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A simple scalable method has been developed to obtain protein hydrolysate from fleshing waste generated during leather processing. UV-Vis, FTIR and Solid State C13 NMR analyses identified that prepared protein hydrolysate is basically collagen hydrolysate. DLS and MALDI-TOF-MS spectra indicated that the prepared protein hydrolysate is mostly comprised of di- and tri-peptides and less poly-dispersed than the standard commercial product. A combination of 0.3% Yeast extract, 1% Protein Hydrolysate (PHz) and 2% Glucose is found to be the most efficient nutrient composition for the fermentative growth of three well-known chitosan producing zygomycetes group of fungi. Mucor sp. showed highest yield of biomass (2.74 g/L) as well as chitosan (335 mg/L). Biomass and chitosan yield for Rhizopus oryzae were found 1.53 g/L; 239 mg/L. Same for Absidia coerulea were 2.05 g/L and 212 mg/L, respectively. This work shows promising prospect of utilization of fleshing waste of leather processing for the low-cost production of industrially important biopolymer chitosan.
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Quitosano , Quitosano/química , Quitosano/metabolismo , Hidrolisados de Proteína/metabolismo , Polímeros/metabolismo , Hongos/metabolismo , FermentaciónRESUMEN
Aim of the study: Activating Transforming factor 3 (ATF3) is a stress induced gene and closely associated with neuro-inflammation while Transforming growth Factor Beta (TGFß) signalling is also reported to be involved in neuro-inflammation and hyper-excitability associated with drug resistant epilepsy. Animal model studies indicate the involvement of ATF3 and TGFß receptors to promote epileptogenesis. Human studies also show that TGFß signalling is activated in MTLE-HS. However, lack of studies on ATF3 and TGFßRI expression in MTLE-HS patients exists. We hypothesize that ATF3 and TGFßRI might be expressed in hippocampi of patients with MTLE-HS and playing role in epileptogenesis.Materials & methods: Protein expression of ATF3 and TGFßRI was performed by western blotting. Localisation of ATF3 was performed by immunohistochemistry and immunoflorescence.Results: Protein expression of ATF3 and TGFßRI was significantly up-regulated in hippocampi of patients as compared to controls. Also ATF3 IR was significantly expressed in hippocampi of patients and ATF3 was expressed predominantly in cytoplasm as compared to nucleus. No correlation was found between ATF3 expression and epilepsy duration and seizure frequency.Conclusions: ATF3 and TGFßRI are both important players in neuro-inflammation and might potentiate epileptogenesis in these patients.
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BACKGROUND AND AIMS: DNA methylation and demethylation play a crucial role in the regulation of gene expression, though their interplay during pathogenesis of hippocampal scelerosis (HS) remains elusive. The present study was designed to investigate the DNA methylation regulated changes in expression of HS patients. METHODS: We performed integrative analysis of genome-wide CpG-DNA methylation profiling and RNA sequencing to profile global changes in promoter methylation and gene expression in HS patients. Real time PCR was performed to validate the findings of methylation and RNA sequencing. RESULTS: A total of 16040 sites showed altered DNA methylation in all the CpG islands. Of these, 3185 sites were in the promoter regions, of which 66 genes showed an inverse correlation between methylation and expression. These genes are largely related to pathways predicted to participate in axon guidance by semaphorins, MAPK, ionotropic glutamate receptor pathway, notch signaling, regulatory activities related to TFAP2A and immune response, with the most distinct ones included TFAP2A, NRP1, SEMA3B, CACNG2, MAP3K11, and ADAM17. CONCLUSION: We performed integrated analysis of genomic methylation signature and differential gene expression patterns of hippocampal tissues resected from patients with HS for the first time. Collectively, our findings implicate DNA methylation as a critical regulator of the pathogenic mechanisms of epileptogenesis associated with HS.
