Multiple infections caused by white spot syndrome virus and Enterocytozoon hepatopenaei in pond-reared Penaeus vannamei in India and multiplex PCR for their simultaneous detection.

White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow-out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV-positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m-PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m-PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively.

A new strain of white spot syndrome virus affecting Litopenaeus vannamei in Indian shrimp farms.

White spot syndrome virus (WSSV)-infected shrimp samples collected from grow-out ponds located at Nellore, Andhra Pradesh, India, showed WSSV negative and positive by PCR using primer sets specific to ORF119 and VP28 gene of WSSV, respectively. This indicated the deletion of genetic fragments in the genome of WSSV. The WSSV isolate along with lab strain of WSSV was subjected to next-generation sequencing. The sequence analysis revealed a deletion of 13,170 bp at five positions in the genome of WSSV-NS (new strain) relative to WSSV-TH and WSSV-LS (lab strain). The PCR analysis using the ORF's specific primer sets revealed the complete deletion of 10 ORFs in the genome of WSSV-NS strain. The primer set was designed based on sequence covering ORF161/162/163 to amplify a product of 2,748 bp for WSSV-LS and 402 bp for WSSV-NS. Our surveillance programme carried out since 2002 revealed the replacement of WSSV-LS by WSSV-NS in Indian shrimp culture system.

Studies on the occurrence of infectious myonecrosis virus in pond-reared Litopenaeus vannamei (Boone, 1931) in India.

Whiteleg shrimp, Litopenaeus vannamei, with clinical sign of muscle opaqueness with reddish colour at the distal abdominal segments were observed in farms located in West Bengal State, India. The mortality of shrimp in all disease outbreak ponds ranged from 20% to 50%, and mortality increased gradually. The RT-PCR assay of these samples using primer sets specific to infectious myonecrosis virus (IMNV) revealed its presence in the disease outbreak ponds. The IMNV infection was reproduced in healthy shrimp by intramuscular injection to satisfy River's postulates. The virus caused mortality in intramuscularly challenged shrimp, but failed to cause mortality by oral route. Tissue distribution of IMNV in infected shrimp by RT-PCR assay revealed the presence of this virus in haemolymph, gill, hepatopancreas and muscle. This study confirms that the disease outbreak which occurred in the shrimp farms located at Purba Medinipur District, West Bengal, India, was due to IMNV.

Tissue distribution of hepatopancreatic parvo-like virus of shrimp in freshwater rice-field crab, Paratelphusa hydrodomous (Herbst).

An attempt was made to determine the replication efficiency of hepatopancreatic parvo-like virus (HPV) of shrimp in different organs of freshwater rice-field crab Paratelphusa hydrodomous (Herbst) using bioassay, PCR, RT-PCR, ELISA, Western blot and q-PCR analyses. Another attempt was made to use this crab as an alternative to penaeid shrimp for the large-scale production of HPV. This crab was found to be highly susceptible to HPV by intramuscular injection. The systemic HPV infection was confirmed by PCR and Western blot analyses in freshwater crab. The expression of capsid protein gene in different organs of infected crab was revealed by RT-PCR analysis. Indirect ELISA was used to quantify the capsid protein in different organs of the crab. The copy number of HPV in different organs of the infected crab was quantified by q-PCR. The results revealed a steady decrease in CT values in different organs of the infected crab during the course of infection. The viral inoculum that was prepared from different organs of the infected crab caused significant mortality in post-larvae of tiger prawn, Penaeus monodon (Fabricius). The results revealed that this rice-field crab could be used as an alternative host for HPV replication and also for large-scale production of HPV.

A novel deep learning model for diabetic retinopathy detection in retinal fundus images using pre-trained CNN and HWBLSTM.

