BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistances. Activation of the PI3K pathway can mediate resistance to these agents, providing a strong rationale for combination therapy in melanoma. Here, a panel of nine low-passage human metastatic melanoma cell lines with BRAF mutations was tested in cell proliferation and protein expression assays for sensitivity to inhibitors of MEK (selumetinib) and BRAF (vemurafenib) as single agents and in combination with inhibitors of pan-PI3K (ZSTK474), pan-PI3K/mTOR (BEZ235), individual PI3K isoforms , or mTORC1/2 (KU-0063794). Selumetinib and vemurafenib potently inhibited cell proliferation in all cell lines, especially in those that expressed low levels of phosphorylated AKT (pAKT). ZSTK474 and BEZ235 also inhibited cell proliferation in all cell lines and enhanced the antitumor activity of selumetinib and vemurafenib in the majority of lines by either interacting synergistically or additively to increase potency or by inducing cytotoxicity by significantly increasing the magnitude of cell growth inhibition. Furthermore, ZSTK474 or BEZ235 combined with selumetinib to produce robust inhibition of pERK, pAKT, and pS6 expression and synergistic inhibition of NZM20 tumor growth. The inhibitors of individual PI3K isoforms or mTORC1/2 were less effective at inhibiting cell proliferation either as single agents or in combination with selumetinib or vemurafenib, although KU-0063794 synergistically interacted with vemurafenib and increased the magnitude of cell growth inhibition with selumetinib or vemurafenib in certain cell lines. Overall, these results suggest that the sensitivity of BRAF-mutant melanoma cells to BRAF or MEK inhibitors is at least partly mediated by activation of the PI3K pathway and can be enhanced by combined inhibition of the BRAF/MEK and PI3K/mTOR signaling pathways.BRAF and MEK inhibitors have improved outcomes for patients with  BRAF mutations leading to constitutive activation of the RAS/RAF/MEK/ERK pathway and increased cell cycle progression, differentiation, survival, migration, and angiogenesis are reported in 40–50% of melanoma cases  δ 9.49 , 8.86 , 8.52 , 8.47 , 8.39 , 8.06–8.09 , 7.95–7.99 , 7.85–7.90 , 7.73 , 7.37 , 4.85 , 3.70 , 2.39 , 1.79 ; Combustion Analysis, Calc. for C32H31N5O7S2: C, 58.1; H, 4.7; N, 10.6. Found: C, 58.0; H, 4.6; N, 10.65%.BEZ235 was prepared as a dimethanesulfonate salt by treating a suspension of the solid in methanol with 2.2 equivalents of methanesulfonic acid, to give a clear solution. Dilution with ethyl acetate gave a precipitate, which was collected by filtration and washed with further ethyl acetate. Recrystallization from methanol–ethyl acetate gave the dimethanesulfonate salt as a pale yellow solid: mp 283–286°C; 2, 5% CO2) at 37°C.A panel of nine melanoma cell lines was chosen from a series of lines generated from surgical samples of metastatic melanoma obtained with appropriate consent from patients throughout New Zealand as previously described . The celTERT was determined in the melanoma cell lines by Sequenom analysis. DNA was extracted using PureLinkTM Genomic DNA kit (Life Technologies), according to manufacturer’s protocol. To remove the EDTA-based elution buffer, DNA was re-precipitated into milliQ water. This was achieved by addition of ethanol and 5M ammonium acetate at −80°C for 2 h and centrifugation at 18,000 × g for 30 min at 4°C. The pellet was resuspended in ethanol and re-centrifuged at 18,000 × g for 10 min at 4°C, prior to resuspension in milliQ water. Extracted DNA was evaluated for gene mutations on the Sequenom MassARRAY® using the MassARRAY OncoCartaTM Panel v 1.0 and the MelaCartaTM Panel v1.0 plus hTERT, according to manufacturer’s protocol. Analysis was carried out using Sequenom MassARRAY Typer Analyzer 4.0 genotyping software. PTEN mutation status was determined by PCR sequencing as described previously  in 1% acetic acid for 30 min in the dark at room temperature. Plates were washed in 1% acetic acid, dried, and incubated with unbuffered Tris base  for 30 min on a plate shaker in the dark to solubilize the stain. The plates were read on a BioTek EL808 microplate reader at an absorbance of 490 nm with a reference wavelength of 450 nm. Absorbances of treated cells were compared to untreated cells at 0 h (100% growth inhibition) and 72 h (0% growth inhibition) after treatment. Growth inhibition above 100% indicated that fewer cells were present than when the compounds were first administered. EC50 values were calculated by fitting the inhibition data to a four-parameter logistic sigmoidal dose–response curve using GraphPad Prism 6.01. Combination indices (CI) were calculated at EC50 using the method of Chou and Talalay  at 25V for 12 min on a BioRad Trans-Blot® TurboTM semi-dry transfer machine. Following protein transfer, membranes were incubated in blocking buffer [Tris-buffered saline with 0.5% Tween®-20 (Serva) and 3% BSA] for at least 30 min then cut and incubated overnight at 4°C with antibodies at 1:1000 dilution (unless indicated) against either pAKT , pERK1/2 (Thr202/Tyr204), pS6 , AKT, ERK1/2, S6 (1:2000), PTEN (1:100), IGF1Rβ, EGFR, Insulin Receptor β, c-MET, ERBB3, MERTK , and β-actin . Membranes were washed then incubated with anti-mouse  or anti-rabbit  goat IgG HRP-conjugated secondary antibody in blocking buffer for 1 h at room temperature. After further washes, the membranes were incubated with BioRad ClarityTM ECL or Perkin Elmer Western Lighting Ultra (pAKT membranes) for 4 min prior to imaging on a LAS-4000 luminescent image analyzer (Fujifilm). After visualization of phosphorylated proteins, membranes were stripped and reprobed with total proteins and β-actin. β-actin was used to confirm equal protein loading in each blot. Each cell lysate was tested in duplicate in two to three independent experiments.Untreated cells for basal protein expression or cells treated with 500 nM of compound for 1 or 24 h were lysed in lysis buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich) on ice for 15–30 min. Cells were centrifuged at 13,000 rpm for 10 min at 4°C to remove insoluble material. Protein concentration of cell lysates was determined by bicinchoninic acid assay (Thermo Scientific) against bovine serum albumin  standards at an absorbance of 562 nm on a BioTek Synergy HT plate reader using KC4 v3.4 software. Forty micrograms of each lysate was loaded onto polyacrylamide gels (10% acrylamide) and separated by SDS-PAGE at 120V for 90 min. Each gel was transferred onto an Immobilin6 NZM20 cells. Treatment was initiated when tumors reached 150 mm3 in volume as measured by electronic calipers. The dosing vehicles used were 0.5% hydroxypropyl methylcellulose with 0.2% Tween 80 (selumetinib and control vehicle), 2% carboxymethylcellulose with 1% Tween 80 (ZSTK474), and 40% PEG-400 in 20% hydroxypropyl-β-cyclodextrin (BEZ235). All vehicle constituents were supplied by Sigma-Aldrich. Treatments were administered daily for 14 days at half the single agent maximum tolerated dose as the free base equivalent by oral gavage at a volume of 10 ml/kg. Animals were monitored daily for bodyweight and any observational signs of toxicity. Tumor growth inhibition (TGI) was calculated by determining the tumor size of drug-treated mice relative to starting size as a percentage of the average relative tumor size of control mice. Synergy was inferred if the TGI of the combination was greater than the product of the TGI values of each single agent. All animal experiments followed protocols approved by the University of Auckland Animal Ethics Committee.Age-matched specific pathogen-free female NIH-III mice were subcutaneously inoculated with 5 × 10BRAF-mutant melanoma cell lines that were both developed and maintained at low-oxygen tension (5%). The mutation status of common oncogenic driver mutations in these lines was determined by Sequenom or PCR profiling. Seven of the cell lines contained V600E mutations in BRAF, while two contained V600K mutations . All PTEN-mutant lines, along with NZM30, lacked PTEN expression. pERK was expressed in all cell lines at variable levels, with the lowest expression detected in NZM7 and NZM43, while pS6 was expressed in all cell lines at similar levels relative to total protein.The basal protein expression of phosphorylated and/or total forms of AKT, PTEN, ERK1/2, and S6 were determined in the cell line