Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of symmetrical dimethylation of arginine residues in proteins. WD repeat domain 77 (WDR77), also known as p44, MEP50, or WD45, forms a stoichiometric complex with PRMT5. The PRMT5/p44 complex is required for cellular proliferation of lung and prostate epithelial cells during earlier stages of development and is re-activated during prostate and lung tumorigenesis. The molecular mechanisms by which PRMT5 and p44 promote cellular proliferation are unknown.Expression of PRMT5 and p44 in lung and prostate cancer cells was silenced and their target genes were identified. The regulation of target genes was validated in various cancer cells during lung development and tumorigenesis. Altered expression of target genes was achieved by ectopic cDNA expression and shRNA-mediated silencing.PRMT5 and p44 regulate expression of a specific set of genes encoding growth and anti-growth factors, including receptor tyrosine kinases and antiproliferative proteins. Genes whose expression was suppressed by PRMT5 and p44 encoded anti-growth factors and inhibited cell growth when ectopically expressed. In contrast, genes whose expression was enhanced by PRMT5 and p44 encoded growth factors and increased cell growth when expressed. Altered expression of target genes is associated with re-activation of PRMT5 and p44 during lung tumorigenesis.Our data provide the molecular basis by which PRMT5 and p44 regulate cell growth and lay a foundation for further investigation of their role in lung tumor initiation.The online version of this article (doi:10.1186/s12885-016-2632-3) contains supplementary material, which is available to authorized users. Protein arginine methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues within target proteins and has been implicated in diverse cellular and biological processes including transcriptional regulation –3, RNA mp44 gene led to growth arrest and differentiation of lung epithelial cells. More important, PRMT5 and p44 are re-expressed in lung cancers and the shRNA-mediated silencing of PRMT5 or p44 expression strongly inhibited proliferation of lung cancer cells in tissue culture and abolished growth of lung tumor xenografts in nude mice [PRMT5 directly methylates epidermal growth factor receptor (EGFR), E2F1, and p53 to promote cell survival and growth –15. Cyclude mice , 28. TheIn searching for molecules that mediate PRMT5/p44 functions in cell growth, we performed DNA microarray analysis with lung adenocarcinoma A549 cells expressing PRMT5 or p44 shRNA and identified a set of genes targeted by both PRMT5 and p44. Altered expression of these genes was observed during mouse lung development and lung tumorigenesis and affected growth of lung cancer cells. Our results demonstrate PRMT5 and p44 regulation of gene expression of growth and anti-growth factors to promote cell growth.A549 and PC14 cells were cultured in minimum essential medium (CellGro) with 10 % (v/v) fetal bovine serum (FBS) (HyClone), 2 % vitamins, 1 % L-glutamine, 1 % non-essential amino acids, and 1 % sodium pyruvate. PC3 and LNCaP cells were cultured in RPMI 1640 medium (CellGro) with 10 % FBS. For cell growth assays, cells were plated on 24-well plates  and counted 6 days later. For bromodeoxyuridine (BrdU) (BD Biosciences) incorporation assays, cells (50–70 % confluence) were plated on a chamber slide  and cultured in the presence of 10 μM BrdU for 4 h. The BrdU-positive cells were detected by immunostaining with the monoclonal anti-BrdU antibody (BD Biosciences) as described previously .Lung tumor samples were obtained from existing pathological specimens at Tangdu Hospital , and the study protocol was approved by its institutional review board . BALB/c www.r-project.org) and comparison was used to select genes differentially expressed at a significance level of p < 0.001. Microarray data presented in this study has been deposited in the GEO public repository (GSE56757).The non-target (NT), p44, and PRMT5 small hairpin RNA (shRNA) and lentivirus production were described previously , 28. Bri4) were plated in 6-well plates and transduced with lentivirus. After 48 h, cells were replated in a 100-mm dish, and 2 days later protein expression was confirmed by Western blot analysis.Human cDNA clones encoding GLI Pathogenesis-Related 1 (GLIPR1), leucine proline-enriched proteoglycan (Leprel1), B-cell translocation gene 2 (BTG2), and V-Erb-B avian erythroblastic leukemia viral oncogene homolog 3 (ErbB3) were purchased from DNASU and subcloned into the lentiviral expression vector . Lentivirus was prepared as described previously . Cells . The cDNA products were PCR-amplified with the RT2 Real-Time SYBR Green qPCR master mix (SuperArray Biosciences) and the gene-specific primer sets . The membranes were washed in Tris-buffered saline with Tween 20  and blocked with 3 % nonfat milk in Tris-buffered saline with Tween 20 for 1 h. The blots were then probed for 2 h with the primary antibody at dilutions of 1:1,000 (anti-p44), 1:1,000 (anti-FGFR3), or 1:5,000  or overnight with the primary antibody at dilutions of 1:1,000 (anti-ErbB3) or 1:1,000 (anti-GLIPR1). The blots were then incubated with a horseradish peroxidase-conjugated secondary antibody for 1.5 h. Immunoreactive proteins were detected using an enhanced chemiluminescence detection system  per the manufacturer’s instructions. Protein concentrations were determined using the Bradford protein assay (Bio-Rad). The protein bands were scanned using a densitometer and the relative intensities were quantified using the ImageJ software program .oC overnight. Cells were collected and stained with propidium iodide (PI). The cell-cycle distributions were determined by flow cytometry analysis as described [Cells (50–60 % confluency) were harvested, washed with PBS, and fixed in 70 % ethanol at 4 escribed .The TUNEL assay was performed with cells cultured in chamber slides using the apoptosis detection kit (Promega) according to the manufacturer’s instructions. Slides were evaluated under a microscope (Olympus IX71) with a digital camera (Retiga 1300) interfaced to a computer with PCI software. The apoptotic cells were quantified as follows. The number of cells of the area (DAPI staining) were captured and counted. There were approximately 100 cells per area and three areas were captured in each slide. The fluorescence-labeled cells (apoptotic cells) from the same area were captured and counted. Three independent experiments were performed and analyzed.t-test was used to determine whether differences between control and experiment samples were statistically significant. P values less than 0.05 were considered statistically significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001.Data are presented as the means of three independent experiments ± the standard deviation. A 2-tailed unpaired student p = 0.01755). These results are consistent with previous findings that p44 and PRMT5 proteins form a stoichiometric complex and co-exist in the cytoplasm [Infection of lung cancer A549 cells with lentivirus expressing PRMT5 or p44 small hairpin RNA (shRNA) resulted in 94 % or 83 % reduction in PRMT5 or p44 mRNA levels, respectively Fig. . The shRytoplasm .Fig. 1SiSilencing PRMT5 or p44 expression almost completely abolished cell growth Fig. . We thenA gene expression profiling analysis was performed on A549 cells expressing p44 shRNA, PRMT5 shRNA or NT shRNA (GSE56757). Gene set enrichment analysis (GSEA) indicates that genes up-regulated by PRMT5 shRNA were over-represented on the gene list, whose expression is negatively associated with cell proliferation   Fig. . We choo01) Fig. . The gen01) Fig. . Among t01) Fig.  and PRMT01) Fig.  in A549 These analyses were extended to the other lung cancer PC14) and two prostate cancer (PC3 and LNCaP) cell lines. PC3 is an androgen-independent cancer cell line and LNCaP is an androgen-dependent cancer cell line, which are broadly used in prostate cancer research . Similar4 and twoIn our previous study , we repoBased on these analyses, we performed Western blot analysis Fig.  for protGLIPR1 gene is associated with prostate cancer and Leprel1 functions as a suppressor of cell proliferation and their down-regulation or silencing was observed in cancers [BTG2 gene encodes an anti-proliferation protein [GLIPR1, Leprel1, and BTG2 genes  was significantly decreased in PRMT5 or p44 silencing cells Figs. . Growth PRMT5 and p44 are re-expressed in lung cancer and are required for lung tumor growth , 28. TheSimilarly, ErbB2 and ErbB3 were not expressed in the benign lung epithelial cells  encode proteins functioning as growth suppressors [DNA microarray assays identified a set of genes whose expression altered when PRMT5 or p44 expression was silenced. RT-PCR and Western blot analyses confirmed these observations. Regulation of identified genes by PRMT5 and p44 was further demonstrated in multiple lung and prostate cancer cell lines and in mouse lungs as well as during lung tumorigenesis. Among this set of genes, three genes , indicating that PRMT5 and p44 positive regulate their expression. This regulation was observed in cancer cell lines and mouse lungs as well as in lung hyperplasia and lung cancer. We previously demonstrated that ectopic expression of FGFR3 partially restored the cell growth defect induced by silencing PRMT5 expression . SimilarIt is noticed that either ErbB3 or FGFR3, even ErbB3 together with FGFR3, failed to fully restore the cell growth inhibition induced by p44 silencing. ErbB2 and ErbB3 are two members of the EGF receptor family , 48. ErbPRMT5 and p44 are highly expressed in prostate and lung cancers and are essential for growth of cancer cells , 33, 50.This study is the first to identify specific transcriptional programs associated with PRMT5 and p44. Our data provides critical downstream cellular factors/pathways and potential molecular mechanisms of PRMT5/p44-driving cellular proliferation.BrbB, V-Erb-B avian erythroblastic leukemia viral oncogene homolog; BrdU, bromodeoxyuridine; BTG2, B-cell translocation gene 2; CEACAM1, carcinoembryonic antigen-related cell adhesion molecule 1; DAB, 3, 3′-diaminobenzidine; EMP1, epithelial membrane protein 1; ERK, extracellular signal-regulated kinase; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FGFR, fibroblast growth factor receptor; GLIPR1, GLI Pathogenesis-Related 1; GSEA, gene set enrichment analysis; Leprel1, leucine proline-enriched proteoglycan; NT shRNA, non-target shRNA; PBS, phosphate-buffered saline; PI, propidium iodide; PI3K, phosphatidylinositol-4, 5-bisphosphate 3-kinase; PRMT5, protein arginine methyltransferase 5; RT-PCR, reverse transcription polymerase chain reaction; SEM, the standard error of the mean; shRNA, small hairpin RNA; TGFβ, transforming growth factor beta; WDR77, WD repeat-containing protein 77