Diabetic retinopathy (DR) is a global visual indicator of diabetes that leads to blindness and loss of vision. Manual testing presents a more difficult task when attempting to detect DR due to the complexity and variances of DR. Early detection and treatment prevent the diabetic patients from visual loss. Also classifying the intensity and levels of DR is crucial to provide necessary treatment. This study develops a novel deep learning (DL) approach called He Weighted Bi-directional Long Short-term Memory (HWBLSTM) with an effective transfer learning technique for detecting DR from the RFI. The collected fundus images initially undergo preprocessing to improve their quality, which includes noise removal and contrast enhancement using a Hybrid Gaussian Filter and probability density Function-based Gamma Correction (HGFPDFGC) technique. The segmentation procedure divides the image into subgroups and is crucial for accurate detection and classification. The segmentation of the study initially removes the optical disk (OD) and blood vessels (BVs) from the preprocessed images using mathematical morphological operations. Next, it segments the retinal lesions from the OD and BV removed images using the Enhanced Grasshopper Optimization-based Region Growing Algorithm (EGORGA). Then, the features from the segmented retinal lesions are learned using a Squeeze Net (SQN), and the dimensionality reduction of the extracted features is done using the Modified Singular Value Decomposition (MSVD) approach. Finally, the classification is performed by employing the HWBLSTM approach, which classifies the DR abnormalities in datasets as non-DR (NDR), non-proliferative DR (NPDR), moderate NPDR (MDNPDR), and severe DR, also known as proliferative DR (PDR). The proposed approach is implemented on APTOS as well as MESSIDOR datasets. The outcomes proved that the proposed technique accurately identifies the DR with minimal computation overhead compared to the existing approaches.Communicated by Ramaswamy H. Sarma.

Convolutional neural network-based sea lion optimization algorithm for the detection and classification of diabetic retinopathy.

AIMS: Diabetic retinopathy (DR) becomes a complicated type of diabetic that causes damage to the blood vessels of the retina's light-sensitive tissue. DR may initially cause mild symptoms or no symptoms. But prolonged DR results in permanent vision loss, and hence, it is necessary to detect the DR at an early stage. METHODS: Manual diagnosing of DR retina fundus image is a time-consuming process and sometimes leads to misdiagnosis. The existing DR detection model faces few shortcomings in case of improper detection accuracy, higher loss or error values, high feature dimensionality, not suitable for large datasets, high computational complexity, poor performances, unbalanced and limited number of data points, and so on. As a result, the DR is diagnosed in this paper through four critical phases to tackle the shortcomings. The retinal images are cropped during preprocessing to reduce unwanted noises and redundant data. The images are then segmented using a modified level set algorithm based on pixel characteristics. RESULTS: Here, an Aquila optimizer is employed in extracting the segmented image. Finally, for optimal classification of DR images, the study proposes a convolutional neural network-oriented sea lion optimization (CNN-SLO) algorithm. Here, the CNN-SLO algorithm classifies the retinal images into five classes (healthy, moderate, mild, proliferative and severe). CONCLUSION: The experimental investigation is performed for Kaggle datasets with respect to diverse evaluation measures to deliberate the performances of the proposed system.

Effectiveness of electroconvulsive therapy in acute management of delirious mania in COVID-19 positive woman in second trimester pregnancy.

Delirious mania has been described as a state of acute excitement, fluctuating sensorium, affective and catatonic symptoms. Electroconvulsive therapy (ECT) despite being an effective treatment modality in such cases, has been under-utilised during pregnancy, mainly due to safety concerns. Here, we report the effectiveness of ECT in acute management of delirious mania in a 24 weeks pregnant woman who also tested COVID-19 positive during hospitalisation. Patient presented with three weeks history of acute manic excitement with period of altered sensorium and catatonic symptoms with no response to trials of two antipsychotic agents. After organic causes ruled out, patient was planned for ECT while ongoing antipsychotic was continued. After the first ECT session, patient tested positive for COVID-19, though asymptomatic and had to be shifted to COVID-19 isolation facility. Complete resolution of psychiatric symptoms occurred after fifth ECT. All five ECT sessions, including those in COVID-19 isolation facility were carried out under supervision of a multidisciplinary team. None of the ECT sessions had any major adverse event. Symptom remission sustained even following ECT discontinuation. No neonatal or maternal adverse effects observed after an uneventful delivery at 35 weeks. Both mother and child continued to maintain well in follow-up period of one year on oral olanzapine. In this unusual concurrent presentation of mania, delirium and catatonic symptoms during second trimester pregnancy, we highlighted the effectiveness and safety of ECT as a viable treatment modality. Additionally, management challenges posed by patient testing COVID-19 positive and then, administering ECT in COVID-19 isolation facility using personal protective equipment by multidisciplinary team has been highlighted.

