Development of probe-based real-time loop-mediated isothermal amplification for detection of Brucella.

AIM: To develop a rapid, sensitive and specific diagnostic assay for the detection of Brucella. METHODS AND RESULTS: The probe-based RT-LAMP was carried out by using a set of four or six primers and different LAMP chemicals to compare its results with real-time PCR. Detection of gene amplification is done within 40 min and can be seen by amplification curve, turbidity and addition of DNA-binding dye at the end of the reaction results in colour difference under normal day light and in UV. The sensitivity of probe-based real-time LAMP assay was found 10-fold higher than Taqman-based qPCR. The specificity of the developed assay was validated by the absence of any cross-reaction with other pathogenic bacteria. CONCLUSION: The developed probe-based RT-LAMP assay is extremely rapid, cost effective, highly specific and sensitive, and has potential usefulness for rapid Brucella surveillance. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed probe-based RT-LAMP is a powerful gene amplification technique which is a specific, fast diagnostic tool for early detection and identification of Brucella.

Comparative efficacy of oestrus synchronization protocols in buffalo (Bubalus bubalis).

This study was conducted to test the efficacy of gonadotropic hormone (GnRH)-based synchronization protocols (Ovsynch, Heatsynch, and Ovsynch Plus) in buffaloes under field condition. Based on anamnesis and transrectal palpation twice at 10-day interval and serum progesterone (P4) concentration, 150 anoestrous buffaloes and delayed pubertal heifers were selected to induce oestrus using GnRH-based protocols. These selected animals were randomly divided into three groups: group I: Ovsynch (n = 50), group II: Heatsynch (n = 50), and group III: Ovsynch Plus (n = 50) regimen. Before treatment initiation, blood samples were collected for P4, beta-hydroxy butyric acid (ß-OHB), and mineral estimation, in addition to the monitoring of oestrus signs. In this investigation, no significant difference (P > 0.05) in oestrus signs was deduced among three groups. Oestrus induction rate (OIR) was comparable (P > 0.05) among the groups (Ovsynch 82%, Heatsynch 86%, and Ovsynch Plus 88%). Conception rate (CR) following fixed time artificial insemination (FTAI) was slightly higher with Ovsynch Plus group (28%) as compared to Ovsynch (24%) and Heatsynch (18%) groups, though non-significant. Furthermore, serum glucose, ß-OHB, macrominerals (calcium, potassium, and magnesium), and trace minerals (copper, zinc, and iron) remained comparable (P > 0.05) among the groups. In conclusion, all the protocols (Ovsynch, Heatsynch, and Ovsynch Plus) are efficient in oestrus induction in anoestrous buffaloes under field condition with Ovsynch Plus protocol resulting in higher CR as compared to other protocols.

16S rDNA analysis of archaea indicates dominance of Methanobacterium and high abundance of Methanomassiliicoccaceae in rumen of Nili-Ravi buffalo.

The molecular diversity of rumen methanogens was investigated using 16S rDNA gene library prepared from the rumen contents of Nili-Ravi buffaloes. Microbial genomic DNA was isolated from four adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the inserts of positive clones were sequenced. A total of 142 clones were examined, and the analysis revealed 46 species level (0.01 distance) operational taxonomic units (OTUs). Twenty six OTUs comprising 89 clones (63% of the total clones) were taxonomically assigned to Methanobacterium genus and the majority of them had highest percent identity with Methanobacterium flexile among cultured methanogens. Five OTUs comprising 27 clones (19% of total clones) were taxonomically assigned to Methanomicrobium genus and these clones showed highest sequence identity with Methanomicrobium mobile. Only two OTUs comprising 6 clones (4% of total clones) were assigned to Methanobrevibacter genus. A total of 17 clones belonging to 10 species level OTUs showed highest percent identity (ranging from 85 to 95%) with Methanomassilicoccus luminyensis and were taxonomically classified as Methanomassiliicocaceae. Out of the 142 rDNA clones, 112 clones, which constitute 79% of the total clones representing 42 OTUs, had less than 98.5% sequence identity with any of the cultured strains of methanogens and represent novel species of methanogens. This study has revealed the largest assortment of hydrogenotrophic methanogen phylotypes ever identified from the rumen of Nili-Ravi buffaloes. The study indicates that Methanobacterium is the most dominant methanogen in the rumen of Nili-Ravi buffalo. This is also the first report on the presence of methanogens phylogenetically close to M. luminyensis, an H2 dependent methylotrophic methanogen, in the rumen of buffaloes at such a high level of abundance.

