Microarray Noninvasive Neuronal Seizure Recordings from Intact Larval Zebrafish

Zebrafish epilepsy models are emerging tools in experimental epilepsy. Zebrafish larvae, in particular, are advantageous because they can be easily genetically altered and used for developmental and drug studies since agents applied to the bath penetrate the organism easily. Methods for electrophysiological recordings in zebrafish are new and evolving. We present a novel multi-electrode array method to non-invasively record electrical activity from up to 61 locations of an intact larval zebrafish head. This method enables transcranial noninvasive recording of extracellular field potentials (which include multi-unit activity and EEG) to identify epileptic seizures. To record from the brains of zebrafish larvae, the dorsum of the head of an intact larva was secured onto a multi-electrode array. We recorded from individual electrodes for at least three hours and quantified neuronal firing frequency, spike patterns (continuous or bursting), and synchrony of neuronal firing. Following 15 mM potassium chloride- or pentylenetetrazole-infusion into the bath, spike and burst rate increased significantly. Additionally, synchrony of neuronal firing across channels, a hallmark of epileptic seizures, also increased. Notably, the fish survived the experiment. This non-invasive method complements present invasive zebrafish neurophysiological techniques: it affords the advantages of high spatial and temporal resolution, a capacity to measure multiregional activity and neuronal synchrony in seizures, and fish survival for future experiments, such as studies of epileptogenesis and development.     

The impact of AlphaFold Protein Structure Database on the fields of life sciences

Arguably, 2020 was the year of high-accuracy protein structure predictions, with AlphaFold 2.0 achieving previously unseen accuracy in the Critical Assessment of Protein Structure Prediction (CASP). In 2021, DeepMind and EMBL-EBI developed the AlphaFold Protein Structure Database to make an unprecedented number of reliable protein structure predictions easily accessible to the broad scientific community. We provide a brief overview and describe the latest developments in the AlphaFold database. We highlight how the fields of data services, bioinformatics, structural biology, and drug discovery are directly affected by the influx of protein structure data. We also show examples of cutting-edge research that took advantage of the AlphaFold database. It is apparent that connections between various fields through protein structures are now possible, but the amount of data poses new challenges. Finally, we give an outlook regarding the future direction of the database, both in terms of data sets and new functionalities.     

GSTP1 I105V polymorphism and susceptibility to colorectal cancer in Kashmiri population

The glutathione S-transferase (GST) enzyme encoded by the GSTP1 gene is one of the critical enzymes involved in detoxification of carcinogens. The substitution of isoleucine to valine residue at position 105 of the GSTP1 protein results in decreased enzyme activity and hence less capability of effective detoxification. Hence, we investigated the role of GSTP1 I105V polymorphism in modulating the risk of colorectal cancer (CRC) associated in a Kashmiri population. We designed a case-control study in which 86 CRC cases were studied for GSTP1 I105V polymorphism against 160 controls taken from the general population employing the polymerase chain reaction-restriction length fragment polymorphism technique. There was no significant association between GSTP1 I105V genotypes and the disease, but the Val/Val genotype was associated with an increased risk with some clinicopathological parameters (odds ratio=1.5; 95% confidence interval=0.55-4.57). This study suggests that the GSTP1 I105V polymorphism may modulate CRC risk in the Kashmiri population.     

Curcumin and its synthetic analogue dimethoxycurcumin differentially modulates antioxidant status of normal human peripheral blood mononuclear cells

Curcumin is a polyphenol derived from the herb Curcuma longa, which has been extensively studied in terms of its antitumour, antioxidant, and chemopreventive activity as well as various other effects. In the present work we compared curcumin with its synthetic analogue dimethoxycurcumin (dimc) in terms of its antioxidant enzyme-modulating effects in human peripheral blood mononuclear cells (PBMC). We found that these compounds modulate antioxidant enzymes differentially. Both curcumin and dimethoxycurcumin effected a decrease in lipid peroxidation status in PBMC, however, curcumin had better activity in this regard. An increase in the activity of catalase was seen in the case of curcumin-treated PBMC, whereas dimc increased catalase activity significantly to almost twofold level. Real time-polymerase chain reaction (RT-PCR) analysis revealed significant up-regulation of catalase at mRNA level post treatment with curcumin as well as dimc, however, dimc had better activity in this regard. Glutathione reductase (GR) activity and reduced glutathione levels increased in the case of peripheral blood mononuclear cells (PBMC) treated with curcumin, however, the trend was reversed with dimethoxycurcumin where, both glutathione reductase activity and reduced glutathione levels were significantly reduced. RT-PCR analysis of glutathione reductase mRNA levels showed decrease in mRNA levels post treatment with dimethoxycurcumin (dimc) further corroborating GR enzyme assay results, however, we could not obtain significant result post curcumin treatment. NFkB reporter assay and western blot analysis of nuclear as well as cytosolic fractions of NFkB revealed that curcumin inhibits NFkB activation whereas inhibition was much less with dimc. It has been reported that curcumin and dimc exerts differential cytotoxicity in normal and tumour cells and the reason for this had been attributed to the differential uptake of these compounds by normal cells and tumour cells. Based on our results we propose that differential modulation of antioxidant enzymes via NFkB pathway could be the reason behind differential cytotoxicity of dimc as well as curcumin in normal cells and tumour cells in addition to differential uptake of these compounds as reported previously.     

Allelic loss at PTEN locus leads to progression of colorectal carcinoma among North Indian patients

We evaluated the loss of heterozygosity (LOH) at 10q^23.3 locus of microsatellite markers; D10S198, D10S192, and D10S541 of PTEN gene in 223 North Indian colorectal cancer (CRC) specimens. DNA was isolated and microsatellite-specific markers polymerase chain reaction was performed. Out of total 223 cases 102 showed LOH for at least one of the locus. In addition, thereto a significant association was found with the clinicopathologic features like grade of differentiation, clinical stage, invasion, lymph node invasion, and the clinical outcome (p?<?0.05). These data argue that the given markers to check the possible LOH of PTEN gene at locus 10q^23.3 could be considered as one of the diagnostic markers in CRC.     

The sea urchin histone gene complement

The only eukaryotic mRNAs that are not polyadenylated are the replication-dependent histone mRNAs in metazoans. The sea urchin genome contains two sets of histone genes that encode non-polyadenylated mRNAs. One of these sets is a tandemly repeated gene cluster with a 5.6-kb repeat unit containing one copy of each of the five alpha-histone genes and is present as a single large cluster which spans over 1 Mb. There is a second set of genes, consisting of 39 genes, containing two histone H1 genes, 34 genes encoding core histone proteins (H2a, H2b, H3 and H4) and three genes expressed only in the testis. Unlike vertebrates where these genes are clustered, the sea urchin late histone genes, expressed in embryos, larvae and adults, are dispersed throughout the genome. There are also genes encoding polyadenylated histone mRNAs, which encode histone variants, including all variants found in other metazoans, as well as a unique set of five cleavage stage histone proteins expressed in oocytes. The cleavage stage histone H1 is the orthologue of an oocyte-specific histone H1 protein found in vertebrates.