Mitochondria play a critical role in maintaining the metabolic balance of the cell. The onset and progression of diabetes have been linked to mitochondrial dysfunction, leading to oxidative stress and dysregulation of metabolic intermediates, ultimately leading to a loss of energy production. Mitochondria play a crucial role in glucose stimulated-insulin secretion in pancreatic β-cells and oxidative phosphorylation in beta cells and skeletal muscles. In type-2 diabetes, impaired oxidative phosphorylation and insulin release is linked to insulin resistance (IR). Given the possible involvement of mitochondrial activity in the pathophysiology of diabetes, it would be highly desirable to investigate possible biomarkers or indicators that may provide details on the onset, severity or progression of the disease. The use of biomarkers is essential both for the diagnosis of mitochondrial diseases and for monitoring their metabolic status. The discovery and characterization of numerous biomarkers that correlate with mitochondrial diseases has led to the development of a number of new biomarkers. Biomarkers associated with human mitochondrial dysfunction are critical for the development of targeted therapies and early diagnosis of diabetes. Based on an investigation of the literature, this perspective outlines the state of knowledge on mitochondrial biomarkers and examines the data supporting their application in the early diagnosis, prognosis, and monitoring of diabetes.

Human disease and biological practices are modelled in zebrafish ( Danio rerio ) at various phases of drug development as well as toxicity evaluation. The zebrafish is ideal for in vivo pathological research and high-resolution investigation of disease progress. Zebrafish has an advantage over other mammalian models, it is cost-effective, it has external development and embryo transparency, easy to apply genetic manipulations, and open to both forward and reverse genetic techniques. Drug screening in zebrafish is suitable for target identification, illness modelling, high-throughput screening of compounds for inhibition or prevention of disease phenotypes and developing new drugs. Several drugs that have recently entered the clinic or clinical trials have their origins in zebrafish. The sophisticated screening methods used in zebrafish models are expected to play a significant role in advancing drug development programmes. This review highlights the current developments in drug discovery processes, including understanding the action of drugs in the context of disease and screening novel candidates in neurological diseases, cardiovascular diseases, glomerulopathies and cancer. Additionally, it summarizes the current techniques and approaches for the selection of small molecules and current technical limitations on the execution of zebrafish drug screening tests.

Exposure to large concentrations of organic PAH (polycyclic aromatic hydrocarbon) compound naphthalene can potentially accumulate inside the body, damage the cells and causes serious health issues. The current understanding of naphthalene exposure to many food fish species is limited. For a comprehensive understanding of naphthalene toxicity to fish health, dose dependent exposure was carried out in Anabas testudineus, an important food fish of Asia. Fish were exposed to sublethal concentrations of naphthalene (4.2, 4.4, 4.6, 4.8 and 5 mg L-1) for a period of 72 h. Activity analysis of lipid peroxidation and antioxidant enzymes in liver and muscles of the Anabas testudineus showed a marked increase in the lipid peroxidation activity in almost all the experimental groups. In contrast, the enzymatic activity of antioxidants like catalase, glutathione peroxidase and non-enzymatic antioxidant glutathione decreased in both liver and muscle tissue. The results imply that increasing doses of naphthalene induced lipid peroxidation and the decreasing levels of antioxidants were incapable of reducing the stress exerted by naphthalene. This study is beneficial for evaluating the effect of LMW (Low molecular weight) naphthalene on fish and highlights the specific biomarker response signifying PAHs toxicity in fish.

Macrobrachium gangeticum (Bate) are commercially valuable prawns that are broadly distributed in the Ganga river that flows from northern to eastern India. We studied embryonic development from cleavage (blastomeres), segmentation, formation of optic vesicle, eye pigment development to larva formation, as these data are largely unknown in this species. We conducted this study using mature M. gangeticum broodstock placed in freshwater tanks. Just after spawning, females were kept in separate aerated tanks, where fecundity was estimated from the total number of eggs. Thereafter, egg samples from berried females were used to study their embryonic stages. The correlation coefficient between size and prawn weight, egg weight, egg number, body weight and egg mass of different sizes of prawns was analysed. Our results show that an increase in prawn size led to an increase in egg weight and egg number while decreases in body weight:egg mass ratio was noticed. Further results indicate that an increase in prawn weight had a positive impact on egg weight and egg number. The embryo development stages were used in this study as a tool to evaluate the development process of the prawn in a hatchery environment.