panel by western blotting Figure  and 6, bBRAF-mutant and PTEN-null cell lines.Several RTKs that can activate PI3K/mTOR or RAS/RAF/MEK/ERK signaling pathways were expressed in the NZM lines Figure . High-EG50’s ranging from 12.0 ± 3.7 to 131 ± 56 nM . There was no clear relationship between basal pERK protein expression and selumetinib or vemurafenib sensitivity in the cell line panel.Each cell line in the panel was exposed to multiple concentrations of selumetinib or vemurafenib to determine the effects of these drugs on cell proliferation. Selumetinib was highly effective at inhibiting cell proliferation in all cell lines, with ECBRAF-mutant melanoma  except NZM3, NZM7, and NZM11, where selumetinib or ZSTK474 alone inhibited growth by >105% .Since the antiproliferative activity of selumetinib and vemurafenib was less pronounced in 50’s ranging from 10.2 ± 2.8 to 163 ± 100 nM , as shown for NZM34   to either vemurafenib or BEZ235 as single agents   was observed with ZSTK474 in the six cell lines that were least sensitive to vemurafenib as a single agent Figure , but sigTo determine if selumetinib in combination with ZSTK474 or BEZ235 can prevent both ERK and AKT signaling, pERK and pAKT expression levels were determined by western blotting 1 and 24 h after 500 nM drug treatment  and NZM12 (EC50 = 5.9 ± 2.7 μM)   and in NZM7, NZM20, and NZM34 cells with selumetinib and KU-0063794 (P < 0.05)  and NZM34 (P < 0.05) cells  relative to control at day 14  and 59.0 ± 7.4% (P < 0.0001) relative to control at day 14 for selumetinib with BEZ235 and ZSTK474, respectively. Treatment with selumetinib and BEZ235 prevented tumors from growing above baseline levels during the treatment period and at day 14 tumor size was significantly reduced from selumetinib (P < 0.05) or BEZ235 (P < 0.0001) treatment alone. All treatments were well tolerated over the dosing period, with minimal weight change seen in all groups  is required for cytotoxic inhibition of growth.Inhibition of PI3K alone with ZSTK474 or PI3K and mTOR with BEZ235 was effective at inhibiting cell proliferation and AKT signaling in all n status , 48, in  deleted . However therapy –31. SyneBRAF-mutant melanoma cell lines, inhibition of all or most isoforms is required due to the ability of isoforms to readily compensate for one another (Isoform-selective inhibitors of PI3K lacked potency at inhibiting cell proliferation on their own, but A66, idelalisib, and AS-252424 were able to significantly increase maximal inhibition when combined with selumetinib, but not vemurafenib, in NZM7 cells. However, since the isoform-selective PI3K inhibitors only enhanced selumetinib activity at concentrations approaching 10 μM, it is possible that these effects could be due to loss of isoform selectivity at these high concentrations. Nevertheless, the activity was less than that observed for ZSTK474, suggesting that although one or more of the PI3K isoforms may play a preferential role in promoting survival in  another , 51. Thi another . By cont another , 33, 48.in vivo, these properties will need to be evaluated in future alongside an assessment of optimal dose levels and scheduling of these agents in combination.Substantial inhibition of pS6 was observed here with selumetinib alone in the cell lines that were most sensitive to selumetinib or vemurafenib and expressed low pAKT, while only minor inhibition was detected in less sensitive lines that expressed moderate to high levels of pAKT. Further inhibition of pS6 was achieved in combination with ZSTK474 and near complete inhibition in combination with BEZ235. These results corroborate with previous findings that mTORC1 activity is primarily regulated by ERK signaling in BRAF or MEK inhibitor sensitive melanomas . HoweverBRAF-mutant melanomas, particularly those that express low pAKT and functional PTEN, can be effectively treated with single agent BRAF or MEK inhibition, for the majority of lines, enhancement of antitumor activity can be achieved by combining BRAF or MEK inhibition with an inhibitor of PI3K/mTOR signaling to block ERK, AKT, and S6 signaling. Despite either inhibition of PI3K or mTOR synergizing with BRAF/MEK inhibition, dual PI3K/mTOR inhibition appears the most promising strategy to enhance sensitivity to BRAF and MEK inhibition and possibly overcome ERK-independent intrinsic or acquired resistance.Overall, these results suggest that while some BRAF/MEK inhibitor sensitive MS, PS, and SJ conceived and designed the experiments. MS, ER, and SK performed the experimental studies. GR performed compound synthesis. BB established the melanoma cell lines. Data were analyzed by MS, SK, and SJ. SJ drafted the figures, tables, and manuscript. BB and PS critically revised the paper and all authors approved the final version.The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fibroblasts colonization into injured areas during wound healing (WH) is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH. Neuropeptides can be produced by skin cells or be released by peripheral nerves into the skin, where they bind to respective receptors stimulating different signalling pathways and cellular responses , 2. Withα, and IL-10, as well as vascular endothelial growth factor (VEGF), while upregulating the survival pathway ERK and epidermal growth factor (EGF) expression [The first report demonstrating NT-positive fibbers in the skin was in 1983 . The intpression .In spite of these results not much has been studied regarding NT effects in fibroblasts. Therefore, in the present study we used a cell line established from the skin of normal newborn human foreskin fibroblasts. Fibroblasts are a heterogeneous population of cells of mesenchymal origin which support the development, repair, and homeostasis of their resident tissue. Dermal fibroblasts play a key role in extracellular matrix (ECM) deposition, epithelial-mesenchymal interactions, and wound healing . They alThe main aim of this work was to unravel the effect of neurotensin and its chemoattractant capacity in newborn foreskin fibroblasts, in both homeostatic and inflammatory conditions, in order to understand the possible physiological role of this neuropeptide in skin wound healing as well as speculate about the role of this neuropeptide in other skin pathologies. Escherichia coli (serotype 026:B6) was obtained from Sigma Chemical Co. , NT was obtained from Bachem , 30% Acrylamide/BisSolution 29 : 1, TEMED, and SYBR Green were obtained from Bio-Rad, and High Capacity cDNA Reverse Transcription kit was obtained from Applied Biosystems.Lipopolysaccharide (LPS) fromβ-actin were purchased from Millipore Corporation . The polyclonal antibodies against NTR1(H-130), NTR2(H-19), and NTR3(H-300) were purchased from Santa Cruz (Frilabo), references sc-15311, sc-31696, and sc-30217, respectively. The alkaline phosphatase-linked secondary antibody and the enhanced chemifluorescence (ECF) reagent were obtained from GE Healthcare . The Vectashield mounting medium was purchased from Vector Inc. , the Alexa Fluor 488 antibody was purchased from Molecular Probes , and the Alexa Fluor 555 phalloidin antibody was purchased from Invitrogen . TRIzol was obtained from Invitrogen; diethyl pyrocarbonate (DEPC) was acquired from AppliChem. Methanol, ethanol, and isopropanol were obtained from Merck. All primers were obtained from MWG Biotech . All other reagents were purchased from Sigma Chemical Co.The protease inhibitor cocktail (Complete Mini) and the phosphatase inhibitor cocktail (PhosSTOP) were obtained from Roche . Bicinchoninic acid (BCA) kit assay was obtained from Novagen. The polyvinylidene difluoride (PVDF) membranes and the antibody against The BJ cell line (ATCC number CRL-2522) was kindly supplied by Paula Marques and João Malva . This cell line was established from skin taken from normal newborn human foreskin fibroblasts. The BJ cell line has the capacity to proliferate to a maximum of 72 population doublings before the onset of senescence.