Identifying discriminative features of brain network for prediction of Alzheimer's disease using graph theory and machine learning.

Alzheimer's disease (AD) is a challenging neurodegenerative condition, necessitating early diagnosis and intervention. This research leverages machine learning (ML) and graph theory metrics, derived from resting-state functional magnetic resonance imaging (rs-fMRI) data to predict AD. Using Southwest University Adult Lifespan Dataset (SALD, age 21-76 years) and the Open Access Series of Imaging Studies (OASIS, age 64-95 years) dataset, containing 112 participants, various ML models were developed for the purpose of AD prediction. The study identifies key features for a comprehensive understanding of brain network topology and functional connectivity in AD. Through a 5-fold cross-validation, all models demonstrate substantial predictive capabilities (accuracy in 82-92% range), with the support vector machine model standing out as the best having an accuracy of 92%. Present study suggests that top 13 regions, identified based on most important discriminating features, have lost significant connections with thalamus. The functional connection strengths were consistently declined for substantia nigra, pars reticulata, substantia nigra, pars compacta, and nucleus accumbens among AD subjects as compared to healthy adults and aging individuals. The present finding corroborate with the earlier studies, employing various neuroimagining techniques. This research signifies the translational potential of a comprehensive approach integrating ML, graph theory and rs-fMRI analysis in AD prediction, offering potential biomarker for more accurate diagnostics and early prediction of AD.

Evaluation of surgically treated primary spinal cord tumors in a single Indian institution: A case series study of 178 patients.

Introduction: Primary spinal cord tumors are rare tumors that are usually heterogeneous having variable histopathological subtypes. Rapidly, growing space-occupying lesions in the spinal canal cause severe loss of function. This study aimed to analyze all adult patients diagnosed with primary spinal cord tumors and to describe their location, symptoms, histopathological types, surgical outcome, complications, recurrence in a single institution, and prognostic factors. Materials and Methods: The study was a retrospective study of 178 patients with spinal tumor who underwent surgical intervention at the department of neurosurgery at a tertiary care institute from 2014 to 2021. Patient demographic characteristics, symptoms, radiological features, spinal level of tumor, spinal compartment, preoperative and postoperative McCormick grade, and complications were recorded, and a comparison of outcomes after surgical intervention was done. Results: In our study, 97 patients were males and the rest of the 81 patients were females. The mean age at the time of surgery was 43.5 years. The thoracic region of the spinal canal was the most involved with 67 (37.64%) cases, whereas intradural extramedullary was the most common (65.73%). Schwannoma was the most common histopathological subtype (46.06%). Total excision was achieved in 154 cases. A total of 37 patients were in Grades 4 and 5 of modified McCormick's grade preoperatively. However, postoperatively, there were only 17 patients in this group. Two patients with cervical intramedullary tumor expired in the postoperative period. Conclusion: Early surgical intervention with sound microneurosurgical skills gives good outcomes independent of the type and location of tumor. Better outcomes are seen in extradural tumors, early surgical intervention, tumors better amenable to total resection, and in patients with better preoperative neurological status.

Comparative evaluation of brain neurometabolites and DTI indices following whole body and cranial irradiation: a magnetic resonance imaging and spectroscopy study.

Understanding early differential response of brain during whole body radiation or cranial radiation exposure is of significant importance for better injury management during accidental or intentional exposure to ionizing radiation. We investigated the early microstructural and metabolic profiles using in vivo diffusion tensor imaging (DTI) and proton magnetic resonance spectroscopy ((1)H MRS) following whole body and cranial radiation exposure of 8 Gy in mice using a 7.0 T animal MRI system and compared profiles with sham controls at days 1, 3, 5 and 10 post irradiation. A significant decrease in fractional anisotropy (FA) values was found in hippocampus, thalamic and hypothalamic regions (p < 0.05) in both whole body and cranial irradiated groups compared with controls, suggesting radiation induced reactive astrogliosis or neuroinflammatory response. In animals exposed to whole body radiation, FA was significantly decreased in some additional brain regions such as sensory motor cortex and corpus callosum in comparison with cranial irradiation groups and controls. Changes in FA were observed till day 10 post irradiation in both the groups. However, MRS study from hippocampus revealed changes only in the whole body radiation dose group. Significant reduction in the ratios of the metabolites myoinositol (mI, p = 0.02) and taurine (tau, p = 0.03) to total creatine were observed, and these metabolic alterations persisted till day 10 post irradiation. To the best of our knowledge this study has for the first time documented a comparative account of microstructural and metabolic aspects of whole body and cranial radiation induced early brain injury using in vivo MRI. Overall our findings suggest differential response at microstructure and metabolite levels following cranial or whole body radiation exposure.