Addition of DNA to the liposomes formed lipoplexes, which were characterized for binding, size, biocompatibility, and transgene efficiency. Lipoplex formulations of suitable size and biocompatibility were assayed for DNA delivery, both qualitatively and quantitatively, using RPE cells and a GFP-encoding plasmid. The retinoic lipoplex formulation with DOPE revealed a transfection efficiency comparable to the known lipid references 3β-[N--carbamoyl]-cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) and GeneJuice. The results demonstrate that cationic polyene phospholipids have potential as DNA carriers for ocular gene therapy.Recent success in the treatment of congenital blindness demonstrates the potential of ocular gene therapy as a therapeutic approach. The eye is a good target due to its small size, minimal diffusion of therapeutic agent to the systemic circulation, and low immune and inflammatory responses. Currently, most approaches are based on viral vectors, but efforts continue towards the synthesis and evaluation of new nonviral carriers to improve nucleic acid delivery. Our objective is to evaluate the efficiency of novel cationic retinoic and carotenoic glycol phospholipids, designated C20-18, C20-20, and C30-20, to deliver DNA to human retinal pigmented epithelium (RPE) cells. Liposomes were produced by solvent evaporation of ethanolic mixtures of the polyene compounds and coformulated with 1,2-dioleoyl- Gene therapy is a promising treatment of several pathologies  and has Successful gene therapy requires nucleic acid carriers of minimal toxicity allowing long and stable gene expression, such as adenovirus and lentivirus . NeverthNonviral carriers are typically cationic polymers or liposomes. Cationic liposomes can easily be produced in large scale , can be Cationic lipids interact with negatively charged nucleic acids through a combination of electrostatic and hydrophobic interactions, resulting in lipoplexes of multilamellar structures . The strThe transfection efficiency and biocompatibility of cationic lipid gene carriers can be altered by modifications of the constituent parts of the lipids, specifically, the lipid backbone (often glycerol), the hydrophilic head group, and the interconnecting linker. Less diversity is reported for the hydrophobic part, which is known to play a significant role in the morphology of the lipoplex . The catDietary carotenoids are known to enhance visual performance and to reduce the risk of AMD progression . The glyβ-[N--carbamoyl]-cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine  (The polyene lipids were formulated with either cholesterol (Chol) or 1,2-dioleoyl-e (DOPE) , and theThe ARPE-19 cell line, a human retinal pigment epithelial cell line, was kindly provided by Dr. Franscisco Ambrósio ; Dulbecco's modified eagle medium (DMEM) culture medium and Dulbecco's modified eagle medium F12 ham (DMEM F12 Ham) culture medium, fetal bovine serum (FBS), trypsin, glutamine, penicillin-streptomycin solution, dichloromethane, thiazolyl blue tetrazolium bromide (MTT), 0.04 N HCl in 2-propanol constituents, dd-water, tris-acetate-EDTA (TAE) constituents, Avertin anesthesia constituents (tribromoethanol), Triton X-100, Mowiol mounting media constituents, sucrose (saccharose), paraformaldehyde, eosin, acetic acid, dibutyl phthalate xylene (DPX), sodium phosphate, potassium phosphate, and goat serum stock were purchased from Sigma-Aldrich . Absolute ethanol was purchased from Merck Millipore .4), and hematoxylin were purchased from Merck . Control cationic lipids 3β-[N--carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC) [sn-glycero-3-phosphoethanolamine (DOPE) were obtained from Avanti Polar Lipids .Plasmid DNA containing the reporter gene green fluorescent protein (GFP) was kindly provided by Dr. Jean Bennett . Agarose was purchased from Invitrogen . GreenSafe and GeneJuice were purchased from NZYTech . For the phosphate buffered saline (PBS), sodium chloride, potassium chloride, 4′,6-diamidino-2-phenylindole (DAPI), and optimal cutting temperature (OCT) cryostat embedding solution were purchased from VWR . Primary antibody Iba1, a microglia marker, was purchased from Wako Pure Chemical Industries (Japan) and secondary antibody Alexa 594 was purchased from Invitrogen . Oxalic acid, potassium permanganate (KMnOne (EPC)  and the Three cationic polyene lipids, C20-18, C20-20, and C30-20, were synthesized from glycol, retinoic acid (C20:5) or C30-acid (C30:9), and choline precursors as reported elsewhere , 15.Ethanolic stock solutions were made separately by first dissolving each cationic lipid and colipid in dichloromethane. The dichloromethane was removed under reduced pressure with a rotary evaporator, and then the residue dissolved in absolute ethanol to a final lipid concentration of 1 mM and subsequently stored at −80°C.Cholesterol and DOPE were employed as neutral colipids . LiposomThe addition of predetermined volumes of negatively charged plasmid DNA to positively charged liposomes resulted in lipoplex formulations of defined nitrogen-to-phosphorus (N/P) molar charge ratios. DNA contained the reporter gene GFP. Lipid concentrations, derived from a 2 mM hydrated stock, were 0.243 mM, 0.162 mM, and 0.081 mM, which correlates with the molar charge ratios of 1.5 : 1, 1 : 1, and 0.5 : 1, respectively. Equal volumes of DNA solution in DMEM or DMEM/F12 Ham culture medium and lipids were mixed and incubated at room temperature for 30 min to allow lipoplex formation.μL. Samples were loaded onto a 1% agarose gel impregnated with GreenSafe and electrophoresed at 70 V in tris-acetate-EDTA (TAE) buffer. The DNA bands were observed using an ultraviolet transilluminator.A gel retardation assay was used to evaluate lipoplex formation, as a neutral or net positive charge associated with the lipid-DNA complex retards migration through agarose gel. Lipoplexes with varying lipid : DNA ratios were prepared as described above in a total volume of 20 Lipoplexes were prepared in phosphate buffered solution (PBS), for subsequent determination of size and zeta potential by dynamic light scattering (DLS) and laser Doppler anemometry, respectively, using a Zetasizer Nanoseries ZS  by diluting the sample 50x with dd-water.dH > 2000 nm may be outside of the instrument's reliable range.The size and size distribution of lipoplexes are indicated as displayed by the instrument. Measurement parameters were as follows: a laser wavelength of 633 nm and a scattering angle of 173° (fixed). The samples were loaded into polystyrene cuvettes and three measurements were performed, for which the mean result was recorded. Particles with PdI > 0.7 and 2. ARPE-19 cells were maintained in DMEM F12 HAM culture medium supplemented with 10% FBS, 1% glutamine, and 1% penicillin/streptomycin at 37°C with 5% CO2. For the cytotoxicity assays, cells were seeded at 35,000 cells per well (D407) or 65,000 cells per well (ARPE-19) in 24-well plates in complete medium and incubated for 24 h at 37°C and 5% CO2. Cell seeding numbers differ with cell line due to differences in cell doubling rate. After 24 h, cells were washed with PBS and 250 μL lipoplex formulations with various N/P molar charge ratios added to each well. After a 4-hour incubation period, 366 μL of culture medium was added. Cells were incubated for 24, 48, and 72 h. Cell viability was determined by the standard MTT -2,5-diphenyltetrazolium bromide) assay. Briefly, after the incubation period, 50 μL of a 5 mg/mL MTT in PBS solution was added per well, followed by a 4 h incubation period, after which the culture medium was removed and 500 μL of a 0.04 N HCl in 2-propanol solution was added per well. After dissolution of the formazan crystals, their absorbance was measured at 570 and 630 nm for each well. Cell viability was obtained by measurement of optical densities and normalization against the positive control, consisting of cells grown in standard culture medium.D407 cells were maintained in DMEM culture medium supplemented with 5% FBS, 1% glutamine, and 1% penicillin-streptomycin solution at 37°C with 5% CO2 for 24 h. Cell seeding numbers differ with cell line due to differences in cell doubling rate. Immediately prior to transfection, cell monolayers were washed with PBS. The lipoplex formulations at various N/P molar charge ratios were added to each well in culture medium up to 2 mL and incubated for 4 h at 37°C, 5% CO2. After this period, the medium containing the lipoplexes was removed, the cells were washed with PBS, and complete growth medium was added and further incubated for 72 h at 37°C, 5% CO2. Subsequently, the cells were suspended using trypsin, washed with PBS, and resuspended in 500 μL of PBS for flow cytometry analysis . Transfection efficiency was assessed by detection of GFP with results normalized for 10,000 events.Transfection was determined using cells seeded at 