Septins are a conserved family of GTP-binding proteins that assemble into cytoskeletal filaments to function in a highly sophisticated and physiologically regulated manner. Originally septins were discovered in the budding yeast as membrane-associated filaments that affect cell polarity and cytokinesis. In the last decades, much progress has been made in understanding the biochemical properties and cell biological functions of septins. In line with this, mammalian septins have been shown to be involved in various cellular processes, including regulation of cell polarity, cytoskeletal organization, vesicle trafficking, ciliogenesis, and cell-pathogen interactions. A growing number of studies have shown that septins play important roles in tissue and organ development and physiology; yet, little is known about their role in the kidney. In the following review, we discuss the structure and functions of septins in general and summarize the evidence for their presence and roles in the kidney.

Metformin, the first-line drug to treat type 2 diabetes (T2D), inhibits mitochondrial glycerolphosphate dehydrogenase in the liver to suppress gluconeogenesis. However, the direct target and the underlying mechanisms by which metformin increases glucose uptake in peripheral tissues remain uncharacterized. Lipid phosphatase Src homology 2 domain-containing inositol-5-phosphatase 2 (SHIP2) is upregulated in diabetic rodent models and suppresses insulin signaling by reducing Akt activation, leading to insulin resistance and diminished glucose uptake. Here, we demonstrate that metformin directly binds to and reduces the catalytic activity of the recombinant SHIP2 phosphatase domain in vitro. Metformin inhibits SHIP2 in cultured cells and in skeletal muscle and kidney of db/db mice. In SHIP2-overexpressing myotubes, metformin ameliorates reduced glucose uptake by slowing down glucose transporter 4 endocytosis. SHIP2 overexpression reduces Akt activity and enhances podocyte apoptosis, and both are restored to normal levels by metformin. SHIP2 activity is elevated in glomeruli of patients with T2D receiving nonmetformin medication, but not in patients receiving metformin, compared with people without diabetes. Furthermore, podocyte loss in kidneys of metformin-treated T2D patients is reduced compared with patients receiving nonmetformin medication. Our data unravel a novel molecular mechanism by which metformin enhances glucose uptake and acts renoprotectively by reducing SHIP2 activity.-Polianskyte-Prause, Z., Tolvanen, T. A., Lindfors, S., Dumont, V., Van, M., Wang, H., Dash, S. N., Berg, M., Naams, J.-B., Hautala, L. C., Nisen, H., Mirtti, T., Groop, P.-H., Wähälä, K., Tienari, J., Lehtonen, S. Metformin increases glucose uptake and acts renoprotectively by reducing SHIP2 activity.

ABSTRACT Calcium channels are necessary for cardiac excitation–contraction (E–C) coupling, but Ca2+ channel composition of fish hearts is still largely unknown. To this end, we determined transcript expression of Ca2+ channels in the heart of zebrafish (Danio rerio), a popular model species. Altogether, 18 Ca2+ channel α-subunit genes were expressed in both atrium and ventricle. Transcripts for 7 L-type (Cav1.1a, Cav1.1b, Cav1.2, Cav1.3a, Cav1.3b, Cav1.4a, Cav1.4b), 5 T-type (Cav3.1, Cav3.2a, Cav3.2b, Cav3.3a, Cav3.3b) and 6 P/Q-, N- and R-type (Cav2.1a, Cav2.1b, Cav2.2a, Cav2.2b, Cav2.3a, Cav2.3b) Ca2+ channels were expressed. In the ventricle, T-type channels formed 54.9%, L-type channels 41.1% and P/Q-, N- and R-type channels 4.0% of the Ca2+ channel transcripts. In the atrium, the relative expression of T-type and L-type Ca2+ channel transcripts was 64.1% and 33.8%, respectively (others accounted for 2.1%). Thus, at the transcript level, T-type Ca2+ channels are prevalent in zebrafish atrium and ventricle. At the functional level, peak densities of ventricular T-type (ICaT) and L-type (ICaL) Ca2+ current were 6.3±0.8 and 7.7±0.8 pA pF−1, respectively. ICaT mediated a sizeable sarcolemmal Ca2+ influx into ventricular myocytes: the increment in total cellular Ca2+ content via ICaT was 41.2±7.3 µmol l−1, which was 31.7% of the combined Ca2+ influx (129 µmol l−1) via ICaT and ICaL (88.5±20.5 µmol l−1). The diversity of expressed Ca2+ channel genes in zebrafish heart is high, but dominated by the members of the T-type subfamily. The large ventricular ICaT is likely to play a significant role in E–C coupling. Summary: Zebrafish heart expresses a diversity of Ca2+ channel genes dominated by the T-type (Cav3.1) subfamily; the associated current (ICaT) is likely to play a significant role in excitation–contraction coupling.