Categorization of hotspots into three types - weak, moderate and strong to distinguish protein-protein versus protein-peptide interactions.

Protein-protein and protein-peptide interactions (PPI and PPepI) belong to a similar category of interactions, yet seemingly subtle differences exist among them. To characterize differences between protein-protein (PP) and protein-peptide (PPep) interactions, we have focussed on two important classes of residues-hotspot and anchor residues. Using implicit solvation-based free energy calculations, a very large-scale alanine scanning has been performed on benchmark datasets, consisting of over 5700 interface residues. The differences in the two categories are more pronounced, if the data were divided into three distinct types, namely - weak hotspots (having binding free energy loss upon Ala mutation, ΔΔG, ∼2-10 kcal/mol), moderate hotspots (ΔΔG, ∼10-20 kcal/mol) and strong hotspots (ΔΔG ≥ ∼20 kcal/mol). The analysis suggests that for PPI, weak hotspots are predominantly populated by polar and hydrophobic residues. The distribution shifts towards charged and polar residues for moderate hotspot and charged residues (principally Arg) are overwhelmingly present in the strong hotspot. On the other hand, in the PPepI dataset, the distribution shifts from predominantly hydrophobic and polar (in the weak type) to almost similar preference for polar, hydrophobic and charged residues (in moderate type) and finally the charged residue (Arg) and Trp are mostly occupied in the strong type. The preferred anchor residues in both categories are Arg, Tyr and Leu, possessing bulky side chain and which also strike a delicate balance between side chain flexibility and rigidity. The present knowledge should aid in effective design of biologics, by augmentation or disruption of PPIs with peptides or peptidomimetics.Communicated by Ramaswamy H. Sarma.

Disallowed spots in protein structures.

Ramachandran (ϕ, ψ) steric map was introduced in 1963 to describe available conformation space for protein structures. Subsequently, residues were observed in high-energy disallowed regions of the map. To unequivocally identify the locations of disallowed conformations of residues, we got 36 noise-free protein structures (resolution ≤1 Å, Rwork/Rfree ≤ 0.10). These stringent criteria were applied to rule out data or model errors or any crystallographic disorders. No disallowed conformation was found in the dataset. Further, we also examined disallowed conformations in a larger dataset (resolution ≤1.5 Å, devoid of any model errors, or disorders). The observed locations of disallowed residues are referred as disallowed spots. These spots include short loops of 3-5 residues, and locations where residues participate in disulfide bonding or intramolecular interactions or inter-molecular interactions with neighboring water, metals or ligands. Conformational sampling revealed that short loops in between secondary structures hardly have any opportunity to relieve from conformational strain. Residues involved in interactions, which provide energetic compensation for high-energy conformational states, were relieved from strain once the causative interaction was removed. The present study aims to identify disallowed spots in the native state of proteins, wherein residues are forced to be trapped in high-energy disallowed conformations. Moreover, it was also observed that pre-Pro, Ser, Asp, trans-Pro, Val, Asn & Gly have higher tendency to occur in disallowed conformation, which could be attributed to factors such as conformational restrictions, residue propensity of secondary structures and compensating sidechain and mainchain interactions, stabilizing turn-mimics.

An exploratory study into the role of dynamic contrast-enhanced (DCE) MRI metrics as predictors of response in head and neck cancers.

AIM: To assess the efficacy of dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) in prediction of the response to treatment in patients with head and neck cancers (HNCs). METHODS AND MATERIALS: Twenty-one patients with advanced HNC, suitable for concurrent chemoradiotherapy underwent DCE MRI, for the quantification of blood flow (BF) and volume (BV). All the patients received radical doses of conventionally fractionated radiotherapy up to a dose of 70 Gy along with concurrent weekly cisplatin. RESULTS: The values of BV and BF were higher in complete responders as compared to partial responders at primary as well as nodes. Both BF and BV were found to be significantly higher in patients with high T-stage as compared to patients with lower T-stage. CONCLUSIONS: DCE metrics can be used as a predictor of response to treatment in locally advanced HNCs after validation of these observations in a larger number of patients.

1-Meth-oxy-4-({[(4-meth-oxy-phen-yl)-sulfan-yl](phen-yl)meth-yl}sulfan-yl)benzene.

The title compound, C(21)H(20)O(2)S(2), forms a propeller-shaped structure with the tetra-hedral C atom as the central hub and meth-oxy-benzene and phenyl residues as radiating blades. Short C-H⋯π contacts are observed.