100,000 cells per well (D407) or 250,000 cells per well (ARPE-19) in 6-well plates in complete growth medium and incubated at 37°C with 5% CO in vivo compatibility of the C20-20/DOPE/DNA (N/P molar charge ratio 0.5 : 1) was evaluated by intravitreal injection of the lipoplexes dispersed in PBS into C57Bl6 mice. C57BL6 mice (6 months old) were housed at controlled temperature with 12 h light/dark cycle and food and water ad libitum. All experimental procedures involving animals were carried out in accordance with the Portuguese and European Union regulations (FELASA) and the Association for Research in Vision and Ophthalmology (ARVO). All experimental injections were performed under anesthesia administered by intraperitoneal injection (250 mg/kg dose). Intravitreal injection was performed under a stereomicroscope (Nikon Stereoscopic Microscope) with a 30-gauge needle. Only one eye was injected; the contralateral eye was left noninjected as control. Fourteen days postinjection, the mice were sacrificed by cervical dislocation and the eyes were enucleated and preserved in PFA 4% at 4°C until further use. The eyes were treated with 30% sucrose solution and embedded in OCT  embedding solution and stored at −80°C. Cryosections of 10 μm thickness were used for detection of Iba1  by immunohistochemistry. Cryosections were permeabilized with PBS/0.1% Triton X-100 and washed twice with PBS. A 1 : 1000 dilution of the Iba1 antibody was used, and the samples incubated overnight at 4°C in the dark. Samples were washed three times with PBS/0.1% Triton X-100 and a 1 : 2000 dilution of the secondary antibody Alexa 594 was used for detection and incubated for 1 h, at room temperature, in the dark. Samples were washed three times with PBS/0.1% Triton x-100, mounted, and visualized by fluorescence microscopy (Zeiss Axio Imager Z2 Fluorescence Microscope). Samples were counterstained with DAPI , a marker for nuclear detection. Cryosections were also used for hematoxylin and eosin staining to analyze the retina for its morphology and major signs of inflammation. Sections were washed with distilled water, stained with hematoxylin for 1 min, washed with distilled water, stained with 1% eosin for 5 min, washed with 0.2% acetic acid, and mounted with DPX . Samples were visualized under bright field (Zeiss Axio Imager Z2 Fluorescence Microscope).TheP < 0.05 considered statistically significant.All figures are representative of at least 3 separate experiments. For comparison of multiple sets of data one-way ANOVA, including the Dunnet post-test, was performed. Results are expressed as mean +/− SEM. Statistical analysis was performed using GraphPad Prism 6 Software with dH (hydrodynamic diameter) = 320–2010 nm, while for those composed of C20-18, C20-20, and C30-20 was dH = 320–2210 nm. The average size of the lipoplexes did not correlate with increasing N/P ratio, with the exception of EPC/DOPE and C20-18/Chol (denoted by ∗), and also did not correlate with formulation of the same lipid with different colipids, Chol, and DOPE, with the exception of EPC and C30-20 lipoplexes (denoted by †). DLS is a technique that depends on the scattering of light with the particles and polydispersity may “hide” real populations due to the presence of large particles. dH values are representative of the sample and will be similar to the mode. However if the sample is polydisperse, dH values represent less accurately the sample and its value is apart from the mode [The size and surface charge of lipid-DNA complexes composed of the polyene lipids were determined using Dynamic Light Scattering (DLS) . The lipthe mode .The surface charge values  and time dependent (duration of the incubation period) as previously described . Lipople in vivo assays for all formulations due to lower cytotoxicity.Based on the cytotoxicity results, the 0.5 : 1 N/P molar charge ratio was subsequently used in transfection assays and in vitro was similar for in vivo, C20-20/DOPE lipoplexes prepared in PBS at a N/P molar charge ratio of 0.5 : 1 were administered by intravitreal injection to the eyes of C57BL6 mice. After 14 days, mice were sacrificed and the eyes were enucleated and used for Iba1 detection by immunohistochemistry or stained with hematoxylin and eosin for assessment of retinal integrity and inflammation. Iba1 is a calcium-binding protein characteristic of microglia that is expressed when the microglia is reactive, that is, in situations where inflammation occurs [ in vivo in the retina of C57Bl6 mice.To evaluate whether the level of toxicity observedn occurs . Our res in vivo study.Administration of drugs by intravitreal injection can result in morphological disruption of the retinal structure, caused by the injection procedure and tissue inflammation. Hematoxylin and eosin staining  showed n in vitro transfection efficiency of the C20-18, C20-20, and C30-20 lipoplexes in retinal cells using a plasmid coding for GFP and assessing its expression qualitatively by fluorescence microscopy and quantitatively by flow cytometry. ARPE-19 cells were transfected with C20-18, C20-20, and C30-20 lipoplexes with either Chol or DOPE as colipid at a N/P molar ratio of 0.5 : 1; transfection was evaluated by GFP expression after 72 h . For polyene and reference lipids alike, all DOPE formulated lipoplexes, with the exception of C30-20, revealed greater transfection efficiency than those formulations with Chol as colipid . As was found with the ARPE-19 cell line, the C20-20/DOPE formulation outperformed the corresponding Chol formulation in the D407 cell line . In addition, C20-20/DOPE revealed a level of transfection competitive to the control GeneJuice, whereas the C30-20 lipid revealed only a low level of transfection with Chol as colipid.Cationic polyene lipids were evaluated for their potential to carry DNA into retinal cells. Transfection efficiency was correlated with molecular structure, lipoplex size, zeta potential, biocompatibility, and the presence of colipids. DOPE and cholesterol increase DNA delivery and thus were employed as colipids. DOPE, often referred to as a “fusogenic lipid,” could alter the packing of the hydrophobic chains within the lipoplex  by promo in vitro cell internalization, although a correlation between size, surface charge, and transfection efficiency is still debatable [The analysis of the physical properties of the lipoplexes, summarized in ebatable .Almofti et al. demonstrated for the cationic lipid DC-6-14 that lipoplex size increased lipoplex transfection . With thZeta potential reflects the surface charge and lipoplex stability: ZP > ±30 mV indicates stable particles, and ZP < ±10 mV suggests unstable particles with a tendency to aggregation [Our lipoplexes presented negative zeta potential values between −30 ± 2 and −66 ± 1 mV. Although a positively charged lipoplex interacts more favorably with the negative charged biological membranes, it is not a necessary condition for transfection  and, as Negative ZP values most likely derive from DNA bound to the outer shell of the lipoplexes and from the phosphate groups associated with the solvent (PBS) that remains close to the surface of the lipoplexes. In vitro testing showed that the lipoplexes have increased in vitro toxicity with increasing N/P molar ratio and incubation period, in line with reference lipids DC-Chol, EPC, and other cationic lipids [ in vivo [ in vivo compatibility of C20-20 with the retinal tissue, as shown by the absence of morphological alterations in the retina and by nonreactive microglia.Successful gene carriers do not only transfect efficiently, but they also need to be biocompatible with the organism and target tissue.c lipids , 23. Tox in vivo . In the  in vivo . In its  in vivo . Our res in vitro gene delivery was evaluated with a plasmid coding for the GFP reporter gene, through detection of GFP-positive cells. C20-20 and the C20-18 formulations showed greater transfection efficiency relative to C30-20 when either DOPE or Chol was used as colipid. All lipid-DOPE formulations showed enhanced transfection efficiency when compared with lipid-Chol formulations. DOPE probably facilitates lipoplex phase transitions from lamellar to inverted hexagonal packing, which allows an effective fusion of the lipoplex and the cellular endocytic membrane [The efficiency ofmembrane , 27. It membrane . HoweverAlthough transfection in ARPE-19 cells was higher for the DC-Chol/DOPE and EPC/DOPE lipoplexes, transfection efficiencies of the C20-18/DOPE and C20-20/DOPE lipoplexes are noteworthy when compared to other investigations reporting considerably lower efficiencies .In an attempt to further compare the relative transfection efficiency of C20-20 and C30-20, we tested the same formulations in D407 cells , another cell line from the retina, but with a faster mitotic rate than ARPE-19. We observed with these cells a greater transfection efficiency than with ARPE-19 cells, which is most likely due to the higher mitotic rate, since during mitosis the nuclear membrane is disaggregated and therefore nuclear penetration of nucleic acids is facilitated.Remarkably, C20-20 displayed greater transgene efficiency than C30-20 in both cell lines tested. The retinoic lipids C20-18 and C20-20 may therefor enter the nucleus via a possible retinoid receptor . Further in vitro to RPE cells and were biocompatible during in vivo applications. These results encourage us to optimize and characterize this process.C20-18/Chol and C20-20/Chol performed comparably to references DC-Chol and EPC. C20-20/DOPE lipoplexes delivered DNA in vitro conditions was, as anticipated, concentration dependent and increased with increasing N/P ratio. Toxicity was also found to increase with increasing incubation period, in line with the formulations for reference lipids DC-Chol and EPC.Cationic liposomes formulated from polyene lipids C20-18, C20-20, and C30-20 and a neutral colipid were combined with a GFP-coded plasmid DNA for studies in retina cells. Gel electrophoresis was used to confirm the formation of lipoplexes in all formulations containing either DOPE or Chol as colipid. The diameters of lipoplexes were relatively large and variable; however, this did not appear to hinder transfection, since for a given cationic lipid larger lipoplexes gave rise to greater transfection . Toxicit in vitro cytotoxicity than C20-18 or C30-20. Nonetheless, C20-20 showed no evidence of inflammation in the mouse retina. In vitro transfection efficiencies of 6% and 8% were achieved with ARPE-19 cells with C20-18/DOPE and C20-20/DOPE, respectively. Furthermore, DNA delivery using C20-20/DOPE outperformed C30-20 in D407 retinal cells and revealed a level of transfection competitive to reference GeneJuice.C20-20 was found to have greater in vivo ocular gene delivery.Overall, C20-20/DOPE gave promising results for ocular gene therapy. Future work is directed to optimize formulations forS1: Transfection efficiency of novel cationic polyene lipids in another retinal cell line, D407. Comparison with Gene Juice, a commercial reagent, shows a similar trend.S2: Flow cytometry scatter plots for cell transfection with the novel cationic polyene lipids.


