SEW2871 reduces seizures via the sphingosine 1-phosphate receptor-1 pathway in the pentylenetetrazol and phenobarbitone kindling model of drug-refractory epilepsy.

Epilepsy is a prevalent neurological disorder characterized by neuronal hypersynchronous discharge in the brain, leading to central nervous system (CNS) dysfunction. Despite the availability of anti-epileptic drugs (AEDs), resistance to AEDs is the greatest challenge in treating epilepsy. The role of sphingosine-1-phosphate-receptor 1 (S1PR1) in drug-resistant epilepsy is unexplored. This study investigated the effects of SEW2871, a potent S1PR1 agonist, on a phenobarbitone (PHB)-resistant pentylenetetrazol (PTZ)-kindled Wistar rat model. We measured the messenger ribonucleic acid (mRNA) expression of multi-drug resistance 1 (MDR1) and multi-drug resistance protein 5 (MRP5) as indicators for drug resistance. Rats received PHB + PTZ for 62 days to develop a drug-resistant epilepsy model. From day 48, SEW2871 (0.25, 0.5, 0.75 mg/kg, intraperitoneally [i.p.]) was administered for 14 days. Seizure scoring, behaviour, oxidative markers like reduced glutathione, catalase, superoxide dismutase, inflammatory markers like interleukin 1 beta tumour necrosis factor alpha, interferon gamma and mRNA expression (MDR1 and MRP5) were assessed, and histopathological assessments were conducted. SEW2871 demonstrated dose-dependent improvements in seizure scoring and neurobehavioral parameters with a reduction in oxidative and inflammation-induced neuronal damage. The S1PR1 agonist also downregulated MDR1 and MRP5 gene expression and significantly decreased the number of dark-stained pyknotic nuclei and increased cell density with neuronal rearrangement in the rat brain hippocampus. These findings suggest that SEW2871 might ameliorate epileptic symptoms by modulating drug resistance through downregulation of MDR1 and MRP5 gene expression.

Neuroprotective effect of Berberine Nanoparticles Against Seizures in Pentylenetetrazole Induced Kindling Model of Epileptogenesis: Role of Anti-Oxidative, Anti-Inflammatory, and Anti-Apoptotic Mechanisms.

There is an unmet need to develop alternative therapeutic strategies to not only restrain seizures but also to alleviate the underlying pathologies and sequelae. Berberine (BBR), an isoquinoline alkaloid, has shown promising effect in the kindling model of epileptogenesis, but due to the poor oral bioavailability its clinical application is limited. So, the present study was designed to study the neuroprotective effect of BBR nanoparticles (enhanced bioavailability as compared to BBR) against seizures in pentylenetetrazole (PTZ) induced kindling model of epileptogenesis. Kindling model was established in male Wistar rats by intraperitoneal (i.p.) administration of PTZ (30 mg/kg) on every alternate day till the animal became fully kindled or till 6 weeks. Three doses of BBR (50, 100, and 200 mg/kg) and nano-BBR (25, 50, 100 mg/kg) were studied for seizure score, percentage of animal kindled, histopathological score, oxidative stress, inflammation, and apoptosis in PTZ treated rats by conducting cytokines, gene expression and protein expression analysis. BBR nanoparticles showed significant effect on the seizure score and percentage of animal kindled, histopathological score, neurobehavioral parameters (Forced swim test, Rotarod), oxidative (MDA, SOD, GSH, GPx) and inflammatory (IL-1beta, TNF-alpha) parameters, apoptotic parameters (Bax and iNOS), and gene (Nrf2, NQO1, HO1) and protein expression (Nrf2) as compared to both PTZ and BBR. BBR nanoparticles showed neuroprotective effect in PTZ induced kindling model of epileptogenesis and proves to be a promising antiepileptogenic therapy for the patients who are at high risk of developing seizures.

Role of Serology, Dietary Assessment, and Fecal Gluten Immunogenic Peptides for Predicting Histologic Recovery in Children with Celiac Disease.