1,1'-[(2-Bromo-phen-yl)-methyl-ene]-dipyrrolidin-2-one.

In the title compound, C(15)H(17)BrN(2)O(2), both pyrrolidinone rings adopt envelope conformations. The crystal packing is characterized by short C-Br⋯O=C inter-actions [Br⋯O = 3.1730 (13) Å], leading to supra-molecular dimers. Inter-molecular C-H⋯O and C-H⋯π inter-actions are also observed.

Minimum MD simulation length required to achieve reliable results in free energy perturbation calculations: case study of relative binding free energies of fructose-1,6-bisphosphatase inhibitors.

In an attempt to establish the criteria for the length of simulation to achieve the desired convergence of free energy calculations, two studies were carried out on chosen complexes of FBPase-AMP mimics. Calculations were performed for varied length of simulations and for different starting configurations using both conventional- and QM/MM-FEP methods. The results demonstrate that for small perturbations, 1248 ps simulation time could be regarded a reasonable yardstick to achieve convergence of the results. As the simulation time is extended, the errors associated with free energy calculations also gradually tapers off. Moreover, when starting the simulation from different initial configurations of the systems, the results are not changed significantly, when performed for 1248 ps. This study carried on FBPase-AMP mimics corroborates well with our previous successful demonstration of requirement of simulation time for solvation studies, both by conventional and ab initio FEP. The establishment of aforementioned criteria of simulation length serves a useful benchmark in drug design efforts using FEP methodologies, to draw a meaningful and unequivocal conclusion.

Correlation of phase values with CT Hounsfield and R2* values in calcified neurocysticercosis.

PURPOSE: To correlate phase and R2* derived from susceptibility-weighted magnetic resonance imaging (MRI) with computed tomography-Hounsfield (CT-HU) values in calcified neurocysticercosis and to evaluate phase imaging in the assessment of calcified neurocysticercosis. MATERIALS AND METHODS: Thirty-five patients with 52 calcified lesions underwent both CT and MRI. Phase and R2* were calculated from multi-echo 3D-T2-star-weighted-angiography data. MRI and CT data were coregistered using mutual information. Spearman's correlation was performed between quantitative phase and CT-HU and R2* values. The Mann-Whitney U-test was used to see differences between CT-HU and R2* values from corresponding positive and negative phase regions. RESULTS: The median values of CT-HU and R2* from regions with positive and negative phase were found to be 142.10 (range: 41.89-491.75) and 68.5/sec (range: 20-110/sec) and 137.30 (range: 30.83-458.88) and 69/sec (range: 0-110/sec), respectively. There was a significant correlation of positive phase values with corresponding CT-HU and R2* values. In addition, there was a significant correlation of R2* and CT-HU with negative phase values. CONCLUSION: We conclude that there is a significant correlation between negative and positive phase with CT-HU and R2* values, suggesting that the CT hyperdense lesion may have both calcium and other minerals, which can be differentiated using phase imaging. Conventional MRI should include phase imaging to detect calcified neurocysticercosis.

4-(4-Chloro-phen-yl)-N-[(E)-4-(dimethyl-amino)-benzyl-idene]-1,3-thia-zol-2-amine.

The title compound, C(18)H(16)ClN(3)S, adopts an extended mol-ecular structure. The thia-zole ring is inclined by 9.2 (1) and 15.3 (1)° with respect to the chloro-phenyl and 4-(dimethyl-amino)-phenyl rings, respectively, while the benzene ring planes make an angle of 19.0 (1)°. A weak inter-molecular C-H⋯π contact is observed in the crystal structure.

3-Ethyl-4-methyl-1H-pyrazol-2-ium-5-olate.

The title compound, C(6)H(10)N(2)O, is a zwitterionic pyrazole derivative. The crystal packing is predominantly governed by a three-center iminium-amine N(+)-H⋯O(-)⋯H-N inter-action, leading to an undulating sheet-like structure lying parallel to (100).

Bis(acetyl-acetonato-κO,O')(pyridine-κN)zinc(II).

In the title compound, [Zn(C(5)H(7)O(2))(2)(C(5)H(5)N)], the metal atom has square-pyramidal coordination geometry with the basal plane defined by the four O atoms of the chelating acetyl-acetonate ligands and with the axial position occupied by the pyridine N atom. The crystal packing is characterized by a C-H⋯O hydrogen-bonded ribbon structure approximately parallel to [10[Formula: see text]].