Saccharomyces cerevisiae isolated from palm wine. Exactly 609 and 406 g each of the fruits in two-mixed and three-mixed fruit fermentation respectively were crushed using laboratory blender, mixed with distilled water (1:1 w/v), and heated for 30 min with subsequent addition of sugar (0.656 kg). The fruit musts were subjected to primary (aerobic) and secondary (anaerobic) fermentation for 4 and 21 days respectively. During fermentation, aliquots were removed from the fermentation tank for analysis. During primary fermentation, consistent increases in alcohol contents (ranging from 0.0 to 15.0 %) and total acidities (ranging from 0.20 to 0.80 %) were observed with gradual decrease in specific gravities (ranging from 1.060 to 0.9800) and pH (ranging from 4.80 to 2.90). Temperature ranged from 27 °C to 29 °C. The alcoholic content of the final wines were 17.50 ± 0.02 % (pawpaw and watermelon), 16.00 ± 0.02 % (pawpaw and banana), 18.50 ± 0.02 % (banana and watermelon wine) and 18.00 ± 0.02 % . The alcoholic content of the wines did not differ significantly (p > 0.05). The pH of all the wines were acidic and ranged from 2.5 ± 0.01 to 3.8 ± 0.01 (p > 0.05). The acid concentration  were within the acceptable limit and ranged from 0.35 ± 0.02 to 0.88 ± 0.01 % (p > 0.05). Sensory evaluation (P > 0.05) rated the wines acceptability as ‘pawpaw and banana wine’ > ‘pawpaw and watermelon’ > ‘pawpaw, watermelon and banana’ > ‘banana and watermelon wine’. This study has shown that acceptable mixed fruit wines could be produced from the fruits with S. cerevisiae from palm wine.Pawpaw, banana and watermelon are tropical fruits with short shelf-lives under the prevailing temperatures and humid conditions in tropical countries like Nigeria. Production of wine from these fruits could help reduce the level of post-harvest loss and increase variety of wines. Pawpaw, banana and watermelon were used to produce mixed fruit wines using  Fruit juices are fermented to produce wine, an alcoholic beverage. Grapes are usually preferred because of the natural chemical balance of the grape juice which aids their fermentation process without the addition of sugars, acids, enzymes, or other nutrients. However, fruits such as banana, cucumber, pineapple and other fruits are used in wine production  is an important staple starchy food in Nigeria. Ripe bananas are consumed raw as a desert fruit. Banana serves as good nutritional sources of carbohydrates, minerals such as potassium and vitamins such as B1, B2, B3, B12, C and E. Following the high nutritional content of banana, it is consumed in large quantity in a variety of ways in Africa. The banana fruit can be eaten raw or cooked , processed into flour or fermented for the production of beverages such as banana juice, beer (e.g. mbege brewed by the Chagga people in the Kilimanjaro region of Tanzania), vinegar and wine  is grown mostly for fresh consumption or for production of latex. C.papaya plants produce natural compounds  in leaf bark and twig tissues that possess both highly anti-tumour and pesticidal properties  is a tropical fruit which grows in almost all parts of Africa and South East Asia  and Raphia palm (RaphiaHookeris and R. vinifera)  Okafor . Palm wins Fleet . The majS. cerevisiae isolated from palm wine.Though, studies have shown that bananas, pawpaw and watermelon (Obaedo and Ikenebomeh M. acuminata), pawpaw (C. papaya L), and watermelon (C. vulgaris L.) were purchased from the local central market (Nkwo Achara) in Uturu, Abia State, Nigeria. Fresh palm wine from Raphia hookeri were obtained from the palm wine tappers in Uturu within 1 h of tapping. The fruits and the palm wine were transported to the laboratory in clean cellophane bags and in an ice box respectively for analysis.Mature ripe banana  and incubated at room temperature for 24 h. Nineteen isolates were obtained and sub-cultured on fresh medium to obtain pure cultures. The yeast cultures were transferred to modified Malt Extract Agar (MEA) containing yeast extract and 2 % glucose and then incubated for 24 h. Out of the 19 isolates, six were identified as a et al. . The difThe isolated organism was multiplied prior to fermentation by culturing them on Malt Extract Broth (MEB) and incubating for 48–72 h at 27.0 °C ± 0.02. The broth cultures of the organism were centrifuged at 500 rpm for 5 min. The sediments were collected and used for must fermentation.2S2O5) was dissolved in 400 ml of water and poured in 100 ml aliquots to each of the mixtures and stirred properly. Sodium metabisulphate serve as a sterilizer and prevents fermentation before the addition of the yeast starter. The sugar concentrations were measured and the musts were mixed in the combination of ‘pawpaw and watermelon’ (30.4 °Brix), ‘pawpaw and banana’ (29.3 °Brix), ‘pawpaw, banana and watermelon’ (32.1 °Brix) and then ‘banana and watermelon’ (31.2 °Brix).The must was prepared for two-mixed fruit and three-mixed fruit fermentation respectively. The fruits were washed thoroughly with distilled water and then peeled. Exactly 609 and 406 g each of the fruit samples, banana, pawpaw and watermelon were weighed for two-mixed fruit and three-mixed fruit fermentations respectively. This was then chopped into smaller pieces using a clean knife before transferring them quantitatively into laboratory blender for crushing. The crushed sample was transferred into a clean new transparent bucket and mixed with distilled water (1:1 w/v). Exactly 0.656 kg of sugar was added to the must followed by vigorous stirring. Exactly 4 g of sodium metabisulphate (Na8 cfu/ml (measured using McFarland standard) of the yeast (S. cerevisiae) isolated from palm wine after centrifugation was added to each of the mixture, stirred properly and allowed to stand for 24 h before use.The yeast starter culture was prepared from a known quantity of the must for fermentation, small quantity of sugar, yeast and a known volume of water. The mixture of all these were treated with yeast nutrients and allowed to stand for 24 h. Approximately 200 ml of water was boiled and allowed to attain 37 °C and 200 ml of each mixture of the must  respectively treated with sugar was added. Exactly 5 g of citric acid was added to each of the preparations and then stirred for proper mixing. Exactly 2 g each of the yeast nutrient namely Potassium phosphate, Ammonium sulphate and Magnesium sulphate was dissolved in 100 ml of distilled water and poured to each of must mixture. Exactly 3.7 ml representing approximately 10The primary fermentation was initiated by the addition of the starter culture. The must was stirred every 12 h with subsequent reading of the specific gravity, pH, temperature and alcohol content for 4 days. After 4 days, the wine was racked into the secondary fermenter. The secondary fermentation was done in an air tight container in which a tube was passed into a clean bottle containing clean water. The essence was to monitor the course of fermentation. This was allowed until completion of fermentation as was evidenced by lack of the appearance of bubbles in the container usually within 3 weeks. Secondary fermentation was done for 21 days. When fermentation stopped, the wine was promptly racked off the lees ensuring minimum exposure to oxygen. After secondary fermentation, the wines were clarified. The clarification/fining were done using bentonite (a clarifying agent). Exactly 500 g of bentonite was dissolved in two litres of boiling water and stirred properly to a gel form. This was allowed to stand for 24 h. Then 150 g of the gel-like bentonite was transferred into each of the wine followed by stirring to dissolve properly. A small quantity of the mixture was collected in a clean bottle which was covered tightly and was used to monitor the process of clarification. This was done for a period of 3 months. Filtration was done after the wines had completed clarification using muslin cloth, sieve and syphon tubes sterilized by 70 % alcohol. The wines was syphoned into the sieve containing four layers of muslin cloth. The residues were removed and the filtrates were allowed to mature for a period of 6 months before other chemical analysis was carried out.Microbial analysis of the fermentation broth was performed as described by Fleet  using NuThe volatile acidity was determined using the method described by McClements , total aThe wines produced were compared for colour, flavour, taste, clarity, and overall acceptability by a panel of twenty judges on a seven point hedonic scale where seven denotes excellent and one very poor.The completely