μg/mL streptomycin, and 30 mM of glucose, in a humidified incubator with 5% CO2/95% air, at 37°C. BJ fibroblasts have a doubling time of about 72 h and were used after reaching 80–90% confluence, which occurred approximately every 7 days. Along the experiments, cells were monitored by microscopic observation in order to detect any morphological change.BJ cells were cultured in endotoxin-free Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) of inactivated fetal calf serum, 3.02 g/l sodium bicarbonate, 100 U/mL penicillin, 100 2 flask) were washed with ice-cold PBS and harvested in a sonication buffer. Cell lysates and protein quantification were performed as previously described [β-actin. The generated signals were analysed using the Image-Quant TL software.The cells  were plated in 60 mm dishes in a final volume of 6 mL and were treated with 10 nM of NT during 30 h, or pretreated with 10 nM of NT during 24 h, and then stimulated with 1 μg/mL of LPS during 6 h, treated for 6 h with LPS, or left untreated (control). Total RNA was isolated from these cells with TRIzol according to the manufacturer's instructions. The RNA concentration was determined by OD260 measurement using a Nanodrop spectrophotometer . RNA was stored in RNA Storage Solution  at −80°C.Cells (8 × 10μL volume containing 2.5 μL cDNA (25 ng), 10 μL 2X SYBR Green Supermix, 2 μL of each primer (250 nM), and 1 μL of H2O PCR grade. Samples were denatured at 95°C for 3 min. Subsequently, 40 cycles were run for 10 sec at 95°C for denaturation, 30 sec at the appropriate annealing temperature, and 30 sec at 72°C for elongation. Real-time RT-PCR reactions were run in duplicate for each sample on a Bio-Rad My Cycler iQ5. After amplification, a threshold was set for each gene and Ct-values were calculated as previously described [One microgram of total RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription (RT), from Applied Biosystems. Real-time RT-PCR was performed in a 20 escribed .Primers were designed using Beacon Designer Software v7.2, from Premier Biosoft International, and thoroughly tested. Primer sequences are given in 5) were cultured in 24-well plates containing circular glass lamella on the bottom. Cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde in PBS for 10 min, and then permeabilized with 0.1% Triton X-100 in PBS containing 200 mM glycine for 5 min. After a blocking step with PBS/1% BSA for 30 min, cells were incubated overnight with antibodies against NTR1, NTR2, and NTR3 (1 : 100 in PBS containing 0.1% BSA). After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody (1 : 500) and Alexa Fluor 555 phalloidin antibody (1 : 500) for 30 min at room temperature. After a washing step, cells were then incubated for 1 min with DAPI (0.1 μg/mL in PBS) and mounted with Vectashield medium to reduce photobleaching. For image acquisition, fluorescence labelling was visualized using a fluorescence microscope—Zeiss Axiovert 200—and images captured with a coupled AxioCam HR camera. In each experiment, the optimal acquisition parameters were defined for the control cells and maintained for all the other conditions within the same experiment.Cells .The expression of neurotensin receptors, namely, NTR1, NTR2, and NTR3, was determined by real-time RT-PCR, Western blot, and immunocytochemistry analyses. BJ cells constitutively express the genes for NTR1, NTR2, and NTR3, with NTR2 being the most abundant, as shown in P < 0.05, n = 3) and 0.7 ± 0.9- (n = 3) fold above control, respectively. However, the expression of NTR3 slightly diminished by 0.2 ± 0.3- (n = 3) fold, as shown in In addition, the expression of neurotensin receptors was studied under inflammatory conditions. The stimulation of BJ cells with LPS for 6 h caused an increase in NTR1 and NTR2 gene expression of 2.9 ± 1.9- .To evaluate the expression of different genes under homeostatic conditions and upon 6 h of LPS exposure, real-time RT-PCR was performed for CCL4, CCL5, CCL11, IL-8, IL-14 ± 1.1 × 104 , 2.8 × 104 ± 1.9 × 104 , and 2.5 × 104 ± 1.7 × 104 , respectively, relative to HPRT1 gene expression, while CCL4 was barely expressed, by 6.4 ± 5.0 (n = 4), relative to HPRT1 gene expression (P < 0.05), IL-1β , CCL4 , CCL11 , IL-8 , and IL-6 , relative to nonstimulated cells . After these incubation periods, total RNA was isolated, quantified, and reverse-transcribed to cDNA to finally perform real-time RT-PCR.To evaluate the effect of neurotensin on key inflammatory cytokines and chemokines, BJ cells were incubated with the neuropeptide alone, or with the inflammatory stimulus of LPS alone, or were incubated with the combination of both neurotensin and LPS . Indeed,P < 0.05, n = 3) and 0.88 ± 0.48-  fold above control, respectively, while decreased CCL4 expression by −0.59 ± 0.27-  fold below the control  fold, relative to the control, as shown in P < 0.05, n = 4), relative to cells treated with NT (10 nM). In contrast, the chemokine CCL11 showed a significant increase in expression of 0.65 ± 0.43-  fold relative to cells treated with NT (10 nM). Furthermore, EGF expression was significantly increased by 0.99 ± 0.785-  fold relative to the control (BJ cells treated with NT (10 nM) significantly increased the expression of EGF and IL-8 by 1.07 ± 0.45- , NT seemed to have the opposite effect in respect to chemokine/cytokine profile, significantly downregulating the chemoattractant CCL4 and proinflammatory cytokine IL-6. Meanwhile, NT also upregulated CCL11. Besides being a chemoattractant, CCL11 may also induce eosinophil degranulation  and IgE-Considering epidermal growth factor, a cell growth and wound healing growth factor, its expression was significantly enhanced, which is in accordance with our previous study performed in dendritic cells . In factThese results report the effect of exogenous NT under both homeostatic (without LPS) and inflammatory conditions (with LPS) in BJ cells. Under inflammatory conditions, NT was able to downregulate the chemoattractant function of fibroblasts, vital for the last phases of wound healing, which include its migration-proliferation and remodelling phases. Downregulation of chemokines triggered by exogenous NT could decrease the inflammatory status of the wound and could beneficially promote migration of fibroblasts to the wound site, with consequent expression of ECM proteins, important for skin repair. Furthermore, EGF-mediated effects on fibroblasts and keratinocytes will be fulcra for the remodelling phase of wound healing. in vitro model has limitations and treatment of fibroblasts in vitro with NT may not mimic its biological processes in their native environment, not taking into account the neuroendocrine, for example, sympathetic and parasympathetic, control of tissues. Thus, these findings should be confirmed with in vivo experiments. Meanwhile, we can hypothesize that in vivo NT administration, under similar conditions, may promote a decreased inflammatory response through these cells, which may be important in the later phases of healing. In vivo studies are needed to further confirm the potential application of NT as a therapy for diabetic foot ulcers.Indeed, delayed wounds like diabetic wound healing, characterized by a proinflammatory cytokine profile , could p


When measured by magnetic resonance imaging (MRI)-based techniques such as the proton density fat fraction (PDFF), 5% macroscopic steatosis corresponds to a PDFF of 6% to 6.4%. In contrast to “Obese/metabolic NAFLD”, NAFLD caused by genetic variants is not associated with insulin resistance. This implies that NAFLD is heterogeneous and that “Obese/Metabolic NAFLD” but not NAFLD due to the PNPLA3 or TM6SF2 genetic variants predisposes to type 2 diabetes and cardiovascular disease.Non-alcoholic fatty liver disease (NAFLD) covers a spectrum of disease ranging from simple steatosis (NAFL) to non-alcoholic steatohepatitis (NASH) and fibrosis. “Obese/Metabolic NAFLD” is closely associated with obesity and insulin resistance and therefore predisposes to type 2 diabetes and cardiovascular disease. NAFLD can also be caused by common genetic variants, the patatin-like phospholipase domain-containing 3 (PNPLA3) or the transmembrane 6 superfamily member 2 (TM6SF2). Since NAFL, irrespective of its cause, can progress to NASH and liver fibrosis, its definition is of interest. We reviewed the literature to identify data on definition of normal liver fat using liver histology and different imaging tools, and analyzed whether NAFLD caused by the gene variants is associated with insulin resistance. Histologically, normal liver fat content in liver biopsies is most commonly defined as macroscopic steatosis in less than 5% of hepatocytes. In the population-based Dallas Heart Study, the upper 95th percentile of liver fat measured by proton magnetic spectroscopy ( Non-alcoholic fatty liver disease (NAFLD) is defined as steatosis not caused by excess alcohol intake (>30 g/day in men and >20 g/day in women), hepatitis B or C, autoimmune hepatitis, iron overload, drugs or toxins . It coveSeveral longitudinal studies have shown that NAFLD increases the risk of and mortality from type 2 diabetes and cardiovascular disease . FibrosiAlthough NAFLD commonly coexists with obesity, insulin resistance and type 2 diabetes , common The ensuing review will focus on defining normal liver fat content and discussing how liver fat content is related to insulin sensitivity in “Obese/Metabolic NAFLD” and the common genetic forms of NAFLD.1H-MRS)  .1H-MRS 1H-MRS stluded 13  subjectsto 15.7%  and 13.9iver fat . 