BACKGROUND: A strict lifelong gluten-free diet (GFD) remains the only treatment of celiac disease (CD). Adherence to gluten-free diet is best reflected by mucosal healing. Noninvasive tools capable of predicting mucosal recovery in CD patients need to be identified. AIMS: To compare the ability of various modalities used to assess compliance to GFD, for predicting persistent mucosal damage in children with CD. METHODS: A prospective, single-center, observational study on children with CD on a GFD was conducted between January 2020 and April 2021. Children with CD on GFD were consecutively enrolled and various modalities used to assess adherence to GFD were compared. RESULTS: One hundred and fifty children (Mean age 12.2 ± 3.6 years, 58% Boys) on GFD (Mean duration 6 ± 3.1 years) were enrolled in the study. Persistent mucosal damage was seen in 88% of the enrolled. Fecal gluten immunogenic peptide (GIP) was positive in 87.8% (129/147). Antibodies to tissue transglutaminase (TGA-IgA) and/or deamidated gliadin peptide (DGP) were positive in 32% (48/150) whereas antibody to synthetic neoepitopes of TGA-IgA was positive in 24.8% (37/149). Non-compliance as assessed by local questionnaire, Biagi score, and dietitian detailed interview were 62.7%, 60%, and 75.3%, respectively. Serology had the highest specificity (83%) and fecal GIP had the highest sensitivity (89%). On logistic regression analysis, only non-compliance by Biagi score predicted poor mucosal recovery. CONCLUSION: Fecal GIP may be sensitive to detect only "one-point dietary transgression." None of the existing modalities used to assess compliance to GFD accurately predict persistent mucosal damage. A subset of patients may develop gluten tolerance.

The gene expression and proteomic profiling of Acanthamoeba isolates.

INTRODUCTION: The free-living protozoan Acanthamoeba can cause severe keratitis known as Acanthamoeba Keratitis (AK) and granulomatous amoebic encephalitis (GAE). The pathogenesis of Acanthamoeba includes intricate interactions between the organism and the host's immune system. The downstream analysis of a well-annotated genome assembly along with proteomic analysis can unravel several biological processes and aid in the identification of potential genes involved in pathogenicity. METHODS: Based on the next-generation sequencing data analysis, genes including lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein were selected as probable pathogenic targets that were validated by conventional PCR in a total of 30 Acanthamoeba isolates. This was followed by real-time PCR for the evaluation of relative gene expression in the keratitis and amoebic encephalitis animal model induced using keratitis (CHA5), encephalitis (CHA24) and non-pathogenic environmental isolate (CHA36). In addition, liquid chromatography-mass spectrometry (LC-MS/MS) was performed for keratitis, encephalitis, and non-pathogenic environmental isolate before and after treatment with polyhexamethylene biguanide (PHMB). RESULTS: The conventional PCR demonstrated the successful amplification of lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein genes in clinical and environmental isolates. The expression analysis revealed phospholipase, lysophospholipase, and mannose-binding genes to be significantly upregulated in the keratitis isolate (CHA 5) during AK in the animal model. In the case of the amoebic encephalitis model, phospholipase, lysophospholipase, S8/S53 peptidase, and carboxylesterase were significantly upregulated in the encephalitis isolate compared to the keratitis isolate. The proteomic data revealed differential protein expression in pathogenic versus non-pathogenic isolates in the pre and post-treatment with PHMB. CONCLUSION: The gene expression data suggests that lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein (MBP) could play a role in the contact-dependent and independent mechanisms of Acanthamoeba pathogenesis. In addition, the proteomic profiling of the 3 isolates revealed differential protein expression crucial for parasite growth, survival, and virulence. Our results provide baseline data for selecting possible pathogenic targets that could be utilized for designing knockout experiments in the future.

Assessment of pathogenic potential of Acanthamoeba isolates by in vitro and in vivo tests.