randomized analysis of variance (ANOVA) was used as described by Winner  to analyThe morphological and physiological characteristics of the yeast isolated from palm wine are represented in Table There were fluctuations in the temperature of the mixed fruit wines throughout the period of fermentation Fig. . These vA steady increase in alcohol content was observed in the mixed fruit wines throughout the period of primary fermentation Fig. . This inFigure After secondary fermentation, the temperature of the wines were observed to range from 27 ± 0.07 °C for pawpaw and banana wine to 28 ± 0.07 °C for pawpaw, banana and watermelon wine and banana and watermelon wine. The pH of the wines maintained an acidic range of 2.7 ± 0.1 for pawpaw and banana wine to 3.9 ± 0.1 for pawpaw and watermelon wine. There were little increases in the alcoholic content of the mixed fruit wines after secondary fermentation. The alcohol content of pawpaw and watermelon wine increased from 15 ± 0.02 % in primary fermentation to 16.5 ± 0.02 % after secondary fermentation, 14 ± 0.02 to 15.2 ± 0.02 %, 15.5 ± 0.02 to 17.5 ± 0.02 % and 15 ± 0.02 to 18 ± 0.02 % for pawpaw and banana wine, pawpaw, banana and watermelon wine and banana and watermelon wine respectively. The highest alcohol content was observed in banana and watermelon wine (18 ± 0.02 %) while pawpaw and banana wine recorded the lowest alcohol content (15.2 ± 0.02 %). In the case of specific gravities, little decreases were also observed in all the wines after secondary fermentation with banana and watermelon wine having the lowest value (0.9770 ± 0.00) and, pawpaw and banana wine having the highest value (0.9800 ± 0.00) while the acid concentrations ranged from 0.34 ± 0.02 (pawpaw and watermelon wine) to 0.86 ± 0.02 % (pawpaw and banana wine)  rated the acceptability of the wines as pawpaw and banana wine > pawpaw and watermelon > pawpaw, watermelon and banana > banana and watermelon wine Table .Table 4SThe fermentation of wine is known to be complex with various ecological and biochemical processes involving yeast strains Fleet . The ferThe mixed fruit wines  produced in the present investigation revealed low pH values (in the range of 2.5–3.8) throughout the fermentation periods and in the final product. Similar observations have been reported for other tropical fruit wines such as tundu wine . This could be attributed to the efficiency of the yeast in fermentation. It also implies that consumers are not exposed to the risk of taking in too much solid into the body. However, reduction in the total solid of the wines could be achieved by further filtrations.S.cerevisiae from palm wine). This trend was consistent in all the wines. In general, the percentage alcohol produced from the respective mixed fruit wines at the end of fermentation by the test yeast were 17.50 ± 0.02 % (pawpaw and watermelon wine), 16.00 ± 0.02 % (pawpaw and banana wine), 18.00 ± 0.02 %  and 18.50 ± 0.02 % (banana and watermelon wine). This finding agree with the work of Bechem et al. (S.cerevisiae) isolated from palm wine are different genetically from the yeast strains that are used to make wine from grapes and have the ability to survive and continue fermentation process up to ethanol concentration of 18 %, making them ideal candidate for producing ethanol for fuel.Remarkable amount of alcohol were produced from the fruit wines during fermentation with the test yeast  compared to the studies on commercial yeast (10.46 %) as reported by Chilaka et al. . High alS.cerevisiae) was the only organism isolated from pawpaw and watermelon wine as well as pawpaw and banana wine while neither pawpaw, banana and watermelon wine nor banana and watermelon wine showed the presence of any microorganism. This is an indication of good quality. This observation may be attributed to low pH values, high acidity and high alcohol contents of the wines which are known to inhibit the growth of pathogens and gives fermenting yeast a competitive advantage in natural environment as reported by Reddy and Reddy , straw yellow (pawpaw and banana wine), dark brown  and cream (banana and watermelon wine). This is an indication that the combination of the fruits served as a good substrate for wine production with pawpaw and banana being the most efficient as shown in this study. The good aroma obtained in the wines could be attributed to high alcohol content in accordance with the report of Clement-Jimenez et al. .Sensory evaluation rated the wines acceptability as pawpaw and banana wine > pawpaw and watermelon > pawpaw, watermelon and banana > banana and watermelon wine. These attributes compared favourably with the reports for other tropical wines  are good substrates for wine production. The biochemical and sensory attributes of the wines were acceptable by the consumers. The study has also given an insight into the efficacy and role of S. cerevisiae from palm wine during alcoholic fermentation of fruits. Pawpaw, banana and watermelon have short shelf-life under the prevailing temperature and humidity condition in Nigeria. Therefore, this study provides an avenue to preserve their nutrients, minerals, vitamins, aroma and taste to the consumers by fermenting them into wines.The present study which was based on the evaluation of three indigenous fruits as substrates for wine production and the efficiency of isolated


The purpose of this study was to determine test–retest reliability for peak barbell velocity (Vpeak) during the bench press (BP) and bench press throw (BPT) exercises for loads corresponding to 20–70% of one-repetition maximum (1RM). Thirty physically active collegiate men conducted four evaluations after a preliminary BP 1RM determination (1RM·bw-1 = 1.02 ± 0.16 kg·kg-1). In counterbalanced order, participants performed two sessions of the BP in one week and two sessions of the BPT in another week. Recovery time between sessions within the same week was 48 hours and recovery time between sessions of different weeks was 120 hours. On each day of evaluation the individual load-velocity relationship at each tenth percentile (20–70% of 1RM) in a Smith machine for the BP or BPT was determined. Participants performed three attempts per load, but only the best repetition (highest Vpeak), registered by a linear position transducer, was analysed. The BPT resulted in a significantly lower coefficient of variation (CV) for the whole load–velocity relationship, compared to the BP . Test–retest intraclass correlation coefficients (ICCs) ranged from r = 0.94–0.85 for the BPT and r = 0.91–0.71 for the BP (p < 0.001). The reduction in the biological within-subject variation in BPT exercise could be promoted by the braking phase that obligatorily occurs during a BP executed with light or moderate loads. Therefore, we recommend the BPT exercise for a most accurate assessment of upper-body velocity. Ballistic exercises (e.g. a bench press throw [BPT] or a jump squat) are preferred during power training as athletes are able to generate higher values of velocity, power, force, and muscle activation in comparison to similar traditional resistance training exercises (e.g. a bench press [BP] or a squat) . The oblDespite the limitations previously discussed, it is common that the BP is the selected exercise to assess and monitor upper-body power . AccordiOn the other hand, retest reliability concerns the repeatability of the observed value when the measurement is repeated . A reliaTo our knowledge, there are no previous studies that compare test-retest reliability between the traditional resistance training exercise (BP) and the ballistic exercise (BPT) which are frequently used to monitor and assess upper-body power. Therefore, the purpose of this study was to determine test–retest reliability for peak barbell velocity during the BP and BPT exercises for loads corresponding to 20–70% of one-repetition maximum (1RM). We hypothesized that BPT might be a more reliable exercise promoted by the unnatural braking phase that obligatorily occurs during a traditional BP. These findings should provide valuable information to coaches and practitioners regarding the most accurate exercise for assessing the effect of a training intervention on the development of maximum velocity with a range of loads commonly employed during power training.−2; 1RM BP: 75.7 ± 13.9 kg) volunteered to participate in this study. Selection criteria included (a) having at least six months of experience in BP training; (b) having no previous experience in BPT training; and (c) not consuming drugs, medications, or dietary supplements which influence physical performance. The study, which was conducted according to the Declaration of Helsinki, was approved by the University of Granada Institutional Review Board and, after being informed of the purpose and experimental procedures, participants meeting all criteria signed a written informed consent form.Thirty physically active collegiate men  during the BP and BPT exercises performed on a Smith machine. After a preliminary testing session , participants visited the laboratory on four separate occasions . They were randomly assigned with half of them performing both sessions of the BP in the first week and two sessions of the BPT in the second week, and vice versa. Individual load-velocity relationships at each tenth percentile (20–70% of 1RM) were determined on each day of evaluation. A linear position transducer  was used to measure barbell velocity with a frequency of 1,000 Hz. Relative (ICC) and absolute consistency (CV) were used to assess the repeatability of Vpeak for each percentage of the 1RM in both exercises. Furthermore, paired samples t-tests and effect size (ES) were employed to establish differences in Vpeak between the BP and BPT.This study used a repeated measures design to compare test–retest reliability for peak barbell velocity (V−1). From