1H-MRS-< 0.001) . These det al. [et al. which uses fast gradient echo techniques [r = 0.77, p < 0.001). The newer MRI-determined proton density fat fraction (PDFF) technique provides a quantitative, standardized and objective MRI measurement of hepatic fat based upon inherent tissue properties [1H-MRS (r = 0.99) [p < 0.0001) [Hepatic steatosis can be diagnosed with MRI using an out-of-phase and in-phase imaging technique developed by Dixon WT et al. . This meet al. ,36,37. Oet al. . Modifiechniques ,39 and coperties ,41. The  = 0.99) ,42 and l 0.0001) ,43,44. W 0.0001) ,46.1H-MRS-measured liver fat content greater than 12.5%. A meta-analysis of 44 studies comprising 4720 subjects concluded that US has a sensitivity of 85% and a specificity of 94% for detecting 20%–30% macroscopic steatosis [Ultrasound (US) is an inexpensive and widely available tool to visualize the liver and its fat content. Hepatic steatosis appears as a diffuse increase in parenchymal brightness and echogenicity on US images, and is often compared to hypoechogenity of the kidney cortex. Most studies score steatosis semiquantitatively as “mild”, “moderate” and “severe” based upon the visual assessment of hepatic echogenicity ,47,48,49teatosis . The senet al. created an equation for accurate quantification of liver fat content using US in Chinese subjects [1H-MRS. The adjusted R2 for the model was 80%. The optimal cut-off for the US-measured liver fat content to diagnose hepatic steatosis was 9.15%, which yielded a sensitivity and specificity of 95% and 100%, respectively. The utility of this technique in other ethnic groups which are more obese than the Chinese in the face of a similar amount of liver fat [Xia MF subjects . A tissuiver fat ,56 remaiHepatic steatosis can also be assessed by using computed tomography (CT) by comparing attenuation of the liver parenchyma to that of the spleen . Tissue i.e., in “Obese/Metabolic NAFLD”, liver fat is closely correlated with direct measures of insulin resistance such as the inability of insulin to suppress hepatic glucose production [In subjects with NAFLD and the metabolic syndrome (MetS), oduction , and indoduction . Indeed,oduction ,64. ThisLipolysis is the main source of fatty acids used for synthesis of intrahepatocellular triglycerides ,66. LiveAny obese person with NAFLD and features of the MetS can be considered to have “Obese/Metabolic NAFLD” irrespective of genetic risk factors. The most recent proposal defines the MetS in 10 different ways . The preApproximately 30% of Europids and several other ethnic groups carry the PNPLA3 I148M variant . The assIn vitro, the PNPLA3 I148M gene variant abolishes intrahepatocellular lipolysis [ipolysis ,77 and bipolysis . The conApproximately 7% of all subjects carry the TM6SF2 E167K variant. This gene variant increases the risk of NAFLD, independent of genetic variation in PNPLA3 at rs738409, obesity and alcohol intake . A recenin vitro studies have examined the mechanism by which the TM6SF2 E167K gene variant could increase liver fat. Recombinant adeno-associated viral vectors expressing short hairpin RNAs were used to reduce Tm6sf2 transcripts in the mouse liver, which increased hepatic triglyceride content three-fold [Four ree-fold . TM6SF2 ree-fold . In anotree-fold . Overexpree-fold . Transieree-fold . In the ree-fold .1H-MRS, MRI, histology or US [gy or US ,99,100 tgy or US ,98,100 ogy or US ,97,99 bugy or US  in TM6SF3 I148M or TM6SF2 E167K gene variants. A total of 124 matched were found. Of these, 22 studies were informative with respect to liver fat content and insulin resistance between genotypes, and were thus included.We performed a systematic search using PubMed and Medline on two topics. For definitions of normal liver fat, we used the following search terms and their combinations: “normal liver fat”, “liver histology”, “liver biopsy” and “liver triglycerides”, “liver H-MRS”, “liver MRI”, “liver MRI-PDFF”, “liver CT”, “liver ultrasound” and received 526 matches. Thirty-three studies included data on normal liver fat content or compared liver fat measured using different techniques. To review the association between insulin resistance and genetic NAFLD, we searched for studies using the following search terms: “PNPLA3” or “TM6SF2” and “insulin resistance”, “euglycemic (hyperinsulinemic) clamp”, “fasting glucose”, “fasting insulin”, “HOMA-IR”, “oral glucose tolerance test” and included studies which compared results between carriers and non-carriers of PNPLA1H-MRS, normal liver fat in the population-based DHS was defined as less or equal than 5.56% [Normal liver fat content based on liver histology can be defined as macroscopic steatosis in less than 5% of hepatocytes. With an 5.56% , which can 5.56% ,32. DefiAlthough NAFLD has often been regarded simply as the hepatic manifestation of the MetS, it is now clear that NAFLD is heterogeneous. While “Obese/Metabolic NAFLD” is associated with NAFLD and features of the MetS and an increased risk of type 2 diabetes and cardiovascular disease, NAFLD caused by I148M variant in PNPLA3 and the E167K variant in TM6SF2 is not accompanied by insulin resistance. Thus, lack of insulin resistance does not exclude NAFLD and not all patients with NAFLD are at increased risk of type 2 diabetes and cardiovascular disease. Given that both the MetS and the genetic variants in PNPLA3 and TM6SF2 are common, there are also many individuals with “double trouble NAFLD” [Although NAFL defined as macroscopic steatosis affecting >5% of hepatocytes predicts fibrosis ,8,9, it


VT), ARF patients showed 30.0% and 27.8% decrease at 1 min and 10 min, respectively. Furthermore, the initial respiratory system compliance (Crs) decreased by 29.6% and 28.5% at 1 min and 10 min, respectively. Under VCV mode, compared with the initial level, 38.6% and 37.5% increase in peak airway pressure (PAP) were found at 1 min and 10 min, respectively. Under PCV mode, the initial PaO2 increased by 6.4% and 10.2 % at 3 min and 10 min, respectively, while 18.9% and 30.6% increase of the initial PaO2 were observed under VCV mode. Summarily, endotracheal suctioning may impair gas exchange and decrease lung compliance in ARF patients receiving mechanical ventilation under both PCV and VCV modes, but endotracheal suctioning effects on gas exchange were more severe and longer-lasting under PCV mode than VCV.This study was conducted to evaluate the effects of open endotracheal suctioning on gas exchange and respiratory mechanics in ARF patients under the modes of PCV or VCV. Ninety-six ARF patients were treated with open endotracheal suctioning and their variations in respiratory mechanics and gas exchange after the suctions were compared. Under PCV mode, compared with the initial level of tidal volume ( Respiratory failure is an acute or chronic condition with impaired gas exchange and pulmonary functions and is characterized by elevated carbon dioxide or decreased oxygen in the arterial blood . RespiraDue to establishment of artificial airway, most acute respiratory failure (ARF) patients receiving mechanical ventilation could not produce sputum. Suctioning could effectively eliminate airway secretions to maintain respiratory function . EndotraThe Ethics Committee of the First Affiliated Hospital of China Medical University approved this study. Written informed consent was acquired from all subjects using the procedures authorized by institutional review boards. Next of kin, caretakers, or guardians consented representing the study participants whose capacity to consent was reduced. The study was executed at the emergency intensive care units (EICU) at the First Affiliated