Acanthamoeba are free-living protozoa present ubiquitously in numerous environmental reservoirs that exist as an actively feeding trophozoite or a dormant cyst stage. The pathogenic Acanthamoeba are known to cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Despite their omnipresence, the number of infections is quite low. The reason behind this low frequency of Acanthamoeba infections could be the existence of many non-pathogenic strains or a successful host immune response to these infections. Studies in the past have proposed a few physiological parameters for the differentiation of pathogenic and non-pathogenic strains. Additionally, in vivo experiments are known to play an essential role in understanding the virulence of parasites, immunological aspects, and disease pathogenesis. The thermotolerance (30 °C, 37 °C, and 40 °C) and osmotolerance (0.5 M, 1 M, and 1.5 M) tests were performed on 43 Acanthamoeba isolates from patients with keratitis (n = 22), encephalitis (n = 5), and water samples (n = 16). In addition, the genotype of 10 Acanthamoeba isolates (keratitis (n = 2), encephalitis (n = 2), water (n = 6)) was determined and were then evaluated for pathogenicity on mouse model by inducing Acanthamoeba keratitis and amoebic encephalitis. The results of the thermotolerance and osmotolerance assays categorized 29/43 (67.4%) isolates as pathogenic, 8 as low pathogenic (18.6%), and the remaining 6 (13.9%) as non-pathogenic. The 10 Acanthamoeba isolates were categorized as T11 (5 isolates), T5 (2 isolates), T4 (2 isolates), and T10 (1 isolate) genotypes. Out of 10 Acanthamoeba isolates, 9 were successful in establishing AK, amoebic encephalitis, or both in the mice model, and a single isolate was found non-pathogenic. Two isolates from water samples were non-pathogenic in the physiological tests but successfully established Acanthamoeba infection in the mice model. The results of the physiological assays and in vivo experiments were analogous for 7 isolates while 1 isolate from the water was low pathogenic in the physiological assays but failed to produce pathogenicity during in vivo experiments. The physiological parameters are not very dependable to test the pathogenic potential of Acanthamoeba isolates, and thus results must always be validated by in vivo experiments. There is no infallible approach for determining the potential pathogenicity of environmental isolates of Acanthamoeba because several parameters regulate the pathogenic potential.

Repurposing of FDA approved drugs against uropathogenic Escherichia coli: In silico, in vitro, and in vivo analysis.

Urinary tract infections (UTIs) are a serious health concern worldwide. Treatment of UTIs is becoming a challenge as uropathogenic Escherichia coli (UPEC), which is the most common etiological agent, has developed resistance to the main classes of antibiotics. Small molecules that interfere with metabolic processes rather than growth are attractive alternatives to conventional antibiotics. Repurposing of already known drugs for treating infectious diseases could be an attractive avenue for finding novel therapeutics against infections caused by UPEC. Virtual screenings enable the rapid and economical identification of target ligands from large libraries of compounds, reducing the cost and time of traditional drug discovery. Moreover, the drugs that have been approved by the FDA have low cytotoxicity and good pharmacological characteristics. In this work, we targeted the HisC enzyme of the histidine biosynthetic pathway as enzymes of this pathway are absent in humans. We screened the library of FDA-approved drugs against HisC via molecular docking, and four hits (Docetaxel, Suramin, Digitoxin, and Nystatin) showing the highest binding energy were selected. These were further tested for antibacterial activity, which was observed only for Docetaxel (MIC value of 640 µg/ml); therefore, Docetaxel was further tested for its efficacy in vivo in murine catheter UTI model and antibiofilm activity using crystal violet staining and scanning electron microscopy. Docetaxel inhibited biofilm formation and reduced the bacterial load in urine, kidney, and bladder. Docking studies revealed that Docetaxel acts by blocking the binding site of HisC to the native substrate by competitive inhibition. Docetaxel may be a potential new inhibitor for UPEC with antibacterial and antibiofilm capability.

Relative refractoriness of breast cancer cells to tumour necrosis factor-α induced necroptosis.