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Epilepsia Refractaria/genética , Epilepsia del Lóbulo Temporal/genética , Hipocampo/metabolismo , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Metilación de ADN , Epilepsia Refractaria/patología , Epilepsia Refractaria/cirugía , Epilepsia del Lóbulo Temporal/patología , Epilepsia del Lóbulo Temporal/cirugía , Femenino , Expresión Génica , Hipocampo/patología , Humanos , Masculino , RNA-Seq , Reacción en Cadena en Tiempo Real de la Polimerasa , Esclerosis , Transducción de Señal , Adulto JovenRESUMEN
Focal cortical dysplasia (FCD) is one of the most common pathologies associated with drug-resistant epilepsy (DRE). The pharmacological targets remain obscured, as the molecular mechanisms underlying FCD are unclear. Implications of epigenetically modulated aberrant gene expression in disease progression are reported in various DRE pathologies except FCD. Here we performed genome-wide CpG-DNA methylation profiling by methylated DNA immunoprecipitation (MeDIP) microarray and RNA sequencing (RNAseq) on cortical tissues resected from FCD type II patients. A total of 19088 sites showed altered DNA methylation in all the CpG islands. Of these, 5725 sites were present in the promoter regions, of which 176 genes showed an inverse correlation between methylation and gene expression. Many of these 176 genes were found to belong to a cohesive network of physically interacting proteins linked to several cellular functions. Pathway analysis revealed significant enrichment of receptor tyrosine kinases (RTK), EGFR, PDGFRA, NTRK3, and mTOR signalling pathways. This is the first study that investigates the epigenetic signature associated with FCD type II pathology. The candidate genes and pathways identified in this study may play a crucial role in the regulation of the pathogenic mechanisms of epileptogenesis associated with FCD type II pathologies.
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Metilación de ADN , Epigénesis Genética , Epilepsia/genética , Epilepsia/metabolismo , Estudio de Asociación del Genoma Completo , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/metabolismo , Transducción de Señal , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Humanos , Regiones Promotoras GenéticasRESUMEN
Transforming growth factor beta (TGFß) signalling cascade has been implicated in enhancing neuronal excitability and excitatory synaptogenesis following blood brain barrier (BBB) damage and inflammation. We aimed to study if TGFß signalling expression is altered in patients with Hippocampal Sclerosis (HS). We probed into the protein expression level of the ligand transforming growth factor beta 1 (TGFß1), transforming growth factor beta receptor II (TGFßRII) and downstream signalling molecule SMAD3 and phosphorylated SMAD3 (pSMAD3) on surgically resected hippocampal samples of thirty-four patients with HS through immuno-blotting. The increase in protein expression level of the ligand TGFß1 was 285 ± 1.15% higher and its receptor TGFßRII was 170 ± 0.98% higher in hippocampus of patients with HS in comparison to the autopsy hippocampal control samples. The expression of the downstream signalling molecules, SMAD3 is 157 ± 0.13% and 106 ± 0.17% higher in patients with HS as compared to both types of non-seizure controls. The expression of active form of SMAD3, pSMAD3 (2.6010 ± 1.2735) was significantly upregulated in hippocampus of patients with HS compared to autopsy hippocampal controls (0.7899 ± 0.3688). While the expression of pSMAD3 (1.527 ± 0.9425) was significantly upregulated in hippocampus of patients with HS with another type of non-seizure control viz. tumour periphery tissue (0.5791 ± 0.2679), hence strongly supporting the altered expression of the pathway. This study provides the first evidence of alteration of TGFß pathway in patients with HS which could be a potential therapeutic target.
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Epilepsia Refractaria/metabolismo , Hipocampo/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Adolescente , Adulto , Anciano , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/cirugía , Niño , Preescolar , Epilepsia Refractaria/cirugía , Femenino , Expresión Génica , Hipocampo/cirugía , Humanos , Inflamación/metabolismo , Inflamación/cirugía , Masculino , Persona de Mediana Edad , Fosforilación , Esclerosis/metabolismo , Esclerosis/cirugía , Transducción de Señal , Adulto JovenRESUMEN
Experimental and clinical evidence have demonstrated aberrant expression of cytokines/chemokines and their receptors in patients with hippocampal sclerosis (HS) and focal cortical dysplasia (FCD). However, there is limited information regarding the modulation of cytokine/chemokine-regulatory networks, suggesting contribution of miRNAs and downstream transcription factors/receptors in these pathologies. Hence, we studied the levels of multiple inflammatory mediators (IL1ß, IL1Ra, IL6, IL10, CCL3, CCL4, TNFα and VEGF) along with transcriptional changes of nine related miRNAs and mRNA levels of downstream effectors of significantly altered cytokines/chemokines in brain tissues obtained from patients with HS (n = 26) and FCD (n = 26). Up regulation of IL1ß, IL6, CCL3, CCL4, STAT-3, C-JUN and CCR5, and down regulation of IL 10 were observed in both HS and FCD cases (p < 0.05). CCR5 was significantly up regulated in FCD as compared to HS (p < 0.001). Both, HS and FCD presented decreased miR-223-3p, miR-21-5p, miR-204-5p and let-7a-5p and increased miR-155-5p expression (p < 0.05). As compared to HS, miR-204-5p (upstream to CCR5 and IL1ß) and miR-195-5p (upstream to CCL4) were significantly decreased in FCD patients (p < 0.01). Our results suggest differential alteration of cytokine/chemokine regulatory networks in HS and FCD and provide a rationale for developing pathology specific therapy.