Hospital of China Medical University.2 < 60 mmHg, PaCO2 < 50 mmHg; (2) type II: PaO2 < 60 mmHg, PaCO2 ≥ 50 mmHg. All patients received endotracheal intubation and continuous mechanical ventilation , continuous monitoring of heart rate (HR), blood pressure (BP), and SpO2 with Philips Intellivue MP60 monitor .This was a single center prospective study of acute respiratory failure (ARF) patients admitted to the EICU between October 2010 and February 2013. Patients met the following criteria to be enrolled in this study; patients lived up to the criteria for the diagnosis of ARF: (1) type I: PaOP > 0.05).In the present study, ninety-six acute respiratory failure (ARF) patients  were enrolled, with a mean age of 62 years (range: 26~84 years). They were divided into two groups: PCV and VCV. PCV group included 49 patients , with a mean age of (57 ± 21) years. And they were with an APACHE II score of (17.6 ± 10.3), a disease course of (4.6 ± 2.0) years. There were 15 patients who had complications, such as compensatory chronic respiratory failure, acidosis, pulmonary encephalopathy, and gastrointestinal bleeding. Compared with PCV, VCV group had 47 patients , with a mean age of (58 ± 26) years. Their APACHE II score was (17.2 ± 10.1) and disease course was (5.1 ± 2.2) years. Twenty among them had complications, including compensatory chronic respiratory failure, acidosis, pulmonary encephalopathy, and gastrointestinal bleeding. As shown in 2O (0.98 kPa) plateau airway pressure (Pplat) by increasing tidal volume (VT) under VCV mode and 10 cm H2O inspiratory pressure under PCV mode, maintaining ventilation and oxygenation for 20 s. According to the clinical need for suctioning, suction indications include visible secretions attached within the patient's endotracheal tube, auscultation of lung breath weak sound and sputum sound, patients with irritability and other poor performances, changes, or abnormities in breathing rate, slowness of heart rate, increase in blood pressure, reduction of ventilation, and detection of SpO2 less than 90%. Endotracheal suctioning was performed when alarm for peak airway pressure (Ppeak) under VCV mode, low tidal volume under PCV, or SpO2 decreased more than 5%. During endotracheal suctioning, endotracheal tube departed from Y-shaped tube. The 12F suction catheter (4 mm exterior diameter) was inserted into endotracheal tube about 30 cm after blocking negative pressure suction and then slowly rotated and the suction catheter was withdrawn at 150 mmHg. Endotracheal tube was connected with ventilator tube for mechanical ventilation after suctioning.The 96 ARF patients were randomly divided into the PCV and VCV groups using a random number generator. Diazepam (10 mg) was injected intravenously before suctioning. A standardized method was used for a standardized lung volume method after 1 h of mechanical ventilation: (1) sufficient suctioning for mouth, nasal, and airway secretions; (2) 10 cm HVT, Ppeak, and Pplat were recorded at 15 min after standardized lung volume (baseline), before suctioning, and 1, 3, 5, and 10 min after suctioning. Total respiratory system compliance (Crs) was reckoned by [Crs = VT/(Ppeak − PEEP(5–8 cm H2O))]. Values of VT, Ppeak, and Pplat were the average of five respiratory cycles. Dynamic changes in SpO2 were monitored. Arterial blood gas analysis was performed at baseline, before suctioning, and at 3 and 10 min after suctioning.The HR, mean arterial pressure (MAP), χ2 test was used to compare frequencies. One-way analysis of variance (ANOVA) and Student's t-test were applied to generally distributed variables, while the Mann-Whitney U test was applied to nonnormal distributed variables. Comparisons between two groups for nominal variables were made by the Fisher exact test. All of the statistical significance tests were two-sided, with a P value less than 0.05 considered as statistically significant. All the statistical analysis was performed by the use of SPSS 18.0 software .Data was presented as mean ± standard deviation (SD), median with interquartile ranges (IQR), or frequencies. A −1, P < 0.05; MAP: 91 ± 14 versus 82 ± 12 mmHg, P < 0.05, resp.). Under VCV mode, the HR and MAP increased by 10.4% and 11.1%, respectively, at 5 min after suctioning compared with the baseline level . No significant differences were found in changes in HR and MAP at 10 min after suctioning compared with the baseline level under both PCV and VCV modes . Furthermore, there were also no significant differences in HR and MAP between PCV and VCV modes at different time points after suctioning .Changes in heart rate (HR) and mean arterial pressure (MAP) before and after suctioning at different time points under PCV and VCV modes are shown in VT), Crs, and airway pressure in ARF patients before and after suctioning at different time points are shown in VT and Crs showed a decrease of 30.0% and 29.6%, respectively, at 1 min after suctioning, compared with the baseline level  . No significant differences were found for changes in the VT and Crs at 10 min after suctioning, compared with baseline level .Changes in tidal volume (Ppeak) and plateau pressure (Pplat) showed an increase of 38.6% and 17.2%, respectively, at 1 min after suctioning, compared with the baseline level , while Crs decreased by 32.5%  , while Crs decreased by 31.7% . Compared with the level before suction, t changes in the Ppeak and Crs at 10 min after suctioning also did not represent notable differences .Under VCV mode, peak airway pressure  . Under VCV mode, compared with the level before suction, PaO2 also showed an increase of 18.9% and 30.6% at 3 and 10 min, respectively, after suctioning . There was no obvious difference in PaO2 between the baseline level and the level at 10 min after suctioning . We have demonstrated that under PCV mode PaO2 had a mean increase (3 min: (6.4 ± 2.6) %; 10 min: (10.2 ± 6.2) %) for ARF patients after suctioning compared with before suctioning, and the increased ratio was lower than under VCV mode .Under PCV mode, compared with the initial level before suctioning, PaO, resp.) . HoweverMechanical ventilation mechanically assists or replaces spontaneous breathing . In geneVT, ARF patients showed a 30.0% and 27.8% decrease at 1 min and 10 min, respectively, after suctions under PCV mode. Alveolar collapse under PCV mode could lead to an increased respiratory resistance when the inspiratory pressure was stabilized on a fixed point, thereby causing VT to reduce [Crs decreased by 29.6% and 28.5% at 1 min and 10 min, respectively, after suctions under PCV mode. The major causes of suctioning effects on respiratory mechanics include alveolar collapse, lung volume, and compliance decrease [Pplat and a 32.5% and 31.7% decrease of Crs at 1 min and 10 min, respectively, after suctions under VCV mode. Given that VT under VCV mode was not changed, alveolar collapse would lead airway pressure to increase accordingly and overexpansion of the open alveolar may also cause increase in Pplat.In the present study, we evaluated endotracheal suctioning effects on gas exchange and respiratory mechanics in 96 ARF patients under PCV and VCV modes. Our results indicated that endotracheal suctioning under PCV and VCV modes impairs both gas exchange and respiratory mechanics in ARF patients receiving mechanical ventilation. Compared with the initial level of o reduce . Furtherdecrease . Under VPpeak under VCV mode and a decreased VT under PCV mode. In addition, our results also suggested that the initial PaO2 increased by 6.4% and 10.2% at 3 min and 10 min, respectively, after suctions under PCV mode, while 18.9% increase and 30.6% increase of the initial PaO2 were observed under VCV mode. There was a significant difference in the increment of the initial PaO2 between PCV and VCV modes. Our results are consistent with multiple previous studies [During mechanical ventilation, the retention of airway mucus causes increased airway resistance, which is manifested as increased airway pressure . However studies –25.In conclusion, our study provides a comprehensive and reliable evidence that endotracheal suctioning of ARF patients receiving mechanical ventilation may impair gas exchange and decrease lung compliance, under both PCV and VCV modes, but the effects of endotracheal suctioning on gas exchange were more severe and longer-lasting under PCV mode, in comparison to VCV mode.


