Understanding the complexity of epimorphic regeneration in zebrafish caudal fin tissue: A transcriptomic and proteomic approach.

The complex epimorphic regeneration of zebrafish caudal fin tissue is hasty and absolute. This study was executed to understand the role of various genes/proteins involved in the regeneration of zebrafish caudal fin tissue through differential transcriptomics and proteomics analysis. Based on our study 1408 genes and 661 proteins were found differentially regulated in the regenerating caudal fin tissue for having at least 1-log fold change. Interleukin, Solute carrier, Protein arginine methyltransferase, Homeobox, Neurotransmitter and several novel genes were found to be associated with regeneration for its differential regulation during the mechanism. Based on the network and pathway analysis the differentially regulated genes and proteins were found allied with activation of cell proliferation, cell viability, cell survival & cell movement and inactivation of organismal death, morbidity, necrosis, death of embryo & cell death. This study has mapped a detailed insight of the genes/proteins expression associated with the epimorphic regeneration more profoundly.

Proteome profile of telencephalon associates attenuated neurogenesis with chronic stress induced mood disorder phenotypes in zebrafish model.

Debilitating mental illness like depression and related mood disorders is due to the disruption in circuitry that controls emotion, motivation, and reward, characterized by disparate phenotypes like decrease in socialization, motivation, threshold for threat apprehension, etc. Chronic stress is a major factor in the etiology of these disorders. Here, using a chronic unpredictable stress (CUS) paradigm the characterization of an array of mood disorder phenotypes in adult zebrafish, in comparison to normal control unstressed fish, was achieved using a battery of behavioral assays including novel ones comprising social interaction test, feed approach test, threat response test and novel tank test. For the predictive validity of the model for mood disorders, the mitigative role of a slow (imipramine) and fast (ketamine) acting antidepressant was assessed. The molecular changes associated with CUS-induced mood disorder phenotype was investigated utilizing a high throughput method called isobaric tag for relative and absolute quantification (iTRAQ) in telencephalon, the region critically associated with the processing of emotional information in the fish brain. Out of 222 proteins identified to be significantly altered, 58 were differentially expressed across the stress and antidepressant-treatment groups at more than one fold (in log2) change. Of these proteins, some were implicated in earlier studies on mood disorders such as CABP1, PER2, mTOR, etc. The enrichment of altered proteins by Ingenuity Pathway Analysis (IPA) led us to mTOR and opioid signaling pathways, the top canonical pathways affected in the fish telencephalon. Interestingly, most of the pathways affected converge at the one controlling cell proliferation thus indicating altered neurogenesis, which was validated using immunohistochemistry for cell proliferation markers BrdU, SOX2, and BLBP. The study concludes that molecules that regulate telencephalon neural progenitor cell proliferation or neurogenesis are crucially involved in chronic stress-induced mood disorders by affecting the circuitry that controls emotion and reward.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced Parkinson's disease in zebrafish.

Parkinson's disease (PD) is the most common age associated neurodegenerative disease, which has been extensively studied for its etiology and phenotype. PD has been widely studied in alternate model system such as rodents towards understanding the role of neurotoxin by inducing PD. This study is aimed to understand the biomechanism of PD in zebrafish model system induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The phenotype and role of various genes and proteins for Parkinsonism were tested and evaluated in this study using behavior, molecular and proteomic approaches. Zebrafish PD model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed a significant level of decrease in the movement with erratic swimming pattern and increased freezing bouts. CHCHD2, EEF2B, LRRK2, PARK7, PARK2, POLG, SNCGB and SYNB genes were differentially regulated at the transcript level in PD zebrafish. Similarly a total of 73 proteins were recognized as differentially expressed in the nervous system of zebrafish due to Parkinsonism based on quantitative proteomics approach. Proteins such as NEFL, MUNC13-1, NAV2 and GAPVD1 were down regulated in the zebrafish brain for the PD phenotype, which were associated with the neurological pathways. This zebrafish based PD model can be used as a potential model system for screening prospective drug molecules for PD.

Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media.

Cryostorage is of immense interest in biomedical research, especially for stem cell-based therapies and fertility preservation. Several protocols have been developed for efficient cryopreservation of cells and tissues, and a combination of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) is commonly used. However, there is a need for an alternative to FBS because of ethical reasons, high cost, and risk of contamination with blood-borne diseases. The objective of the present study was to examine the possibility of using buffalo (Bubalus bubalis) ocular fluid (BuOF) to replace FBS in cryomedia. Frozen-thawed cells, which were cryopreserved in a cryomedia with BuOF, were assessed for viability, early and late apoptosis, and proliferation. Three cell lines (CHO, HEK, and C18-4), mouse embryonic stem (mES) cells, and primary cells, such as mouse embryonic fibroblast (MEF) cells, human peripheral blood mononuclear cells (hPBMCs), and mouse bone marrow cells (mBMCs), were cryopreserved in cryomedia containing 10% DMSO (D10) with 20% FBS (D10S20) or D10 with 20% BuOF (D10O20). For all three cell lines and mES cells cryopreserved in either D10S20 or D10O20, thawed cells showed no difference in cell viability or cell recovery. Western blot analysis of frozen-thawed-cultured cells revealed that the expression of Annexin V and proliferating cell nuclear antigen (PCNA) proteins, and the ratio of BAX/BCL2 proteins were similar in all three cell lines, mES cells, and hPBMCs cryopreserved in D10S20 and D10O20. However, initial cell viability, cell recovery after culture, and PCNA expression were significantly lower in MEF cells, and the BAX/BCL2 protein ratio was elevated in mBMCs cryopreserved in D10O20. Biochemical and proteomic analysis of BuOF showed the presence of several components that may have roles in imparting the cryoprotective property of BuOF. These results encourage further research to develop an efficient serum-free cryomedia for several cell types using BuOF.