Myofibrils made up of actin, myosin, and associated proteins generate the contractile force in muscle, and, consequently, mutations in these proteins may lead to heart failure. Septins are a conserved family of small GTPases that associate with actin filaments, microtubules, and cellular membranes. Despite the importance of septins in cytoskeleton organization, their role in cardiomyocyte organization and function is poorly characterized. Here, we show that septin 7 is expressed in both embryonic and adult zebrafish hearts and elucidate the physiological significance of sept7b, the zebrafish ortholog of human septin 7, in the heart in embryonic and larval zebrafish. Knockdown of sept7b reduced F-actin and α-cardiac actin expression in the heart and caused disorganization of actin filaments. Electron microscopy of sept7b-depleted larvae showed disorganization of heart myofibrils and partial detachment from Z-disks. Functional studies revealed that knockdown of sept7b leads to reduced ventricular dimensions, contractility, and cardiac output. Furthermore, we found that depletion of sept7b diminished the expression of retinaldehyde dehydrogenase 2, which catalyzes the synthesis of retinoic acid necessary for heart morphogenesis. We further observed that the sept7b and retinoic acid signaling pathways converge to regulate cardiac function. Together, these results specify an essential role for sept7b in the contractile function of the heart.NEW & NOTEWORTHY Knockdown of the zebrafish ortholog of human septin 7 (sept7b) destabilizes cardiac actin and reduces ventricular dimensions, contractility, and cardiac output in larval zebrafish, indicating that sept7b is essential for cardiac function. We further found that sept7b and retinoic acid signaling pathways converge to regulate cardiac function. These data prompt further studies defining the role of sept7b in cardiomyopathies.

Novel physiological challenges in different environments can promote the evolution of divergent phenotypes, either through plastic or genetic changes. Environmental salinity serves as a key barrier to the distribution of nearly all aquatic organisms, and species diversification is likely to be enabled by adaptation to alternative osmotic environments. The threespine stickleback (Gasterosteus aculeatus) is a euryhaline species with populations found both in marine and freshwater environments. It has evolved both highly plastic and locally adapted phenotypes due to salinity-derived selection, but the physiological and genetic basis of adaptation to salinity is not fully understood. We integrated comparative cellular morphology of the kidney, a key organ for osmoregulation, and candidate gene expression to explore the underpinnings of evolved variation in osmotic plasticity within two populations of sticklebacks from distinct salinity zones in the Baltic Sea: the high salinity Kattegat, representative of the ancestral marine habitat; and the low salinity Bay of Bothnia. A common-garden experiment revealed that kidney morphology in the ancestral high-salinity population had a highly plastic response to salinity conditions whereas this plastic response was reduced in the low-salinity population. Candidate gene expression in kidney tissue revealed a similar pattern of population-specific differences, with a higher degree of plasticity in the native high-salinity population. Together these results suggest that renal cellular morphology has become canalized to low salinity, and that these structural differences may have functional implications for osmoregulation.

Rosuvastatin based novel indole derivatives designed as potential anti-cancer agents were synthesized via a newly developed ligand-free, simple, straightforward and inexpensive one-pot method. The methodology involved a Cu-catalyzed coupling-cyclization of a rosuvastatin based alkyne with o-iodoanilides in the presence of CuI and K2CO3 in PEG-400. Three of the synthesized compounds showed promising anti-proliferative activities against cancer cell lines and an increase of p21 mRNA expression and apoptotic effects in zebrafish embryos/larvae.