Candida bombicola ATCC 22214, its characterization and potential applications in enhancing oil recovery were studied at laboratory scale. The seed media and the production media were standardized for optimal growth and biosurfactant production. The production media were tested with different carbon sources: glucose (2%w/v) and corn oil (10%v/v) added separately or concurrently. The samples were collected at 24 h interval up to 120 h and checked for growth (OD660), and biosurfactant production [surface tension (ST) and interfacial tension (IFT)]. The medium with both glucose and corn oil gave better biosurfactant production and reduced both ST and IFT to 28.56 + 0.42mN/m and 2.13 + 0.09mN/m, respectively within 72 h. The produced biosurfactant was quite stable at 13–15% salinity, pH range of 2–12, and at temperature up to 100°C. It also produced stable emulsions (%E24) with different hydrocarbons . The produced biosurfactant was extracted using ethyl acetate and characterized as a mixture of sophorolipids (SPLs). The potential of SPLs in enhancing oil recovery was tested using core-flooding experiments under reservoir conditions, where additional 27.27% of residual oil (Sor) was recovered. This confirmed the potential of SPLs for applications in microbial enhanced oil recovery.Biosurfactant production using  Surfactants are amphiphilic molecules consisting of both hydrophilic and hydrophobic domains that partition preferentially at the interface between fluid phase with different degree of polarity and hydrogen bonding (such as oil and water or air and water). Surfactants intercede in nearly every product and aspect of day to day life, with a total world production exceeding 13 million tons per year . Nearly Candida bombicola . C. bombicola was isolated from bumblebee honey , matrix-assisted laser desorption ionization – time of flight-mass spectroscopy (MALDI-TOF-MS) and nuclear magnetic resonance (NMR) spectroscopy (1H and 13C NMR). The stability of the biosurfactant under extreme conditions of pH, temperature and salinity was also investigated and finally we tested its efficacy in enhancing oil recovery using core-flood experiments.In this paper we investigated the effect of carbohydrate and oil based media on biosurfactant production by All of the experimental data were expressed in terms of arithmetic averages obtained from at least three independent replicates, with standard deviation (±).All chemicals and reagents used were of analytical grade, media constituents for microbial studies were of microbiology grade, and the hydrocarbons had minimum of 99% purity. All chemicals, reagents and hydrocarbons were purchased from Sigma–Aldrich Co. LLC. Corn oil was purchased from local market. Light and heavy crude oil were kindly provided by local oil company, Petroleum Development Oman (PDO).C. bombicola ATCC22214 was sub- cultured from the stock culture on freshly prepared potato dextrose agar slants (PDA), composition (g/l): potato extract, 4.0; dextrose, 20.0; agar, 15.0) and glucose yeast peptone agar slants (GYPA), composition (g/l): glucose, 100; yeast extract, 30; peptone, 50; agar, 20; and incubated at (25°C) for 48 h. The agar slants were preserved at 5°C.The yeast Two types of seed media were used for developing the seed culture: potato dextrose broth (PDB) and glucose-yeast extract-urea broth (GYUB), composition (g/l): glucose, 50; yeast extract, 5; urea, 0.5. Seed media were prepared and distributed 50 ml each in 250 ml Erlenmeyer flasks and autoclaved at 121°C and 15 psi for 15 min. Seed media flasks were inoculated with a loop full of the microorganism freshly grown on GYPA or PDA agar slant, and incubated for 48 h at 25°C; 160 rpm in a rotary incubator shaker.660), pH, ST, and IFT.Three different production media were tested containing (g/l): yeast extract, 10; urea, 1; and different carbon sources. The pH was adjusted to 4.0 using 1M HCl, before autoclaving. Glucose and/or corn oil was used as carbon sources, as: only glucose (2% w/v), only corn oil (10% v/v) or both glucose (2% w/v) and corn oil (10% v/v). Glucose and corn oil were autoclaved separately and added to pre-sterilized production media. The flasks were inoculated with 2% (v/v) pre-grown seed media (GYUB) and incubated in a rotary shaker at 25°C, 160 rpm for 120 h. Samples were withdrawn every 24 h for different analysis: growth  was used to measure ST and IFT. The IFT was measured against hexadecane as the embedding phase as reported by C. bombicola was studied under various environmental parameters including wide range of temperature, pH and different salt (NaCl) concentrations. The study was carried out by changing the levels of one parameter while keeping other parameters constant using cell-free biosurfactant broth centrifuged at 10,000 × g for 15 min. ST and IFT were measured after each treatment to test their effect on biosurfactant activity.The stability of biosurfactant produced by For the temperature stability test, 25 ml of the cell-free broth were added in 50 ml glass tubes and tightly closed with stopper to prevent evaporation. The tubes were incubated at different temperatures  each for 30 min, cooled down to room temperature and the biosurfactant activity was measured.Different concentrations of salt (NaCl) were added  into 25 ml cell-free broth, dissolved completely and the effect on biosurfactant activity was measured.For the pH stability study, 25 ml of cell-free broth was adjusted using either 1N HCl or 1N NaOH to desired values  and the effect on biosurfactant activity was measured.24) was analyzed using previously reported procedures . The solution was mixed by vortexing for 2–3 min at high speed and was left to stand for 24 h. After 24 h the height of the emulsion was measured and the emulsification index was given as the percentage of the height of the emulsified layer divided by the total height of the liquid column multiplied by 100.The emulsification index  to obtain cell-free broth. The cell-free broth (1L) was extracted twice with an equal volume of ethyl acetate, shaken vigorously in a separation funnel. The bottom aqueous layer and the top ethyl acetate layers were collected separately. The aqueous portion was re-extracted further twice with ethyl acetate. Ethyl acetate extracts were combined and the solvent was evaporated under vacuum using Rotavapor  to give crude biosurfactant along with residual oil. The residual oil was removed by washing thrice with n-hexane. Crude biosurfactant was recovered by evaporating the n-hexane  spotting device . The plates were developed using an automatic developing chamber ADC 2  with remote operation from winCATS software, containing solvent systems – MP1: Chloroform: Methanol: Acetic acid (95:5:5) and MP2: Chloroform: Methanol: Water (65:15:2). The documentation and evaluation of the TLC plate was done using TLC visualizer (CAMAG) under direct UV 254 and UV 366 nm light, capturing the images. The separated bands were extracted and eluted by TLC-MS interface , based on the coordinates determined by the TLC scanner 4. The TLC-MS interface head  was connected to the pump  and the extraction was performed at a flow rate of 10 μl/min, with methanol: acetonitrile (50% diluted with water) – 1:10. The interface outlet was directly connected with the ESI – MS , using Mass Lynx V4.0 software. The experimental conditions were: capillary voltage, 3.0 kV; cone voltage, 35 V; lens voltage, 0.0 V; source block temperature, 100°C; desolvation temperature, 120°C; analyzed under both positive and negative modes.Biosurfactant was separated and analyzed using an automated HPTLC system  in the Central Analytical and Applied Research Unit (CAARU), Sultan Qaboos University, Oman. Twenty five microliter samples were spotted onto a 10 cm × 10 cm pre-coated silica gel HPTLC plate  containing green fluorescent FAll MALDI-TOF experiments were performed at CAARU, Sultan Qaboos University, on UltraFlextreme  operating in positive reflectron mode in the m/z range of 50–2000 Da. Stainless steel MTP 384 target plate was used for all the molecular weight analysis. Dihydroxy benzoic acid (DHB) dried droplet protocol described in Bruker manual was employed for sample preparation and spotting. Two micro liter of 2, 5-DHB matrix (20 mg/ml) in TA 30 (30:70 v/v ACN:TFA 0.1%TFA) was premixed with 2 μl of the sample solution. One micro liter of the mixture was applied to the ground steel target plate, dried at room temperature. The spectra were acquired using FlexControl software v3.3 . A SmartBeam-II laser, set at a frequency of 1000 Hz, was used for ionization. The laser strength was optimized at 25–35%. A summed spectrum was obtained for each MALDI-spot. Peaks were detected using the SNAP peak detection algorithm and a baseline subtraction was carried out using “TopHat” algorithm. The MALDI-TOF spectra were externally calibrated using a commercially available peptide mix . FlexAnalysis Software v3.3  was used for visualization and initial data processing.1H) NMR experiment was run using zg30 pulse program operating at 176.08 MHz. Acquisition parameters were as follows: 90° proton pulse width of 8.00 μs, relaxation delay of 1 s, 16 scans. The proton decoupled 13C NMR experiments were carried out using composite pulse decoupling scheme operating at 176.08 MHz. Acquisition parameters were as follows: 90° proton pulse width of 8.00 μs, relaxation delay of 2 s, 512 scans. The Spectra were recorded in CDCl3 at 298K and processed using TOPSPIN 3.2 software.The NMR experiments were carried out in Bruker Avance III HD 700 MHz spectrometer equipped with 5 mm TCI H/C/N cryoprobe. The proton (3): Sodium, 25.083; Calcium, 3.762; Magnesium, 0.878; Iron, 0.045; Chloride, 47.722; Sulfate, 0.247; Bicarbonate, 0.079. Formation water was filtered prior to use, by Millipore