Flight for fish in drug discovery: a review of zebrafish-based screening of molecules.

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.

Lack of CD2AP disrupts Glut4 trafficking and attenuates glucose uptake in podocytes.

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.

Sept7b is essential for pronephric function and development of left-right asymmetry in zebrafish embryogenesis.

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.

Towards reaching the target: clinical application of mesenchymal stem cells for diabetic foot ulcers.

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.

Targeting nonhealing ulcers of lower extremity in human through autologous bone marrow-derived mesenchymal stem cells.

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.