Necroptosis, a recently identified programmed cell death pathway, has attracted attention as an alternative route to target apoptosis-resistant cancer cells. The status of the necroptosis pathway in different subtypes of breast cancer has not been well explored. Stimulating the cells by TNF-α can trigger cell survival or death depending on the combination of downstream players involved. In this work, we attempted to induce necroptosis in them using a combination of TNF-α and Z-VAD-FMK with and without chemotherapy. Cell viability, apoptosis, and necroptosis were assessed using MTT and Annexin-V/PI assays, respectively. Gene and protein expression was analysed by qPCR and immunophenotyping. Both the cell lines were resistant to induction of cell death by necroptosis. There was no enhancement in cell death when chemotherapeutic drugs were combined with necroptosis induction. Expression studies showed reduced translational expression of key necroptosis molecules like RIP kinases and MLKL in breast cancer cells compared to positive control cell line L929. Also, cell survival molecules were expressed more in MDA-MB-231 in contrast to death pathway molecules which were expressed more in T47D cells. In this work, the two breast cancer cell lines were observed to be resistant to TNF-α induced necroptosis with or without chemotherapy. Expression of key necroptosis players revealed relative insufficiency of the molecular machinery involved in the above pathway. In our opinion this may be the cause for resistance to necroptosis and novel strategies to upregulate these molecules need to be developed to sensitize the breast cancer cells towards cell death by necroptosis.

Clinical, biochemical, and serologic predictors of drug reaction with eosinophilia and systemic symptoms syndrome: A prospective case-control study.

BACKGROUND: Detailed scoring systems such as the European Registry of Severe Cutaneous Adverse Reactions (RegiSCAR) score for validating a diagnosis of drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome are available, but there is no rapid, easy tool to identify DRESS at presentation. OBJECTIVE: To identify the clinical, biochemical, and serologic markers predicting the DRESS syndrome and its severity. METHODS: In this prospective observational study, 25 patients with the DRESS syndrome and 25 control patients with maculopapular drug rash were recruited. Baseline clinical, biochemical, and serologic markers, such as high-sensitivity C-reactive protein (hsCRP), erythrocyte sedimentation rate, and thymus and activation-regulated chemokine (TARC) levels, were recorded and their utility in identifying the DRESS syndrome at presentation and predicting severity was analyzed. RESULTS: The effectiveness of TARC level (>613.25 pg/mL), total body surface area (TBSA, >35%), hsCRP (>5 mg/L), eosinophils (>6%), absolute eosinophil count (>450 cells/mm3), and aspartate transaminase (>92 U/L) were statistically similar to the effectiveness of the RegiSCAR DRESS validation score (≥2) in diagnosing the DRESS syndrome. A combination model (TBSA at baseline, eosinophil count, and hsCRP) at the cutoff of 6.8 had a sensitivity of 96% and a specificity of 100%. Baseline serum TARC levels did not predict the DRESS severity or outcome. LIMITATIONS: Small sample size. CONCLUSION: The combination of TBSA involvement, eosinophil count, and hsCRP levels can predict the DRESS syndrome at presentation.

Metformin ameliorates the status epilepticus- induced hippocampal pathology through possible mTOR modulation.

The initial precipitating injury such as SE progresses to chronic epilepsy through multiple epileptogenic processes. Early epileptogenic events are generally characterized by neuroinflammation, neurodegeneration and abnormal neurogenesis in the hippocampus. Metformin has exhibited anti-inflammatory and neuroprotective properties in numerous studies. The current study attempts to investigate the effect of metformin on seizure-induced inflammation and neuronal degeneration, and the involvement of the mTOR pathway. Status epilepticus (SE) was induced in male Wistar rats with systemic administration of Lithium (127 mg/kg) and Pilocarpine (30 mg/kg). In test rats, Metformin 100 mg/kg or 200 mg/kg was administered orally for 7 days, followed by SE induction. Results indicate that metformin did not alter the SE profile significantly which was evident by the behavioural scoring and electroencephalogram (EEG) recordings. However, metformin 200 mg/kg attenuated the SE-induced glial activation (p < 0.01), up regulated mRNA levels of proinflammatory cytokines (p < 0.001) and chemokines (p < 0.001) and enhanced BBB permeability (p < 0.05). In addition, metformin ameliorated the insult-induced region-specific neuronal damage (p < 0.01) and restored the hippocampal neuronal density. Metformin significantly inhibited phosphorylated S6 ribosomal protein (phospho-S6rp) (p < 0.05), thus demonstrating that the beneficial effects might be partly mediated by the mTOR pathway. The study thus reiterates that mTOR signalling is one of the mechanisms involved in inflammation and neurodegeneration in early epileptogenesis following SE.

TCF 4 tumor suppressor: a molecular target in the prognosis of sporadic colorectal cancer in humans.