Comparative analysis of cultural isolation and PCR based assay for detection of campylobacter jejuni in food and faecal samples.

In the present study, the efficacy of polymerase chain reaction (PCR) based on mapA gene of C. jejuni was tested for detection of Campylobacter jejuni in naturally infected as well as spiked faecal and food samples of human and animal origin. Simultaneously, all the samples were subjected to the cultural isolation of organism and biochemical characterization. The positive samples resulted in the amplification of a DNA fragment of size ~589 bp in PCR assay whereas the absence of such amplicon in DNA extracted from E. coli, Listeria, Salmonella and Staphylococcus confirmed the specificity of the primers. Of randomly collected 143 faecal samples comprising human diarrheic stools (43), cattle diarrheic faeces (48) and poultry faecal swabs (52) only 4, 3 and 8, respectively, could be detected by isolation whereas 6, 3 and 10, respectively, were found positive by PCR. However, among food samples viz. beef (30), milk (35), cheese (30), only one beef sample was detected both by culture as well as PCR. Additionally, PCR was found to be more sensitive for C. jejuni detection in spiked faecal and food samples (96.1% each) as relative to culture isolation which could detect the organism in 86.7% and 80% samples, respectively. The results depicted the superior efficacy of PCR for rapid screening of samples owing to its high sensitivity, specificity and automation potential.

Phenylbenzopyrone of Flavonoids as a Potential Scaffold to Prevent SARSCoV-2 Replication by Inhibiting its MPRO Main Protease.

BACKGROUND: In December 2019, an outbreak of a pneumonia-like illness, Corona virus disease 2019 (COVID-19), originating from Wuhan, China, was linked to novel coronavirus, now termed SARS-CoV-2. Unfortunately, no effective drugs or vaccines have been reported yet. The main protease (MPRO) remains the most validated pharmacological target for the design and discovery of inhibitors. OBJECTIVE: The purpose of the study was to find a prospective natural scaffold as an inhibitor for MPRO main protease in SARS-CoV-2 and compare it with repurposed antiviral drugs lopinavir and nelfinavir. METHODS: Natural compound libraries were screened for potential scaffold against MPRO main protease. Molecular dynamics simulation, MM-GBSA and principal component analyses of enzyme- ligand complexes were carried out with the top-ranking hits and compared with the repurposed antiviral drugs lopinavir and nelfinavir. RESULTS: The structure-based virtual screening indicated phenylbenzopyrone of flavonoids as one of the top-ranking scaffolds that have the potential to inhibit the main protease with the Oglycosidic form, performing better than the corresponding aglyconic form. Simulation studies indicated that glycosidic form of flavonoid is a more suitable inhibitor with compounds rutin, procyanidin B6, baicalin and galloylquercetin, demonstrating high affinity and stability, and rutin, emerging as one of the best candidate compounds. Interestingly, rutin was reported to have inhibitory activity against similar protease (3Cprotease of enterovirus A71) and implicated in lung fibrosis. CONCLUSION: The present study on flavonoids, possessing a potential scaffold for inhibiting main protease activity for all betacoronavirus is an attempt to provide new and safe drug leads within a reasonably short period.

(2E)-1-(1,3-Benzodioxol-5-yl)-3-(2-bromo-phen-yl)prop-2-en-1-one.

The mol-ecule of the title compound, C(16)H(11)BrO(3), is essentially planar with a maximum deviation of 0.178 (4) Šand the configuration of the keto group with respect to the olefinic double bond is typically s-cis. In the crystal structure, inter-molecular Br⋯O inter-actions [3.187 (3)Å] give rise to chains parallel to the b axis. Adjacent chains are further linked along the a axis by C-H⋯π inter-actions. The crystal studied was a racemic twin with a 0.595 (13):0.405 (13) ratio.

(7E)-5-Benzyl-7-(2-chloro-benzyl-idene)-3-(2-chloro-phen-yl)-2-phenyl-3,3a,4,5,6,7-hexa-hydro-2H-pyrazolo-[4,3-c]pyridine.

In the title 2H-pyrazolo-[4,3-c]pyridine derivative, C(32)H(27)Cl(2)N(3), the dihydro-pyrazole ring adopts an envelope conformation and the piperidine fused ring a twisted-chair conformation. Two short intra-molecular C-H⋯Cl contacts are observed. The crystal packing is characterized by dimeric C-Cl⋯π inter-actions involving the 5-benzyl ring, with Cl⋯centroid and closest atomic Cl⋯π distances of 3.778 (2) and 3.366 (4) Å, respectively.