Department of Chemistry, Osmania Univer shivakumarkota@yahoo.co.in; Tel: +91 40 2 Department of Pathology, University of Hel Central Laboratory for Stem Cell Research a Colleges of Medical Sciences, Kanchanbagh, Dr Reddy's Institute of Life Sciences, H Gachibowli, Hyderabad-500 046, India. E +91 40 6657 1500 † Electronic supplementary information (E spectral data for all new compounds 10.1039/c6ra20148b Cite this: RSC Adv., 2016, 6, 100487

Protection or restoration of pancreatic β-cell mass as a therapeutic treatment for type 1 diabetes requires understanding of the mechanisms that drive the specification and development of pancreatic endocrine cells. Septins are filamentous small GTPases that function in the regulation of cell division, cytoskeletal organization and membrane remodeling, and are involved in various tissue-specific developmental processes. However, their role in pancreatic endocrine cell differentiation remains unknown. Here we show by functional manipulation techniques in transgenic zebrafish lines that suppression of sept7b, the zebrafish ortholog of human SEPT7, profoundly increases the number of endocrine progenitors but limits their differentiation, leading to reduction in β- and α-cell mass. Furthermore, we discovered that shh (sonic hedgehog) expression in the endoderm, essential for the development of pancreatic progenitors of the dorsal pancreatic bud, is absent in larvae depleted of sept7b. We also discovered that sept7b is important for the differentiation of ventral pancreatic bud-derived cells: sept7b-depleted larvae exhibit downregulation of Notch receptors notch1a and notch1b and show precocious differentiation of NeuroD-positive endocrine cells in the intrapancreatic duct and gut epithelium. Collectively, this study provides a novel insight into the development of pancreatic endocrine progenitors, revealing an essential role for sept7b in endocrine progenitor differentiation.

The adapter protein CD2-associated protein (CD2AP) functions in various signaling and vesicle trafficking pathways, including endosomal sorting and/or trafficking and degradation pathways. Here, we investigated the role of CD2AP in insulin-dependent glucose transporter 4 (Glut4, also known as SLC2A4) trafficking and glucose uptake. Glucose uptake was attenuated in CD2AP(-/-) podocytes compared with wild-type podocytes in the basal state, and CD2AP(-/-) podocytes failed to increase glucose uptake in response to insulin. Live-cell imaging revealed dynamic trafficking of HA-Glut4-GFP in wild-type podocytes, whereas in CD2AP(-/-) podocytes, HA-Glut4-GFP clustered perinuclearly. In subcellular membrane fractionations, CD2AP co-fractionated with Glut4, IRAP (also known as LNPEP) and sortilin, constituents of Glut4 storage vesicles (GSVs). We further found that CD2AP forms a complex with GGA2, a clathrin adaptor, which sorts Glut4 to GSVs, suggesting a role for CD2AP in this process. We also found that CD2AP forms a complex with clathrin and connects clathrin to actin in the perinuclear region. Furthermore, clathrin recycling back to trans-Golgi membranes from the vesicular fraction containing GSVs was defective in the absence of CD2AP. This leads to reduced insulin-stimulated trafficking of GSVs and attenuated glucose uptake into CD2AP(-/-) podocytes.

The conserved septin family of filamentous small GTPases plays important roles in mitosis, cell migration and cell morphogenesis by forming scaffolds and diffusion barriers. Recent studies in cultured cells in vitro indicate that a septin complex of septin 2, 7 and 9 is required for ciliogenesis and cilia function, but septin function in ciliogenesis in vertebrate organs in vivo is not understood. We show that sept7b is expressed in ciliated cells in different tissues during early zebrafish development. Knockdown of sept7b by using morpholino antisense oligonucleotides caused misorientation of basal bodies and cilia, reduction of apical actin and the shortening of motile cilia in Kupffer's vesicle and pronephric tubules. This resulted in pericardial and yolk sac edema, body axis curvature and hydrocephaly. Notably, in sept7b morphants we detected strong left-right asymmetry defects in the heart and lateral plate mesoderm (situs inversus), reduced fluid flow in the kidney, the formation of kidney cysts and loss of glomerular filtration barrier function. Thus, sept7b is essential during zebrafish development for pronephric function and ciliogenesis, and loss of expression of sept7b results in defects that resemble human ciliopathies.