Membrane Filtration Unit (0.45 μm). The crude oil used for core-flood experiments was of API 36.51°. For all core-flooding experiments, cleaned Berea cores were saturated with filtered formation brine using vacuum desiccators for 24 h and pore volume (PV) was determined using the dry and wet weights of the cores. The cores were then flooded with crude oil at 24 cm3/h until no more water was produced. The oil initially in place (OIIP) was determined, which was indicated by the volume of water displaced. The cores were subjected to water-flooding at 24 cm3/h until no further oil was produced. The residual oil was calculated by measuring the amount of oil produced from the water-flood. Then, 5 PV of the cell-free supernatant (biosurfactant broth) was injected as a tertiary recovery stage and extra oil recovery was determined (Berea sandstone cores (1.5 inch diameter × 3 inch long) were used for core-flooding experiments. The average porosity and permeability of Berea core-plugs were 20% and 250–350 mD respectively. The formation water and crude oil used in these experiments were obtained from an Omani oil field of interest which has an average reservoir temperature of 60°C. The salinity of the formation water was between 7 and 9% its chemical composition was  and hence selected as a seed medium for inoculum preparation. To study the biosurfactant production, two different carbon sources (glucose and corn oil) were tested either alone or as a mixture of both. Growth (OD660) and biosurfactant production (ST and IFT) were studied from all three different production media up to 120 h. In glucose-based production medium both ST and IFT decreased after 72 h, and reached to 33.08 ±0.05 mN/m and 1.63 ±0.29 mN/m from 54.66 ±0.26 mN/m and 22.84 ±0.36 mN/m respectively, and remained almost stable up to 120 h . Maximum growth was observed at 96 h (OD660 = 2.508) and didn’t increase further till 120 h . However no change in pH was observed until 120 h . In corn oil-based production medium maximum reduction in ST and IFT was observed after 96 h, and reached to 30.80 ±0.19 mN/m and 4.26 ±0.54 mN/m from 57.81 ±0.79 mN/m and 25.26 ±0.94 mN/m respectively, and remained almost stable up to 120 h . Maximum growth was observed at 96 h (OD660 = 2.556) and decreased thereafter . The pH decreased after 72 h and the medium became acidic . In production medium containing ‘both glucose and corn oil’ as carbon sources, maximum reductions in ST and IFT were observed at 48 h and after 72 h reached to 28.56 ± 0.42 mN/m and 2.13 ± 0.09 mN/m from 53.69 ± 0.36 mN/m and 23.37 ± 0.41 mN/m respectively. The IFT was further reduced to 1.23 ± 0.02 mN/m after 96 h . Maximum growth was observed at 72 h (OD660 = 3.372) and decreased thereafter . The pH remained acidic throughout the experiments .Two different media GYUB and PDB were tested as seed media of which GYUB supported better growth , reduction in ST and IFT were observed earlier as compared to production media containing either glucose or corn oil alone. At 48 h ST and IFT were reduced to 36.25 ±0.43 and 5.43 ±0.36 mN/m, respectively in medium containing ‘both glucose and corn oil.’ Whereas, in medium containing ‘either glucose’ or ‘only corn oil,’ STs and IFTs were reduced to ∼46.51 ±0.33 and ∼18.66 ±0.15 mN/m, respectively. Other researchers also reported that the highest production of SPLs was achieved when glucose was combined with a lipophilic carbon source . SPLs fe-alkanes . However-alkanes , lactose-alkanes , fructos-alkanes . The rep-alkanes , vegetab-alkanes  or anima-alkanes . Continu-alkanes  or fatty-alkanes , has bee and Na+ , 1997. Wex situ microbial enhanced oil recovery (MEOR) applications, biosurfactant must be stable at range of high temperature (≥50°C), effective at wide range of pH and salt concentration between 7 and 9% to ensure its applicability to induce oil recovery . Thermal stability profile  reveals that at different temperatures (40–100°C) the biosurfactant showed stability at all temperatures tested. These results indicated that the produced biosurfactant is suitable for MEOR applications as it was stable under extreme condition such as salinity, pH and temperature. This study showed that biosurfactants was quite stable at most salt concentration tested (13–15%), over a wide range of pH values tested (2–12), and at different temperatures (40–100°C). It is comparable with other types of biosurfactants reported by several researchers  to be used for enhanced oil recovery (EOR) purpose. For recovery . As showearchers .Figure 3, biosurfactant emulsified a variety of hydrocarbons. It gave high %E24 with heavy crude oil (68.75%), and with n-hexadecane, light crude oil, n-tetradecane, n-pentane, n-tridecane and 2,2,4,4,6,8-heptamethylnonane also formed stable emulsions, with %E24 between 34 and 35%. Whereas it showed lower %E24 (23.86–29.55%) against hexane, 1-methylnaphthalene and heptane. Higher %E24 with ‘heavy-crude oil’ can potentially help in heavy-oil recovery. Bacillus subtilis B30 in minimal medium or molasses medium, which showed around 15–55% emulsification of various hydrocarbons including light or heavy oils. 24 of novel Bacillus strain 139SI as 69% with crude oil, thus aiding in bioremediation of oil polluted water. We also observed similar results with biosurfactant produced by C. bombicola.Emulsification is one of the features of biosurfactant which aids in enhancing the trapped oil from the oil wells. As shown in The biosurfactant was extracted and purified by solvent extraction with ethyl acetate followed by washing with hexane which resulted in 2.42 g/l of crude product as yellowish brown powder , and the extracted bands showed mass in the range of 300–795 . It is reported that typical structure of biosurfactant produced by C. bombicola – SPLs consists of a sophorose sugar β-glycosidically linked to terminal  hydroxylated fatty acid with chain lengths of 16–18, as a mixture of compounds. Where precursor fatty acids added to the production medium leads to formation of different forms of SPLs, which contains both acid and lactone SPLs, but lactones frequently represent the largest fraction of the product  were detected . Where major ions at m/z 711 and m/z 729 can be attributed to the [M+ + Na+] adduct ions for the lactone and free acid forms of the major diacetylated SPLs respectively  can be attributed to the mass difference between the free acid form and the ester-linked lactone form of SPLs respectively. Thus the mass of produced SPLs can be calculated as from ∼646 to 706. Starmerella (Candida) bombicola yeast clade, using high-throughput MALDI-TOF-MS analysis. They reported SPLs production using oleic acid as fatty acid precursor along with other components in the production medium, and thus observed only oleic acid containing SPLs. Whereas we have used corn oil as fatty acid precursor and observed other minor SPLs derived from other types of acids present in corn oil, as sodium adducts [M+ + Na+] derived from palmitic , linoleic , and trace amounts of stearic (m/z 713) and arachidic (m/z 759) acid . The mass difference (18 Da) between the two sets of ions derived from palmitic and linoleic acids are again indicative of the free acid and lactone forms of the minor SPLs respectively. Similar type of SPLs ions were identified and reported previously from C. bombicola and other yeast species using fast atom bombardment MS or MALDI-TOF MS . Multiple signals of protons of (-CH3) below 1.0 ppm and of (-CH2) between 1.0 and 1.4 ppm revealed the existence of a linear alkane, and signals at 5.3–5.4 ppm revealed presence of (-CH = CH-) group in the fatty acid chain. The signal at 5.3–5.4 ppm is attributable to an unsaturated hydrocarbon moiety, also consistent with the oleic acid derived SPLs signatures in MALDI-TOF-MS analysis. Protons of (-CH2) bonded to carboxylic group of fatty acid resonated at ∼1.99 ppm, and ∼2.09 ppm revealed the presence of (-COCH3) group in biosurfactant. Resonance of protons belonging to sophorose moiety resulted in peaks within the region 4–4.5 ppm, and the other protons of sugar were resonated at 3.18–3.8 ppm.The structures of biosurfactants were further confirmed by 13C NMR spectrum of the biosurfactant showed the presence of two (=CH-) groups in the fatty acid chain moiety corresponding to signals at 130.41 ppm and 128.99 ppm; and few more (=CH-) groups in the fatty chain moiety were resonated between 128 and 130 ppm, which may be probably due to contribution from other interfering group of biosurfactant in the sample . In addition, several (-CH2-) groups in the fatty chain moiety were also resonated at 20∼35 ppm. The spectrum also revealed signals of glucose- C-1″ at 101.24 ppm, glucose-C-6′, and glucose-C-6″ at 64.08, 62.29 ppm; the other carbon atoms of glucose were resonated between 70∼78 ppm. The peaks derived from the carbonyl groups (-CO-) were shown at 170∼177 ppm. The chemicals shifts were comparable to previous reports  was calculated to be 55–60% after oil flooding. It was found that after injecting 5–8 PV of formation water, no more oil was produced and residual oil saturation (Sor) was about 20%. Extra oil recovery was observed after injecting 4–5 PV SPLs, where 27.27% of Sor was produced . An increase in oil recovery in ex situ MEOR experiments was detected following SPLs injection. This is similar to reports shown by other researchers for different types of biosurfactants  or used waste frying oil (as fatty acid supplement) be used as raw material for SPLs production at large scale fermentation  trapped between the pores of Berea sandstone cores, which highlight the potential applications of SPLs in MEOR.This study shows that corn oil could be used as a carbon source along with glucose to produce SPLs by the yeast 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.