Biosynthesis of silver nanoparticle using extract of Zea mays (corn flour) and investigation of its cytotoxicity effect and radical scavenging potential.

Silver nanoparticles (AgNPs) possesses a number of exceptional pharmaceutical properties and applications as compared with other types of metallic nanoparticles. Currently, AgNPs was biosynthesized using an aqueous extract of Zea mays L. (corn flour) powder. Further, the effect of concentration of reagents, extract, temperature and time of synthesis was also studied along with the cytotoxicity and radical scavenging potential. UV-vis spectra of AgNPs gave a surface plasmon resonance at ~420 nm. The absorption peak became sharp with the increase in time. AgNPs with monodispersed and aggregated spherical shape was observed by SEM image followed by its confirmation via strong signal in silver region of EDX spectrum. The XRD spectra confirmed its crystallinity and face-centered cubic structure. FT-IR spectra reveal the presence of phytocompounds in the synthesis of AgNPs. Further, the AgNPs exhibited strong cytotoxicity potential against HepG2 cells and its viability declined with an increase in the concentration of AgNP with respect to the control cells. It also demonstrated reasonable radical scavenging potential in terms of DPPH and ABTS scavenging, and reducing power tests. Taken together, these results of the current investigation stated that AgNPs could be beneficial in biomedical applications particularly for treatment of cancer disease along with its applications in pharmaceutical industries for the formulation of new drugs.

Microwave assisted acid hydrolysis for bioethanol fuel production from sago pith waste.

Microwave assisted acid hydrolysis (H2SO4 and HCl with >0.5 mol/L) to produce bioethanol from sago pith waste (SPW) was studied. The energy consumption for microwave hydrolysis at different energy inputs and acid concentration were calculated. The overall energy consumption for bioethanol fuel production from SPW was assessed. A maximum of 88% glucose yield and 80% ethanol yield (3.1 g ethanol per 10 g SPW) were obtained using 1.0 mol/L H2SO4. Microwave hydrolysis using 1.0 mol/L H2SO4 consumed the minimum energy of 8.1 kJ to produce 1 g glucose from SPW when energy input was fixed at 54 kJ (900 W for 1 min). In general, 1 g glucose can produce 16 kJ. The overall energy consumption for fuel grade bioethanol production from SPW was 31.77 kJ per g ethanol, which was slightly higher than the lower heating values of ethanol (26.74 kJ/g ethanol).

Novel Diazenyl Containing Phenyl Styryl Ketone Derivatives as Antimicrobial Agents.

BACKGROUND: Antibiotics play an important role in the treatment of infections to the hu-mans and at the same time, irrational, frequent prescription of higher antibiotics, change in gene com-position of microorganisms are all the reasons behind the development and introduction of new anti-biotics against different microorganisms. OBJECTIVE: In this project, an attempt has been made to synthesize some derivatives of diazenyl con-taining phenyl styryl ketones and also their in vitro screening was conducted against Mycobacterium tuberculosis, Escherichia coli, Klebsiella pneumonia, Bacillus subtilis, Staphylococcus aureus, As-pergillus niger and Candida albicans. METHODS: Ten molecules were synthesized which are diazenyl containing chalcones. 4-aminoacetophenone was diazotised and piperidine was coupled with the formed diazonium chloride. Further, the acetoxy group underwent Claisen-Schmidt condensation with differently substituted al-dehydes to form the final compounds- the chalcones. The proposed chemical structures were con-firmed by different spectroscopic techniques like FTIR, 1H NMR and Mass spectroscopy. TLC was used to know that the reactants were exhausted and the formation of the product occurred. Sharp melting point of the compounds concludes the purity. RESULTS: The MIC of the compounds 3CP, 3DP, 3EP and 3GP is 20 times the MIC of the standard fluconazole drug against Aspergillus niger. The compound 3GP is as equipotent as the standard drug Pyrazinamide with MIC of 3.12 µg/ml against Mycobacterium tuberculosis. CONCLUSION: The results are quite promising which on further studies may lead to drug molecules against different microorganisms. Especially, 3EP can be considered as a broad spectrum agent due to its potent activity against different microorganisms like Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and Candida albicans.