BACKGROUND: A huge array of function is played by the Wnt/ß-catenin signaling pathway in development by balancing gene expression through the modulation of cell-specific DNA binding downstream effectors such as T-cell factor/lymphoid enhancer factor (TCF/LEF). The ß-catenin/TCF-4 complex is a central regulatory switch for differentiation and proliferation of intestinal cells (both normal and malignant). Thus, in the present study we evaluated each of 60 cases of sporadic adenocarcinoma, alongside adjoining and normal mucosa specimens of colorectum in humans, for mutation and expression analysis of the gene coding for TCF-4 protein. METHODS: DNA sequencing following PCR amplification and SSCP analysis (single strand conformation polymorphism) was employed to detect TCF-4 gene mutations in the case of exon 1. Quantitative real-time (qRT) PCR, immunohistochemistry (IHC), confocal microscopy and western blot analysis were used to detect TCF-4 gene/protein expression. RESULTS: Sequencing analysis confirmed 5/60 patients with a point mutation in exon 1 of the TCF-4 gene in tumor samples. mRNA expression using qRT-PCR showed approximately 83% decreased TCF-4 mRNA expression in tumor tissue and adjoining mucosa compared to normal mucosa. Similarly, a significant decrease in protein expression using IHC showed decreased TCF-4 protein expression in tumor tissue and adjoining mucosa compared to normal mucosa, which also corresponds to some important clinicopathological factors, including disease metastasis and tumor grade. Mutational alterations and downregulation of TCF-4 mRNA and hence decreased expression of TCF-4 protein in tumors suggest its involvement in the pathogenesis of CRC. CONCLUSIONS: A remarkable decrease in TCF-4 mRNA and protein expression was detected in tumorous and adjoining tissues compared to normal mucosa. Hence the alterations in genomic architecture along with downregulation of TCF-4 mRNA and decreased expression of TCF-4 protein in tumors, which is in accordance with clinical features, suggest its involvement in the pathogenesis of CRC. Thus, deregulation and collaboration of TCF-4 with CRC could be a concrete and distinctive feature in the prognosis of the disease at an early stage of development.

Chemopreventive role of olive oil in colon carcinogenesis by targeting noncoding RNAs and methylation machinery.

Epigenetic therapy induced by dietary components has become a strong interest in the field of cancer prevention. Olive oil, a potent dietary chemopreventive agent, control colon cancer, however, its role in epigenetic therapy remains unclear. Thus, we aimed to investigate the effect of olive oil in a preclinical model of colon cancer by targeting genetic and epigenetic mechanisms. DMH was used to induce colon cancer in rats; while olive oil was given to separate group of rats along with DMH treatment. Tumor burden and incidence in DMH and DMH + olive oil-treated rats was observed by macroscopic examination and histoarchitectural studies. Potent anti-inflammatory, anti-angiogenic and pro-apoptotic activity of olive oil was explored by gene expression and immunohistochemical studies. The effect of olive oil on epigenetic alterations was examined by detecting promoter methylation with MS-HRM and dysregulation of miRNA by TaqMan MicroRNA Assay. We observed that olive oil administration lowered tumor incidence and inhibited the development of tumors in DMH-treated rats. Olive oil markedly decreased the expression of inflammatory and angiogenic markers and restored the expression of pro-apoptotic markers in DMH-treated rats. Furthermore, the inverse relationship between gene expression and DNA methylation, deviant miRNA pattern and miRNA silencing mediated by aberrant DNA methylation was also seen in DMH-treated rats, which was potentially reversible upon olive oil treatment. Our study concludes that olive oil may play a role in the epigenetic therapy by altering NF-κB and apoptotic pathways via targeting noncoding RNAs and methylation machinery that affecting epigenome to prevent colon carcinogenesis.

Role of IL-17A receptor blocking in melanocyte survival: A strategic intervention against vitiligo.

