potential amplitude and motor CV. All patients were injected with 40 mg triamcinalone at the wrist. After 1 month, symptomatic improvement from baseline was documented using visual analog score of 100. Electrophysiology was repeated. Paired t tests were done between baseline electrophysiology parameters and those obtained 1 month after steroid injection for significant improvement. Subgroup analyses were performed in hands with mild to moderate (Grade 3 or less by Bland's classification) and severe disease (Grades 4 and 5). RESULTS: All patients showed a symptomatic improvement. Distal motor latency showed most consistent improvement irrespective of the severity of CTS. In mild to moderate CTS (Grade 3 or less of the classification given by Bland) sensory parameters were recordable and showed significant improvement in addition to distal motor latency. In the subcategory of severe CTS (Grades 4 and 5 of Bland) where sensory parameters are not recordable distal motor latency and the motor CV showed a significant improvement. CONCLUSION: One month after local steroid injection among the electrophysiological parameters studied distal motor latencies showed most consistent and recordable improvement across the severity spectrum of CTS. This can be used as a single objective parameter to follow-up patients after a local steroid injection to document improvement or relapse. They can also be considered as objective parameter to follow-up patients after surgery.

Impact of statin use on biochemical recurrence in patients treated with radical prostatectomy.

BACKGROUND: The impact of statin use on biochemical recurrence (BCR) in patients treated with radical prostatectomy (RP) remains controversial. METHODS: We retrospectively evaluated 6842 patients who underwent RP for clinically localized prostate cancer (PC) between 2000 and 2011. Uni- and multivariable cox regression models addressed the association of statin use with BCR. RESULTS: Overall, 2275 (33.3%) patients used statins. Statin users were older and had a higher rate of positive surgical margins than patients not using statins (P-values 0.05). Within a median follow-up of 25 months (interquartile range: 8-42 months), 778 (11.4%) patients experienced BCR. Actuarial estimate 5-years BCR-free survival was 82%±1 for patients without statin use and 84±1% for patients using statins (P=0.05); statin use was not associated with BCR (hazard ratio: 0.88, 95% confidence interval: 0.76-1.03, P=0.10) after adjusting for the effects of standard clinicopathologic features. CONCLUSIONS: In PC patients undergoing RP, statin use was not independently associated with lower risk of BCR.

Molecular cloning, sequencing, and biological characterization of GRA4 gene of Toxoplasma gondii.

In the present study, GRA4 (dense granule antigen) gene of Toxoplasma gondii was cloned, sequenced, and biologically characterized. The nucleotide sequence data obtained were analyzed and submitted in GenBank database (accession no. EU660037). Analysis of nucleotide sequence of GRA4 gene revealed 99.2 % homology with the published sequence (accession no. M76432). The gene segment (open reading frame) of 1,054 bp was further amplified and re-cloned in expression vector pET-32a. The recombinant protein obtained following the expression in prokaryotic system had a molecular mass of approx. 50 kDa and showed good immunoreactivity with T. gondii sera collected from infected goats. The immunization study of the recombinant protein performed in laboratory mice and live challenge with T. gondii revealed a high level of IgG response against the tachyzoite lysate antigen (TLA) by an indirect ELISA. Protection against T. gondii challenge infection was not evident in immunized mice except for the prolongation of survival period by 2 days. Humoral immune response profile revealed initially a high level of IgG antibody, but at 1 week post-challenge, a sudden drop in the level of the antibody was appreciable. Cytokine profiling by enzyme-linked immunosorbent spot method revealed relatively high level of IFN-γ production by the rodent spleen cells followed by IL-10 and IL-4. Increase in IFN-γ production by spleen cells of immunized mice following TLA stimulation suggested direct correlation to the up-regulated Th1 cells. However, the present immunization trial failed to show any positive relationship with the protection of mice following T. gondii challenge infection.

Botulinum toxin for meralgia paresthetica in type 2 diabetes.

Botulinum toxin has been used for a variety of neuropathic conditions in diabetes mellitus. Meralgia paresthetica is a mononeuropathy of femoral nerve seen in diabetes and obesity with an unclear etiopathogenesis. We studied the role of botulinum toxin in resistant cases of meralgia paresthetica in type 2 diabetes.

Babesia bigemina infection in yak (Poephagus grunniens L.): molecular detection and characterization.

Yaks contribute significantly in the Himalayan high land economy. Specific information on prevalence of babesiosis in yaks is lacking. A fast and reliable PCR assay targeting Babesia bigemina small subunit ribosomal RNA sequence (SS rRNA) was laboratory standardized for molecular detection of B. bigemina in yaks. Restriction digestion of the PCR amplified 675 bp target sequence with Vsp I confirmed the prevalent species of Babesia as B. bigemina. Nucleotide sequencing and phylogenetic analysis of PCR amplified 675 bp SS rRNA sequence revealed a close genetic relationship with other bovine isolates of B. bigemina. A PCR based survey involving 94 blood samples of yak from the National Research Centre on Yak, Dirang, Arunachal Pradesh detected infection in 5.32% of yak blood samples, which was significantly higher in comparison to microscope based detection of infection in 2.13% blood smears. This is the first report on sensitive PCR based detection of B. bigemina infection in yaks and PCR-RFLP and nucleotide sequence analysis based molecular characterization of the B. bigemina isolated from yaks.

The trifecta outcome in 300 consecutive cases of robotic-assisted laparoscopic radical prostatectomy according to D'Amico risk criteria.

BACKGROUND: To analyze the trifecta outcome (continence, potency, and cancer control) in 300 cases of robotic-assisted laparoscopic radical prostatectomy (RARP). METHODS: A prospective assessment of outcomes in 300 consecutive patients that underwent a RARP performed by a single surgeon. Patients were grouped according to D'Amico risk criteria: Group I consisted of 'low-risk' cases (n = 64), Group II consisted of 'intermediate-risk' cases (n = 88), and Group III consisted of 'high-risk' cases (n = 148). Patients were evaluated for perioperative complications and the trifecta outcome. RESULTS: The operation time, blood loss, post-operative stay, duration of urethral catheterization, and perioperative complication rate were similar among all groups. The incidence of bilateral neurovascular bundle (NVB) preservation was significantly decreased with the increasing risk of cases (P < 0.001). The continence rates at the 1-week, 1-month, 3-month, 6-month, and 12-month follow-ups did not differ significantly between groups. The potency rates at the 12-month follow-up were not significantly different. The positive surgical margin and positive lymph node metastasis rate increased with the increasing risk of cases (P < 0.001). The biochemical recurrence rate (BCR, PSA >0.2 ng/mL) was 3.1, 11.36, and 19.59% in Groups I, II and III, respectively (P = 0.004). The trifecta outcome for RARP with bilateral NVB preservation showed no significant differences among groups. CONCLUSIONS: Undergoing a RARP is safe and feasible in high-risk prostate cancer patients. Compared to low-risk and intermediate-risk groups, the high-risk group had a significant higher incidence of positive surgical margin, positive lymph node metastasis, and BCR rate.