Indian consensus on gastroesophageal reflux disease in adults: A position statement of the Indian Society of Gastroenterology.

The Indian Society of Gastroenterology developed this evidence-based practice guideline for management of gastroesophageal reflux disease (GERD) in adults. A modified Delphi process was used to develop this consensus containing 58 statements, which were generated by electronic voting iteration as well as face-to-face meeting and review of the supporting literature primarily from India. These statements include 10 on epidemiology, 8 on clinical presentation, 10 on investigations, 23 on treatment (including medical, endoscopic, and surgical modalities), and 7 on complications of GERD. When the proportion of those who voted either to accept completely or with minor reservation was 80% or higher, the statement was regarded as accepted. The prevalence of GERD in India ranges from 7.6% to 30%, being < 10% in most population studies, and higher in cohort studies. The dietary factors associated with GERD include use of spices and non-vegetarian food. Helicobacter pylori is thought to have a negative relation with GERD; H. pylori negative patients have higher grade of symptoms of GERD and esophagitis. Less than 10% of GERD patients in India have erosive esophagitis. In patients with occasional or mild symptoms, antacids and histamine H2 receptor blockers (H2RAs) may be used, and proton pump inhibitors (PPI) should be used in patients with frequent or severe symptoms. Prokinetics have limited proven role in management of GERD.

Alterations found in pl6/Rb/cyclin D1 pathway in the dysplastic and malignant cervical epithelium.

Cervical cancer is the one of the most common cancers affecting the south Indian women. The objective of the present study was to analyze the pl6/Rb/cyclin D1 pathway in the normal (n=30), dysplastic (n=56), and malignant cervical epithelium (n=142) using immunohistochemistry. The positive immunoreactivity for p16 was as follows: CIN I--1/12 (8.3%), CIN II--2/8 (25%), CIN III--31/36 (86.1%), and in invasive tumors-121/142 (85.1%); for cyclin D1 it was CIN I--4/12 (66.6%), CIN II--5/8 (62.5%), CIN III--0%, and invasive tumors--5/142 (3.5%); and for pRb it was CIN I--9/12 (75%), CIN II--5/8(62.5%), CIN III--1/36 (97.2%), and in invasive tumors--41/142 (28.8%). Expression of cyclin D1 and p16 in the CINs were mutually exclusive and the correlation between the two biomarkers was found to be statistically significant (p = 0.0009). There was downregulation of pRb in invasive cancers, with only 32% (39/121) of the p16-positive tumors being positive for pRb (p = 0.032). Analysis of the pattern of expression of these biomolecules showed increased p16-positive phenotypes and decreased cyclin D1- and pRB-positive phenotype among the invasive tumors compared to low-grade CIN lesions.

Ontogenetic changes in the expression of immune related genes in response to immunostimulants and resistance against white spot syndrome virus in Litopenaeus vannamei.