that position, every participant was instructed to perform a purely concentric action (as quickly as possible) to regain the initial position. The duration of eccentric and isometric phases was administered by auditory feedback through an ad hoc audio file, while the concentric phase was always performed explosively, at the maximum possible speed. The only difference in the execution between the BP and BPT was that during the BPT participants accelerated the bar during the entire range of movement with the intention of throwing it as high as possible, whereas in the BP the bar had to be voluntarily decelerated at the end of the range in order to not throw it.Participants arrived at the laboratory after refraining from strenuous exercise for a minimum of 48 hours. As soon as they arrived, their body height  and mass  were assessed. Before the commencement of the 1RM determination, participants selected the grip width that was the most comfortable, which was measured and kept constant throughout all testing sessions . The warParticipants visited the laboratory on four separate occasions during two consecutive weeks. In one week participants performed the two BP sessions, and in the other week they performed two BPT sessions. To compensate for a possible training effect, participants were counterbalanced. Recovery time between sessions within the same week was 48 hours and recovery time between sessions of the other week was 120 hours. Every session for the same participant was carried out at the same time of the day (to control circadian variation in performance) and under similar environmental conditions (22–23°c and 60% humidity). All tests were performed on a Smith machine  in which the barbell was attached to both ends, with linear bearings on two vertical bars allowing only vertical movements. According to peak, was considered for subsequent analysis. Recovery time between sets was at least 5 min. Two trained spotters were present on each side of the bar during the BPT and BP protocols to ensure safety, and to strongly encourage the participants throughout the test. Vpeak, which was defined as the maximum instantaneous value achieved during the concentric phase at a given load, was evaluated using a linear position transducer  at a sampling rate of 1,000 Hz. This device was interfaced to a personal computer and custom software  was used to automatically calculate the variable of interest (Vpeak).Each testing session was preceded by a 10 min standardized warm-up, which included dynamic stretching, arm and shoulder mobilization and one set of six repetitions performed in an explosive manner with an external load of 17 kg in the exercise evaluated (BP or BPT). After warming up, participants rested for 5 min before starting the incremental loading test. Then, individual load-velocity relationships at each tenth percentile (20–70% of 1RM) were determined. Participants performed three repetitions (as quickly as possible) at each load, but only the best repetition, according to the criteria of the highest V2,1 (two-way random effect model). To examine absolute reliability, pairwise comparisons were first applied with the paired t-test to assess any significant differences in Vpeak between trials 1 and 2 for each of the intensities (20–70% of 1 RM) analysed. The magnitude of between-session differences was also expressed as a standardized mean difference . The criteria to interpret the magnitude of the ES were as follows: <0.2 = trivial, 0.2–0.6 = small, 0.6–1.2 = moderate, 1.2–2.0 = large, and >2 = very large (−1) and as a percentage of the participants’ mean scores (CV) to assess absolute consistency. The Pearson product moment correlation coefficient (r) was used to determine the relationship of Vpeak between the BP and BPT for the same load. Finally, paired samples t-tests and ES were employed to establish differences in Vpeak between the BP and BPT. Significance was accepted at p ≤ 0.05. All statistical analysis was performed using SPSS version 20.0 .Data are presented as mean ± standard deviations (SD). Data normality was assessed using the Shapiro-Wilk test. A two-way  repeated measures ANOVA was used to examine peak barbell velocity data for BP and BPT exercises. When appropriate, Bonferroni post hoc comparisons were performed. Relative reliability analysis was examined by the ICCry large . Additiop = 0.768 for the BP and p = 0.490 for the BPT), no main effect for trial (p = 0.142 for the BP and p = 0.991 for the BPT), but there was a significant main effect for % 1RM (p < 0.001 for the BP and BPT). Bonferroni comparison showed that Vpeak was higher at 20% of 1RM for both the BP and BPT, and significantly decreased with each subsequent intensity examined .The results from the two-way repeated measures ANOVA indicated there was no significant trial x % 1RM interaction  and intensities (20–70% of 1RM) analysed. Furthermore, the ES of both exercises were trivial (<0.2) in all loads. On the other hand, despite the fact that the results of relative  and absolute consistency  showed good reliability of both exercises, the CV for the whole load–velocity relationship was significantly lower for the BPT compared to the BP  (The results from each of the paired samples t-tests  indicate= 0.040) .peak were obtained in the BPT for all loads employed (p < 0.001). However, differences between both exercises tended to decrease as the load increased  and for each specific load (The aim of the present study was to compare test-retest reliability of peak barbell velocity in the two exercises most frequently used to assess upper-body power. Our results indicate that both exercises have high reliability in terms of relative (ICC) and absolute consistency (CV). As indicated by fic load , suggestpeak between trials 1 and 2 for any of the exercises (BP or BPT) and intensities (20–70% of 1RM) analysed. However, the absence of significant differences does not necessarily imply good reliability, precisely because a large amount of random error between tests may promote the absence of these differences  and absolute (CV) consistencies. We think that these results could be promoted mainly for four reasons: (a) the type of exercise: free weight vs. Smith machine. The Smith machine employed in our study increases reliability by allowing only vertical movements; (b) the number of repetitions performed per load: single repetitions vs. the best of three repetitions. The greater number of repetitions performed in our study increases the chances of obtaining the maximum performance from the individual; (c) the pattern of movement: eccentric-concentric vs. eccentric-isometric-concentric. To impose a momentary pause between the eccentric and concentric actions allows more reproducible, consistent measurements  relationship is one of the tests most commonly used to assess the effects of a training intervention . Surprisurements ; (d) thepeak between both exercises (BP and BPT) has a tendency to increase as the load is increasing. Therefore, it seems that the BPT is more reliable even in subjects without previous experience in this exercise.On the other hand, this is the first study that compares test-retest reliability between the traditional resistance training exercise (BP) and the ballistic exercise (BPT) most frequently used to monitor and assess upper-body power. A good measurement is required to have both concurrent validity and retest reliability . On the peak in loads ranging from 20 to 70% of 1RM. As expected, differences between both exercises tend to decrease as the load is increased (It has been known for some time that the BPT allows for the development of higher values of velocity, power, force, and muscle activation in comparison to BP exercise . Our resncreased  confirmincreased .peak values between exercises and was also a key factor to overcome the learning process.In order to mitigate the effects of potentiation or fatigue the sequence of loads during loaded tests tend to be randomized . NotwithIn summary, due to the higher reliability scores found in the BPT, we recommend this exercise for the assessment and monitoring of upper-body velocity even in subjects without previous experience in this exercise. Although the BP also has good reliability, it does not seem appropriate to choose this exercise since athletes cannot develop their fullest potential. It should be noted that our results may not be representative of other exercises used to assess lower-body power. Consistent with other studies, our results seem to show lower reliability during low-velocity movements . Furtherpeak between the BP and BPT at 20–30% of 1RM could support this claim. Based on these results, we recommend the BPT exercise for the accurate assessment of upper-body velocity, even in subjects without previous experience in this exercise.A good measurement requires both validity and retest reliability . As indi