Cytokines regulate immune response and inflammation and play an important role in depigmentation process of an autoimmune disease, vitiligo. We sought to determine how inflammatory cytokines influence the progression of vitiligo, and based on that, we develop a logical therapeutic intervention using primary melanocyte culture. Melanocytes were cultured and exposed to IL-17A, IL-1ß, IFN-γ and TGF-ß for 4 days. Melanocytes proliferation, tyrosinase assay and melanin content were measured. Real-Time PCR was used to analyse mRNA expression of genes specific for melanocytes growth and pigmentation. Anti-IL-17A receptor antibody was used to block IL-17A receptors expressed on melanocytes. Protein expression of MITF and TYR was assessed by immunofluorescence and Western blotting. A gradual decline in the melanocyte population, melanin content and tyrosinase activity was observed after different cytokine treatment. The expression of MITF and its downstream genes after blocking with anti-IL-17RA, an increased melanin content, increased expression of TYR, MITF along with its downstream genes, and cell proliferation was observed.

Low spontaneous apoptosis index at diagnosis predicts a high-risk phenotype in paediatric acute lymphoblastic leukaemia.

Background & objectives: Significance of apoptosis as a prognostic marker is less well studied in paediatric acute lymphoblastic leukaemia (ALL) cases. Hence, a prospective study, involving 30 paediatric ALL cases, was done to assess the clinical relevance of in vivo apoptosis. Methods: Peripheral blood mononuclear cells from all patients were subjected to annexin V/propidium iodide staining to detect the degree of apoptosis [apoptotic index (AI)] at day 0 and day 35 post-induction chemotherapy. In addition, Bax and Bcl2 apoptotic protein expressions were studied at day 0 and their relative fluorescence mean intensity (RFMI) ratios were calculated. Results: Mean age of patients was 5.1 years. Of the 30 cases, 21 (70%) were at standard-risk, five (17%) at intermediate and four (13%) at high risk. Majority (83%) were B-ALL. Day 8 absolute blast count was >1000/µl in seven (23%) and <1000/µl in 23 of 30 (77%) cases. Day 35 marrow was M1 in 23 (92%) and M2 in two of 25 (8%) cases. AI at day 0 and day 35 ranged from 0.9 to16.6 per cent and 1.4 to 62.8 per cent with a mean of 5.90 and 19.64 per cent, respectively. The Bax/Bcl2 ratio ranged from 0.2 to 3.5 with a mean of 0.83. The ratio was predominantly anti-apoptotic, i.e. <1 (77%). A significant association was noted between low AI at day 0 and high total leucocyte count (P=0.02), T-cell phenotype (P=0.043) and high-risk as per NCI category (P=0.025). Significant increase (>30%) in day 35 AI was seen in only six cases. Interpretation & conclusions: Our study showed that low AI at day 0 was associated with a high-risk clinical phenotype in paediatric ALL. However, studies on larger group, especially with longer follow up or study of relapse cases, will help draw conclusions regarding apoptosis assessment in paediatric ALL.

Increased advanced glycation end product specific fluorescence in repeatedly heated used cooking oil.

Repeated heating of cooking oils is known to cause their degradation and generation of toxins. Dietary Advanced glycation end products (dAGEs) are formed when the foods are cooked in dry heat at very high temperatures. dAGEs are believed to contribute significantly to total pool of AGEs in body. In this study, cooking oil samples used for frying snacks were collected from 102 shops. AGEs were extracted using Aqueous-TCA-chloroform method. Fluorescent AGE levels were determined using a fluorescence spectrophotometer and compared with AGEs in corresponding fresh oil samples collected from same shops. Palm oil was most commonly (62.5%) used for cooking. Most of the samples were subjected to several rounds of heating (1-6). AGE specific fluorescence (ASF) in used oil (range = 8.5-745.11) samples was found to be significantly higher in 88/102 as compared to the corresponding fresh oil samples. Treatment with inhibitors like lime concentrate and vitamin C decreased ASF (10/14 and 10/11 samples respectively) of the used oils. The results suggest that cooking oil subjected to repeated heating can contribute to increase in fluorescent AGEs in diet. Simple practices like liberal use of common household substances like lime concentrate may help to reduce these in fried food.

Frequent activation of the ß-catenin gene in sporadic colorectal carcinomas: A mutational & expression analysis.