In recent years, researchers have focused on viral and plant immunostimulants which could have beneficial effects in disease prevention and control in shrimp culture. At present, the application of the recombinant VP28 protein (r-VP28) and herbal immunostimulant has been considered as a more effective approach to prevent white spot syndrome (WSS) by enhancing the immune response in shrimp. In the present study, expression of selected immune related genes in response to r-VP28 and herbal immunostimulant mix (HIM) were separately studied qualitatively and quantitatively by RT-PCR and real time PCR, respectively during ontogenetic development from nauplius to juvenile stage in Litopenaeus vannamei. The mRNA expression level of immune related genes such as anti-lipopolysaccharides (ALF), Lysozyme, cMnSOD, Crustin, Prophenoloxidase, Tumor necrosis factor receptor-associated factor 6 (TRAF6) and Haemocyanin were found to be up-regulated significantly in different ontogenetic development stages of shrimp fed with r-VP28 and HIM formulated diets. Relative percent survival (RPS) was determined in shrimp fed with immunostimulants formulated diets after oral challenge with WSSV. The survival of WSSV challenged shrimp was found to be higher in immunostimulants treated groups when compared to untreated group. The results of PCR, ELISA and real time PCR revealed the absence of WSSV in WSSV-challenged shrimp after 20 days of treatment with immunostimulants. Among these immunostimulants, HIM was found to be more effective when compared to r-VP28. After a survey of literature, we are of the opinion that this might be the first report on the expression of immune genes during ontogenetic development of L. vannamei in response to immunostimulants.

Type 1 (11;22)(q24:q12) translocation is common in Ewing's sarcoma/peripheral neuroectodermal tumour in south Indian patients.

The Ewing's sarcoma family can present diagnostic difficulties. In the past the basis of diagnosis has been a exclusion. Identification of a specific translocation especially t(11;22) (EWS-FLI 1 fusion gene), which is seen in nearly 85 percent of Ewing's sarcoma cases can help in precise diagnosis. We have carried out a study on twenty patient samples diagnosed to have Ewing's sarcoma/peripheral neuroectodermal tumour (PNET)/small round cell malignant tumour. The study involved RT-PCR analysis for the fusion transcript, followed by sequencing to identify the specific type of fusion. Ninety percent (18/20) of the samples tested were found to be t(11;22) translocations involving EWS-FLI 1 genes. Sixty-one percent (11/18) were found to be type 1 fusion and seven were type 2 (39 percentage). This is the first study in India with quantitative information about the types of EWS-FLI 1 translocations present in Ewing's family of tumours in south Indian patients.

Variant of mitochondrial ribosomal protein s28 (mrps28) gene is differentially expressed in response to radiation in a cervical carcinoma derived cell line.

Gene expression before and after radiation treatment (10 Gy) in a cervical carcinoma derived cell line SiHa was studied. Differential display performed on non-irradiated and irradiated cells (10 Gy) using a combination of 3 anchor primers and 16 arbitrary primers yielded 20 differentially expressed fragments. Differential expression of one of the fragments differentially expressed in the irradiated sample was also confirmed in Northern blots. Cloning and sequencing of the fragment revealed it to be similar to a splice variant of the MRPS28 gene. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using primers designed to amplify the splice variant and confirmed the expression. In conclusion, the study shows that the splice variant kDec03 of the MRPS28 gene is differentially expressed in response to radiation in SiHa cells.

CDC27 protein is involved in radiation response in squamous cell cervix carcinoma.

In the present study, an attempt was made to identify genes involved in radiation response in cervix carcinoma. Differential display technique was used to study the expression profiles of tumour biopsy samples obtained from patients, responding and not responding to treatment. The samples were obtained prior to radiotherapy and subsequent to treatment with Tele-radiation at 10 Gray (Gy). One of the differentially expressed cDNAs, when sequenced was identified to be CDC27. Immuno-histochemical analysis of pre- and post-treated tumour samples from fifteen patients showed the downregulation of expression of CDC27 protein in seven patients. Down-regulation was associated with poorer response to radiotherapy. Cervical cancer cell lines SiHa and C33A were irradiated and their nuclei were stained for expression of CDC27 and analyzed using fluorescent-activated cell sorting (FACS). Down-regulation of CDC27 protein in the irradiated SiHa cell line was associated with greater survival fraction, compared to the irradiated C33A cell line, which had only slight fall in the level of CDC27 protein. This is the first study to suggest a role for CDC27 in radiation response. However, a larger cohort is needed to further confirm the value of CDC27 protein as a predictive marker, for radiation response in cervix cancer.