Skin tissue homeostasis is maintained by a balance between the proliferation and differentiation of epidermal stem cells (EpSCs). EpSC proliferation and differentiation are complex processes regulated by many factors and signaling pathways. This study aimed to explore the connection between the Nanog and the Wnt/β-catenin pathway in the proliferation and differentiation of EpSCs.Our results demonstrated that during the study period, EpSC underwent differentiation when incubated in the presence neuropeptide substance P (SP), there was an opposing expression trend of Nanog and β-catenin after SP treatment, which could be antagonized by the Wnt antagonist, Dkk-1. The transduced EpSCs had a greater proliferative ability than the SP treatment group and they did not undergo differentiation upon SP treatment. More important, β-catenin expression was down-regulated but phosphorylated β-catenin expression and phosphorylated GSK-3β expression was up-regulated upon Nanog overexpression.These results strongly suggest that Nanog plays an important role in maintaining the proliferation and differentiation homeostasis of EpSCs by promoting β-catenin phosphorylation via GSK-3β to inhibit the activity of the Wnt/β-catenin signaling pathway. This is important for precise regulation of proliferation and differentiation of EpSC in the application of tissue engineering. Skin is the largest organ of the human body; it plays a key role in protecting the body against pathogens. The homeostasis of skin tissue is maintained by rare but pluripotent epidermal stem cells (EpSCs) and their progeny, transient amplifying (TA) cells , 2. As pTranscription factors and signals determine whether EpSCs undergo self-renewal or differentiation. Several studies have established the central role of Nanog in maintaining pluripotency and preventing differentiation –7. Nanogin vitro. Subsequently, the expression of β-catenin and Nanog were evaluated in SP treated EpSCs using western blotting and PCR. In addition, the effect of Nanog on β-catenin expression was investigated in EpSCs using a lentivirus infection and/or SP treatment.In this study, we evaluated the relationship between the Wnt/β-catenin signaling pathway and Nanog in the proliferation and differentiation of EpSCs. Rat EpSCs were infected with lentivirus overexpressing Nanog. SP,a neuropeptide present in primary sensory neurons,belongs to the tachykinin family and can activate Wnt/β-catenin signaling pathway . Transdu-7 M SP treatment or Wnt agonist treatment. Differentiated EpSCs showed the special morphologies as long spindle or polygonal . However, β-catenin expression increased significantly from days 3–12 compared to day 0 (P < 0.05)   Figure A and 4B.To reveal the effect of Nanog overexpression on β-catenin and confirm the lentivirus itself do not affect the expression of Nanog or β-catenin, we infected EpSCs with lentivirus loaded with or without Nanog. Nanog and β-catenin expression at mRNA were detected at different time point (day 1- day 12) Figures A and 5B.To explore how Nanog affects the Wnt/β-catenin signaling pathway, EpSCs infected with lentivirus encoding Nanog were incubated in the presence or absence of SP. Nanog, β-catenin and c-myc in Wnt/β-catenin signaling pathway were detected both at mRNA and protein level. At the mRNA level, Nanog expression in transduced cells in the absence or presence of SP was significantly higher than the control or SP-only treatment groups Figure A P < 0.B P < 0.C. The weTo demonstrate the change in β-catenin expression in detail, total β-catenin,phosphorylated β-catenin, total GSK-3β protein and phosphorylated GSK-3β protein were also measured using western blotting Figure A. FigureAs the primary barrier in the human body, skin protects humans from infection, radiation and wounds. However, skin, especially the epidermis, may be injured by external insults. The proliferation and differentiation of EpSCs and their progeny of TA cells greatly affect skin repair , 19, 20.Nanog is a pluripotent state-specific transcription factor that plays a critical role in regulating cell fate during embryonic development, maintaining pluripotent epiblasts and preventing differentiation , 25. Nan-7 M SP caused a rapid proliferation of EpSCs into TA cells in the first twelve days, which subsequently had a limited proliferative ability . GSK-3β is phosphorylated and results in β-catenin phosphorylation and its subsequent degradation by the ubiquitin-mediated pathway , 37. In In summary, our study showed that Nanog and the Wnt/β-catenin signaling pathway are involved in the regulation of EpSC proliferation and differentiation, which is necessary to maintain EpSCs homeostasis in the skin. The underlying mechanism is that Nanog inhibits the activity of the Wnt/β-catenin signaling pathway via GSK-3β mediated β-catenin phosphorylation. This finding represents an important step towards understanding the precise regulation of EpSC proliferation and differentiation, which has important applications in tissue engineering.g. To select stem cells, 1x106 dissociated epidermal cells were plated onto collagen type IV  (100 μg/ml)-coated dishes at room temperature for 15 min. The unattached cells were removed, and the rapidly adherent epidermal cells were cultured with keratinocyte serum-free medium  supplemented with epidermal growth factor (EGF) , bovine pituitary extract , and 0.05 mM calcium chloride  at 37°C under 5% CO2 in a humidified incubator for two days before replacing the medium. The medium was changed every other day.Sprague–Dawley (SD) male rats were purchased from HUST Laboratory Animal Center . The Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, approved all animal care and experimental procedures. The EpSCs isolated procedure was regarded as a standard protocol which was first repoted by Liu Y . Skin tiThe coding sequence of rat Nanog was PCR amplified from GV208: rat-Nanog using primers with AgeI/AgeI overhangs and was cloned into pTZ58 . The AgeI/AgeI fragment was then sub-cloned into pUbi (AgeI/AgeI) and pEGFP-C1  (AgeI/AgeI) to generate Ubi-Nanog-EGFP encoding plasmids, respectively. To produce lentivirus, the pBABE-puro plasmids were co-infected along with the helper plasmids into 293 T cells, and the medium was harvested 36 h and 72 h after infection. EpSC infection was performed by incubating the cells in virus-enriched medium for 12 h, which included 4 μg/ml polybrene. Transduced cells were identified for EGFP expression under a fluorescence microscope. The transduced EpSCs were divided into two groups: the first group of EpSCs was treated with SP for 12 days after infection; the second group of EpSCs was cultured for 12 days. EpSCs were also infected with control lentivirus vector and cultured for 12 days. EpSCs without any treatment were used as control.4 cells/well) for 24 h. The cells were rinsed five times with PBS, fixed with 4% paraformaldehyde solution for 15 min at room temperature, and permeated with 0.5% Triton X-100-PBS solution for 5 min. After blocking with 3% BSA in PBS for 30 min, the cells were respectively incubated with primary antibodies to anti-CD 34 antibody  and anti-β1 integrin antibody  at 4°C overnight. The primary antibody binding was detected via the corresponding goat anti-rabbit IgG: Cy3 and goat anti-mouse IgG FITC . Staining was examined under a fluorescence microscope.The isolated cells were cultured in a 6-well plate (104 cells/well) for 24 h. The cells were treated with 10-7 M SP or LiCl, a Wnt agonist, to induce differentiation for 12 days. The cells were rinsed five times with PBS, fixed with 4% paraformaldehyde solution for 15 min at room temperature, and permeated with 0.5% Triton X-100-PBS solution for 5 min. The cells were incubated with primary antibodies to anti-cytokeratin 18 antibody (CK18)  at 4°C overnight. Subsequent procedures were performed according to the SP-9000 Histostain-Plus Kit instructions . The cells were observed under a microscope .The isolated cells were cultured in a 6-well plate (104 cells/well) and treated with collagen type IV. The cells were then divided into four groups: the first group was composed of cells without any treatment, the second group was composed of cells infected with control lentivirus vector, the third group was composed of transduced cells, and the forth group was composed of cells that were exposed to 10-7 M SP. Every other day after day 2 until day 12 post-treatment, 10 μL of CCK-8  solutions were added to each well, and the samples were incubated for 2 h. The specific absorbance at 450 nm (A450) was determined using a Micro-plate Reader . All of the experiments were repeated independently at least three times, and each measurement was the average of 5 duplicate wells.EpSCs were plated in 96-well plates  containing 50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA and a protease inhibitor cocktail. The protein concentration was determined using the bicinchoninic acid protein assay . The proteins were separated by SDS-PAGE, transferred to nitrocellulose, blocked with bovine serum albumin and incubated with the following primary antibodies: rat anti-β-catenin  diluted 1:1000, rat anti-p-β-catenin  diluted 1:1000, rat anti-c-myc  diluted 1:1000, rat anti-Nanog  diluted 1:2000, rat anti-GSK3β  diluted 1:1000, rat anti-p-GSK3β  diluted 1:1000 or rat anti-GAPDH  diluted 1:1000 that was used as a loading control. The membrane was washed and incubated with the respective secondary antibodies conjugated with peroxidase. Chemiluminescence  was used to detect protein.260 and A260/A280 ratios, respectively. Total RNA was used to synthesize cDNA with a Super-Script II cDNA synthesis kit . The related genes of the Wnt/β-catenin signaling pathway and Nanog were assessed by quantitative real-time PCR using a SYBR-Green Master mix . Glyceraldehyde phosphate dehydrogenase (GAPDH) was selected as an internal control. The primer sequences are: Nanog (accession no. NM_001100781) forward, CCGTTGGGCTGACATGAGCGT and reverse, GGCAGGCATCGGCGAGGAAT; β-catenin (accession no. NM_053357) forward, AGCCCGTTGTACCGCTGGGA and reverse, CGCTGGGATGCCGCCAGATT; c-myc (accession no. NM_012603) forward, CATCATCCAGGACTGTATGTG and reverse, TGGAATCGGACGAGGTA; GAPDH (accession no. NM_017008) forward, TATGACTCTACCCACGGCAAGT and reverse, ATACTCAGCACCAGCATCACC.Total RNA was extracted using the TRIzol reagent , according to the manufacturer’s protocol. Using a spectrophotometer , the RNA concentration and purity were determined by AAll experiments were repeated independently at least three times. The data are presented as the mean ± standard deviation (SD). The results were analyzed using SPSS 18.0 statistical software. Statistically significant differences were identified using Student’s t-test and an analysis of variance (ANOVA). The significance level was set to P < 0.05.