ß-catenin (CTNNB1), an oncogene/onco-protein and an adhesion molecule is a key effector in colorectal cancer (CRC). Its activation, and subsequent up-regulation of Wnt-signaling, is an important event in the development of certain human cancers including CRC. Mutations in the ß-catenin gene in the region of serine-threonine glycogen kinase (GSK)-3ß phosphorylation target sites have been identified in colorectal cancer in humans. In the current study, we investigated 60 sporadic colorectal adenocarcinomas along with adjoining and normal mucosa cases in humans for ß-catenin mutations. Thirteen of sixty colorectal tumors from humans had point mutations with a frequency of 21.66% at codons 24, 26, 27, 32, 34, 35, 41, 42,43, 46, 49, 54, 55, or 67 sites which are mutated in colorectal cancer and some of these sites in other cancers. Thus, there appears to be a key involvement of ß-catenin activation in human colorectal carcinogenesis. mRNA expression analysis using q-Real Time PCR showed 21.5-fold up-regulation of ß-catenin mRNA in tumor tissue compared to normal and adjoining mucosa. Protein expression analysis using immunohistochemistry, confocal microscopy, and Western blot confirmed aberrant accumulation of ß-catenin protein along the nucleus and cytoplasm following mutation. The observed mutations and up-regulation of mRNA in tumors, and the increased expression of ß-catenin protein in CRC suggest that these alterations are early and prognostic events in sporadic colorectal carcinogenesis in humans. © 2015 Wiley Periodicals, Inc.

Antiapoptotic and neuroprotective role of Curcumin in Pentylenetetrazole (PTZ) induced kindling model in rat.

Kindling, a sub threshold chemical or electrical stimulation, increases seizure duration and enhances accompanied behavior until it reaches a sort of equilibrium state. The present study aimed to explore the effect of curcumin on the development of kindling in PTZ kindled rats and its role in apoptosis and neuronal damage. In a PTZ kindled Wistar rat model, different doses of curcumin (100, 200 and 300 mg/kg) were administrated orally one hour before the PTZ injections on alternate day during the whole kindling days. The following parameters were compared between control and experimental groups: the course of kindling, stages of seizures, Histopathological scoring of hippocampus, antioxidant parameters in the hippocampus, DNA fragmentation and caspase-3 expression in hippocampus, and neuron-specific enolase in the blood. One way ANOVA followed by Bonferroni post hoc analysis and Fischer's Exact test were used for statistical analyses. PTZ, 30 mg/kg, induced kindling in rats after 32.0 ± 1.4 days. Curcumin showed dose-dependent anti-seizure effect. Curcumin (300 mg/kg) significantly increased the latency to myoclonic jerks, clonic seizures as well as generalized tonic-clonic seizures, improved the seizure score and decreased the number of myoclonic jerks. PTZ kindling induced a significant neuronal injury, oxidative stress and apoptosis which were reversed by pretreatment with curcumin in a dose-dependent manner. Our study suggests that curcumin has a potential antiepileptogenic effect on kindling-induced epileptogenesis.

An image analysis approach to characterize micronuclei differences in different subtypes of breast cancer.

BACKGROUND: Micronuclei (MN) have been used as screening, diagnostic and prognostic markers in multiple cancer types, including breast cancer (BC). However, the question that the MN present in all subtypes of BC are similar or different remains unanswered. We thus hypothesized that MN present in different subtypes of BC may differ in their contents which may be visible as differences in their morphologic and morphometric features. This study was thus carried out with the aim to identify the differences between MN morphometry, complexity, and texture in different subtypes of BC, such as estrogen and progesterone receptor-positive (ER+/PR+; MCF-7, T-47D), human epidermal growth factor receptor-positive (Her2 +;SKBR3) and triple-negative BC (TNBC; MDA-MB-231, MDA-MB-468) cell lines (CLs) by ImageJ software. METHODS: For analysis of MN dimensions, MN irregularity, and texture, we used morphometry and two mathematical computer-assisted algorithms, i.e., fractal dimension (FD) and grey level co-occurrence matrix (GLCM) of ImageJ software. RESULTS: MN area and perimeter values showed differences in the size of MN in different subtypes of BC, with the largest MN in TNBC CLs. GLCM parameters (entropy, angular second moment, inverse difference moment, contrast, and correlation) showed highly heterogenous texture in case of TNBC MN as compared to the others. FD analysis also revealed more complexity and irregularity in MN found in TNBC cells. CONCLUSION: The study for the first time showed morphometric, architectural and texture related differences amongst MN present in different subtypes of BC. The above may reflect differences in their composition and contents. Further, these differences may point towards the distinct mechanisms involved in the formation of MN in different subtypes of BC that need to be explored further.