PCR-heteroduplex analysis of TCR gamma, delta and TAL-1 deletions in T-acute lymphoblastic leukemias: implications in the detection of minimal residual disease.

Detection of MRD remains one of the major goals in the treatment of acute lymphoblastic leukemia (ALL). We have used the polymerase chain reaction (PCR)-heteroduplex (HD) analysis to assess and confirm the clonal expansion of T cell receptor (TCR) gamma and delta gene rearrangements in 24 T-ALL patients at diagnosis. 52.4% revealed Vdelta1-Jdelta1; 48% Vdelta2-Ddelta3; 62.5% Vgamma1-Jgamma1 and 46% both Vdelta1-Jdelta1 and Vgamma1-Jgamma1 clonal rearrangements. 6/24 patients had TAL-1 deletion. These clonal markers were used to monitor MRD in remission/relapse bone marrow samples for periods ranging from 6 to 75 months after diagnosis. Patients who relapsed and died revealed a continuous PCR-HD positivity in their clinical remission bone marrow samples. HD analysis established identical diagnostic clone at relapse. Patients who are in long-term clinical and morphological remission achieved PCR-HD negativity in their 8-12 months bone marrow remission samples and continue to be PCR-HD negative. MRD monitored in six patients with two diagnostic PCR--HD positive clonal markers reveal an identical pattern ensuring circumvention of false positive and negative results. Thus, we conclude that PCR followed by HD analysis is a useful technique to monitor MRD in remission/relapse samples in ALL patients.

Prevalence of C-erbb3 expression in squamous-cell carcinomas of the cervix as determined by the monoclonal-antibody rtj2.

The c-erbB3 protein is a member of the type 1 tyrosine kinase growth factor receptor family. We report on the production and characterisation of a monoclonal antibody, RTJ2, raised against a synthetic peptide from the cytoplasmic domain of the molecule. The antibody is specific for c-erbB3 protein as determined by Western blotting, immunoprecipitation and immunocytochemistry. Using this monoclonal antibody we have evaluated the prevalence of expression of the c-erbB3 protein in 72 squamous cell carcinomas of the cervix. 17 of the 72 (23.6%) tumours were found to overexpress c-erbB3 protein relative to the levels seen in the normal epithelium. Overexpression was not correlated with stage, grade or histologic subtype of the tumours. The high incidence of c-erbB3 overexpression relative to the normal tissue suggests a possible role for c-erbB3 in the oncogenesis of cervical epithelium.

Oral cancer in Southern India: the influence of body size, diet, infections and sexual practices.

Between 1996 and 1999, we carried out a study in Southern India on risk factors for oral cancer. The study included 591 incident cases of cancer of the oral cavity (282 women) and 582 hospital controls (290 women). Height was unrelated to oral cancer risk. Body mass index (weight in kilograms/height in metres squared) was inversely associated with risk (P for trend<0.001). Paan chewers with low BMI were at particularly high risk. Risk was increased among subjects consuming meat (odds ratio (OR) 1.54, 95% confidence interval (CI) 1.00-2.37), ham and salami (OR 4.40, 95% CI 2.88-6.71) two or more times per week. Frequent consumption of fish, eggs, raw green vegetables, cruciferous vegetables, carrots, pulses, apples or pears, citrus fruit, and overall consumption of vegetables and fruit decreased oral cancer risk (P for trend for each of these items less than or equal to 0.001). The risk associated with low consumption of vegetables was higher among smokers than among non-smokers. Men, but not women, who practised oral sex had an increased oral cancer risk (OR 3.14, 95% CI 1.15-8.63). Women with more than one sexual partner during life were at increased oral cancer risk (OR 9.93, 95% CI 1.57-62.9).