HDZIV genes expresses in different banana tissue, however at varying levels. The transcript levels of some of the banana HDZIV genes were also detected in banana fruit pulp, suggesting their putative role in fruit attributes. A large number of genes of this family showed modulated expression under drought and salinity stress. Taken together, the present work lays a foundation for elucidation of functional aspects of the banana HDZIV encoding genes and for their possible use in the banana improvement programs.The homeodomain zipper family (HD-ZIP) of transcription factors is present only in plants and plays important role in the regulation of plant-specific processes. The subfamily IV of HDZ transcription factors (HD-ZIP IV) has primarily been implicated in the regulation of epidermal structure development. Though this gene family is present in all lineages of land plants, members of this gene family have not been identified in banana, which is one of the major staple fruit crops. In the present work, we identified 21 HDZIV encoding genes in banana by the computational analysis of banana genome resource. Our analysis suggested that these genes putatively encode proteins having all the characteristic domains of HDZIV transcription factors. The phylogenetic analysis of the banana HDZIV family genes further confirmed that after separation from a common ancestor, the banana, and poales lineages might have followed distinct evolutionary paths. Further, we conclude that segmental duplication played a major role in the evolution of banana HDZIV encoding genes. All the identified banana  The protein sequences, thus retrieved were aligned with that of maize, rice and Arabidopsis. Protein sequences containing MEKHLA domain were removed. Further refinement of gene models of banana HDZIV proteins was carried out by retrieving corresponding genomic sequences and by using Fgenesh programme (www.omictools.com/fgenesh-s1037.html). Finally, manual inspection of intron and exon boundaries was carried out. The prediction of molecular weight and PI was done by using tools available at Expasy bioinformatics resource portal. The prediction of Arabidopsis homologs of banana HDZIV genes was carried out by BLAST P searches in The Arabidopsis Information Resource . Identification of conserved motif among banana HDZIV proteins was done by using MEME tool (http://meme-suite.org/tools/meme). The identified motifs, as represented by logos were manually inspected for presence of elements, representing conserved domains of HDZIV proteins.The proteins corresponding to the HDZ IV genes of maize, rice and Arabidopsis, as described in Javelle et al.  were usehttp://gsds.cbi.pku.edu.cn/. For identification of conserved motifs in 3′UTR regions  , genomic information was not available for the upstream region, and therefore was not included in the analysis. Further, in the case of MaHDZIV1 (locus id GSMUA_Achr1P18760_001), the genomic information was available for 1056 bps only. The upstream regions were analyzed using Plant Care database and cis-regulatory elements were retrieved  was used for gene expression analysis in the present study. The vegetative tissues such as different developmental stages of leaves, different layers of pseudostem, bract, fruit fingers, fruit peel, and fruit pulp etc. were harvested from field grown banana plants and stored at −80°C, following freezing in liquid nitrogen. The in-vitro tissues  were developed using male flowers as explants and as per previously described protocol  was used for evoking salinity stress in banana plantlets as described by Sreedharan et al.  50°C, 2 min; step (2) 95°C, 10 min; step (3)  × 40 cycles.The quantitative real-time PCR expression was carried out with an ABI 7700 Sequence Detector . The transcripts were quantified by SYBR Green chemistry. The amount of cDNA was normalized by using amplification of housekeeping banana To comprehensively identify banana HDZIV genes, blast searches in banana genome database was carried out using known HDZIV proteins from Arabidopsis 16 genes), maize (17 genes), and rice (11 genes) as query sequences. The banana protein sequences, thus retrieved were analyzed by carrying out multiple sequence alignment with other known HDZIP proteins. However, some of the sequences were found to have deleted regions of varying amino acid length suggesting requirement of further refinement in gene model prediction. Therefore, corresponding genomic sequences were retrieved and gene models were modified using online tool (Fgenesh program) followed by manual analysis of intron–exon boundaries , a phylogenetic tree was constructed using NJ method. Certain functionally characterized HDZIV proteins from tomato, cotton and wheat were also included in the analysis. The HDZIV proteins were grouped in four distinct clades, named as I to IV Figure . Most ofThe deduced amino acid sequences corresponding to the banana HDZ proteins were further analyzed for comprehensive prediction of conserved motifs using MEME suite Figure . A totalIn order to gain information regarding gene structure, genomic regions corresponding to banana HDZIV genes were analyzed for the architecture of introns and exons. The numbers of exons and introns among banana HDZIV genes varied between 8 to 11 and 7 to 10, respectively Figure . FurtherMaHDZIV14, MaHDZIV15, MaHDZIV6, MaHDZIV11 MaHDZIV4, and MaHDZIV2  were identified to be up-regulated following drought stress suggesting that these could be functional orthologs of AtHDG11. The ZmHDZIV4 gene of maize has been implicated in trichome and in regulation of some aspects of anther cell wall division and proliferation , pectin-related enzymes, and cellulases in Arabidopsis roots (Xu et al., The HDZIV gene family expresses primarily in epidermal cells and regulate differentiation aspects in this tissue domain (Chew et al., Large numbers of banana HDZIV genes were reported to be up-regulated by drought and salinity stress. Various reports suggest that HDZIV transcription factors regulate cuticle deposition, root hair development and stomata differentiation (Takada and Iida, cis-acting elements associated with plant growth, development, and stress response were identified in the proximal promoters of the banana HDZIP genes (Table The transcriptional regulation is one of the most important mechanisms to regulate HDZIV expression (Chew et al., es Table . The liges Table . Presences Table .MaHDZIV1, MaHDZIV8, MaHDZIV2, MaHDZIV9, MaHDZIV3, and MaHDZIV20 were commonly up-regulated by salinity and drought stresses. These genes can be used for the development of stress tolerant banana varieties using biotechnological tools. Future research involving development of banana lines with modified expression of these HDZIV genes should elucidate the function of individual gene family members. We believe that elucidation of functions of these genes should provide novel molecular tools to improve important agronomic traits in banana.The expression analysis in tissues as well as under different stresses suggests their possible role in development, fruit ripening, and stress response. Our expression analysis suggested that few genes, i.e., AP and PT conceived the idea. AP, PM, AA, NK, SS, DL, ST carried out various experiments. AP, PT, PM, MA analyzed the data. AP, PM, and PT wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.