Aspirin as a potential drug repurposing candidate targeting estrogen receptor alpha in breast cancer: a molecular dynamics and in-vitro study.

Estrogen receptor alpha (ERα) is expressed by 70% of breast cancers (BCs). Any deregulation in ERα signaling is crucial for the initiation and progression of BC. Because of development of resistance to anti-estrogenic compounds, repurposing existing drugs is an apt strategy to avoid a long drug-discovery process. Substantial epidemiologic evidence suggests that Aspirin use reduces the risk of different cancers including BC, while its role as an adjuvant or a possible antineoplastic agent in cancer treatment is being investigated. In this study, we attempted to explore possibilities of ERα inhibition by Aspirin which may act through competitive binding to the ligand binding domain (LBD) of ERα. A list of 48 ERα-LBD crystal structures bound with agonists, antagonists, and selective ER modulators (SERMs) was thoroughly analysed to determine interaction patterns specific to each ligand category. Exhaustive docking and 500 ns molecular dynamics (MD) studies were performed on three ERα - Aspirin complexes generated using agonist, antagonist, and SERM-bound crystal structures. Besides, three ERα crystal structures bound to agonist, antagonist, and SERM respectively were also subjected to MD simulations. Aspirin showed good affinity to LBD of ERα. Comparative analyses of binding patterns, conformational changes and molecular interaction profiles from the docking results and MD trajectories suggests that Aspirin was most stable in complex generated using SERM bound crystal structure of ERα and showed interactions with Gly-521, Ala-350, Leu-525 and Thr-347 like SERMs. In addition, in-vitro assays, qPCR, and immunofluorescent assay demonstrated the decline in the expression of ERα in MCF-7 upon treatment with Aspirin. These preliminary bioinformatical and in-vitro findings may form the basis to consider Aspirin as a potential candidate for targeting ERα, especially in tamoxifen-resistant cancers.Communicated by Ramaswamy H. Sarma.

Unlocking the Therapeutic Potential: Sitagliptin's Multifaceted Approach in Drug-Resistant Epilepsy through a Novel Mechanism Inhibiting Protein Kinase C-γ and a Long-Term Potentiation Pathway.

Drug-resistant epilepsy is a prominent challenge in chronic neurological disorders. Valproate, commonly used to treat epilepsy, can fail due to various side effects and interactions, necessitating the exploration of alternative treatments. Our study primarily investigated sitagliptin's potential as a therapeutic agent for drug-resistant epilepsy. Employing computational modeling and enzyme assay testing, three lead compounds, emixustat, sitagliptin, and distigmine bromide, were evaluated against the target enzyme protein kinase C-γ. In vivo, experiments on a pentylenetetrazolium-induced lamotrigine-resistant epilepsy model were conducted to test sitagliptin's antiseizure effects, compared with the standard phenobarbital treatment. Emixustat and sitagliptin showcased strong inhibitory properties, while distigmine bromide was less effective in the enzyme assay. Mechanistic insights revealed sitagliptin's ability to modulate the seizure grade and first myoclonic jerk latency via oxidative stress markers, like reduced glutathione and glutathione peroxidase emphasizing its antioxidative role in epilepsy. Additionally, it demonstrated anti-inflammatory effects by significantly reducing proinflammatory markers interleukin-1ß and interleukin-6. The modulation of key genes of the long-term potentiation pathway, particularly protein kinase C-γ and metabotropic glutamate receptor 5, was evident through mRNA expression levels. Finally, sitagliptin showed potential neuroprotective properties, limiting pentylenetetrazolium-induced neuronal loss in the hippocampal region. Collectively, our findings suggest sitagliptin's multidimensional therapeutic potential for drug-resistant epilepsy specifically via a long-term potentiation pathway by inhibiting protein kinase C-γ.