Mesenchymal stem cells (MSCs) hold great promise for therapeutic application in non-healing ulcers and tissue regeneration because of their multi-lineage differentiation potential. MSCs delivered may migrate to the sites of injury and improve wound healing by stimulating angiogenesis and promoting revascularization. The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide. It is associated with peripheral neuropathy and peripheral arterial occlusive disease (PAOD), which predispose patients to develop non-healing foot ulcers following minor trauma. A high rate of amputation exists among diabetic patients due to non-healing foot ulcers, which are a significant burden for the society despite new therapeutic protocols developed. In recent years, stem cell transplantation has been considered as a new therapeutic option for diabetic foot ulcers (DFUs). The regeneration potential of MSCs has been demonstrated in the experimental and clinical trials. Here we review the potential efficacy and systematic use of MSCs for the treatment of non-healing DFUs, current advances, MSC delivery systems, and possible options to enhance the therapeutic potential of stem cell for wound healing.

This study aimed at developing a suitable cryopreservation protocol for embryonic stem (ES)-like cells of a tiny freshwater fish Leopard danio (Brachydanio frankei). Embryonic stem (ES)-like cells derived from blastomeres of the early blastulae stage of the developing embryo were cultured in vitro in a medium containing Leibowitz-15 supplemented with 10% foetal bovine serum, leopard danio embryo extract, sodium bicarbonate, sodium selenite, basic fibroblast growth factor, epidermal growth factor and leukaemia inhibitory factor. The ES-like cells showed properties similar to ES cells in other species. They were morphologically small, round to polygonal and present in patches and extensively expressed alkaline phosphatase and stage-specific embryonic antigen. The toxicity and chilling sensitivity of these cells were determined using ethylene glycol (EG), propylene glycol (PG) and glycerol as cryoprotective agents at molar concentrations of 0.6, 1.0, 1.4, 1.8 and 2.0. Among them, 1.8 M EG showed 70% significant viable ES-like cells (P < 0.05). The post-thawed cells retained similar properties of non-cryopreserved ES-like cells with a viability rate of 65%. Similarly, blastomeres cryopreserved following the slow cooling rate with EG and PG yielded a viability of more than 70%.

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) represent a promising population for supporting new concepts in cellular therapy. This study was undertaken to assess the efficacy and feasibility of autologous BM-derived MSCs in the treatment of chronic nonhealing ulcers (diabetic foot ulcers and Buerger disease) of the lower extremities. A total of 24 patients with nonhealing ulcers of the lower limb were enrolled and randomized into implant and control groups. In the implant group, the patients received autologous cultured BM-derived MSCs along with standard wound dressing; the control group received only the standard wound dressing regimen, followed up for at least a 12-week period. Wound size, pain-free walking distance, and biochemical parameters were measured before therapy and at every 2-week interval following intervention. The implant group had significant improvement in pain-free walking distance and reduction in ulcer size as compared to those in the control group. In the implant group for Buerger disease, the ulcer area decreased from 5.04 +/- 0.70 cm(2) to 1.48 +/- 0.56 cm(2) (p < 0.001), whereas the pain-free walking distance increased from 38.33 +/- 17.68 m to 284.44 +/- 212.12 m (p < 0.001). In the diabetic foot ulcer group, the ulcer size decreased from 7.26 +/- 1.41 cm(2) to 2 +/- 0.98 cm(2) (p < 0.001) at 12 weeks. Mononuclear cells were cultured for a minimum of five passages and characterized by cell-surface markers showing CD90+, CD105+, and CD34(-). There was no significant alteration in the biochemical parameters observed during the follow-up period, indicating normal liver and renal function following intervention. Biopsy microsection of implanted tissues showed development of dermal cells (mainly fibroblasts), including mature and immature inflammatory cells. The study indicates that autologous implantation of BM-derived MSCs in nonhealing ulcers accelerates the healing process and improves clinical parameters significantly.

During development of animals the primordial genii cells (PGCs) become gonads in adults. Primordial germ cell localization and characterization is a prerequisite for germplasm conservation and other biotechnological applications in economically valued species. As PGC of any Indian carps has not studied earlier. a preliminary study was done to locate primordial germ cells morphologically and their migration pattern in the histological sections of different developmental stages of Labeo rohita. Genii cells were first detected at late morula stage (characterized by deep staining) and later on characterized by larger faintly stained cells having a centrally placed nucleus. The migration starts from the posterior sides of morula stage to the ventero-lateral positions above the yolk syncytial layer (YSL) Lip to the tail bud stage and finally reaches the gonadal ridge (i.e., above the gut and in the sides of vertebral column).