Starch from maize kernels has diverse applications in human and animal diets and in industry and manufacturing. To meet the demands of these applications, starch quantity and quality need improvement, which requires a clear understanding of the functional mechanisms involved in starch biosynthesis and accumulation. In this study, a recombinant inbred line (RIL) population was developed from a cross between inbred lines CI7 and K22. The RIL population, along with both parents, was grown in three environments, and then genotyped using the MaizeSNP50 BeadChip and phenotyped to dissect the genetic architecture of starch content in maize kernels.qSTA9-1) to 10.6 % (qSTA4-1) of the starch variation. The QTL interval was further reduced using the bin-map method, with the physical distance of a single bin at the QTL peak ranging from 81.7 kb to 2.2 Mb. Based on the functional annotations and prior knowledge of the genes in the top bin, seven genes were considered as potential candidate genes for the identified QTLs. Three of the genes encode enzymes in non-starch metabolism but may indirectly affect starch biosynthesis, and four genes may act as regulators of starch biosynthesis.Based on the genetic linkage map constructed using 2,386 bins as markers, six quantitative trait loci (QTLs) for starch content in maize kernels were detected in the CI7/K22 RIL population. Each QTL accounted for 4.7 %  contains supplementary material, which is available to authorized users. Maize is a leading crop worldwide because of its diverse functions as a source for human food and animal feed and as a raw material for industry and manufacturing. With a growing world population and need for biofuel, increasing maize grain yield is necessary to meet the market demand. Starch is the major component of maize kernels, accounting for 70 % of the kernel weight. In addition, starch is increasingly used as a renewable chemical feedstock for the conversion of other products, such as high fructose corn syrup, polymer-based fibers and fuel ethanol . Therefod-glucose, amylose and amylopectin, in the maize endosperm. Although starch metabolism is complex, it is clear that four classes of enzymes, adenosine diphosphate glucose pyrophosphorylases (AGPases), starch synthases (SSs), starch branching enzymes (SBEs) and debranching enzymes (DBEs), play critical roles in starch biosynthesis. Maize mutants have been used to isolate genes encoding key enzymes in starch metabolism, such as Shrunken1 (sh1), Shrunken2 (sh2), Brittle2 (bt2), agpsemzm, agpllzm, Waxy1 (wx1), SS1, Sugary2 (su2), Dull1 (du1), SS2b-2, SS2c, SS3b-1, SS3b-2, SS4, SBEIa, SBEIIa, Amylose extender1 (ae1) and Sugary1 (su1) [sh1 gene encodes the major isoform of sucrose synthase and provides an important link in sucrose-starch conversion reactions, as sucrose synthase catalyzes the reversible reaction between sucrose and uridine diphosphate-glucose [sh2, bt2, agpsemzm and agpllzm encode the large or small subunits of AGPase, which converts glucose-1-phosphate to ADP-glucose, the precursor for starch synthesis [wx1, encoding granule-bound SS I, is solely responsible for amylose production, whereas SS1, Su2, Du1, SS2b-2, SS2c, SS3b-1, SS3b-2 and SS4, encoding four types of soluble SS, are responsible for amylopectin production [SBEIa, SBEIIa and ae1 encode the SBE isoforms Ia, IIa and IIb, respectively [su1 encodes a DBE of the isoamylase type, and mutant su1 kernels contain the highly branched, water-soluble phytoglycogen and constitute the original sweet corns [Starch is deposited as water-insoluble semicrystalline granules, which are chemically comprised of two homopolymers of α-ynthesis –7. wx1, oduction –13. SBEIectively –16, whicet corns . These a3 population, which was derived from a cross of two lines divergently selected for protein content after 76 generations in the Illinois long-term selection experiment [QTL mapping is a classical method for identifying loci for quantitative traits of interest without prior genetic knowledge. A variety of QTLs for the starch content in maize kernels have been identified in different biparental populations since the first study in the Illinois High Protein × Illinois Low Protein Fperiment –32. Amonperiment . This inQTL mapping is firstly suggestive to identify loci for complex quantitative traits, although, the resolution is rather low, often ranging from 10 to 30 cM . IncreasIn this study, a maize CI7/K22 recombinant inbred line (RIL) population was developed and genotyped using the Illumina MaizeSNP50 BeadChip, which contains 56,110 SNPs. The kernel starch contents of this RIL and the parental lines were evaluated after being grown in three environments. The objectives were to (1) construct a high-density genetic linkage map using the inferred bins as markers, (2) dissect the genetic architecture of starch content in maize kernels of the CI7/K22 RIL population, (3) narrow down the position of the identified QTLs using the SNP bin map and (4) mine the candidate genes associated with starch content in the refined QTL interval.P = 0.09). There were moderately positive starch content correlations among the three environments, with correlation coefficients ranging from 0.57 to 0.66  estimate of the starch content was high (82.1 %), indicating that much of the phenotypic starch content variation in the RIL population was genetically determined.The low-starch inbred line CI7 has ~0.1 %, 3.5 % and 7.1 % lower starch content values than K22 in Beijing in 2013, Hainan in 2013 and Neimeng in 2014, respectively. Taken together, no significant difference was observed in the starch content between the two parents, CI7 and K22 , were polymorphic between the two parents. The missing rate of these SNPs ranged from 0 to 15.31 %, with an average of 1.18 %, the heterozygosity ranged from 0 to 15.64 %, with an average of 3.69 % and the minor allele frequency ranged from 0.27 to 0.50, with an average of 0.45 in the CI7/K22 RIL population  to 10.6 % (qSTA4-1). Alleles from K22, the high-starch parent, at all of the mapped loci except qSTA4-1, had increasing effects on the starch content. The largest QTL, qSTA4-1, was located on chromosome 4 and was flanked by PZE104103541 and PZE104106157. The CI7 allele at this locus had an additive effect of 0.54 % for increased starch content. The second largest QTL for starch content, qSTA10-1, located in the genomic region between bins SYN23550 and SYN22965, explained 9.1 % of the phenotypic variation, with an additive effect of 0.50 % on chromosome 10. The next two QTLs for starch content, qSTA5-1 and qSTA5-2, were both located on chromosome 5 and explained 7.7 % and 5.3 % of the phenotypic variation, respectively.Based on a linkage map of 1,719.7 cM, QTLs for starch content were first identified using the BLUP value across the three environments. In total, six QTLs controlling starch content were detected in the CI7/K22 RIL population at an empirical threshold logarithm of odds (LOD) value of 3.1 after 1,000 permutations , indicating that the genetic component of starch content in the CI7/K22 RIL population is mainly characterized by additive gene actions.qSTA1-1) to 2.2 Mb (qSTA5-1), with each bin encompassing 2 (qSTA1-1) to 45 (qSTA10-1) genes, based on the annotated genes in the B73 reference genome Version 5b.60 (http://ensembl.gramene.org/Zea_mays/Info/Index). The functional annotations of all 144 genes indicated that seven genes, ZmGAL (GRMZM2G127123), ZmTPS (GRMZM2G151044), ZmKCS (GRMZM2G569948), ZmWRKY78 (GRMZM2G073272), ZmSnRK1I (GRMZM2G119769), ZmSnRK1 (GRMZM2G157743) and ZmMYB132 (AC206901.3_FG005), were most likely to be the candidate genes for the six QTLs  synthase in the trehalose metabolic pathway, which shares some common intermediate products, such as glucose, with starch metabolism [ZmTPS expression will influence starch metabolism. In addition, T6P, a reactant in the reverse reaction catalyzed by T6P synthase, is a sugar signal that is indispensable for carbohydrate utilization and starch pathway regulation in Arabidopsis [Arabidopsis [ZmTPS was a strong candidate gene for qSTA4-1.For  in milk , 58. Thetabolism . Thus, abidopsis , 61. Leabidopsis . Thus, ZqSTA5-1 was the largest among those of the six identified QTLs and harbored 44 genes in the spanning genomic region  is an alternative and efficient strategy for improving starch quantity and quality when QTLs or genes have been identified. Multiple traits have been improved by MAS in maize, such as head smut resistance , provita linkage –74. The We identified starch-associated QTLs in a RIL population using a high-density linkage map, refined the identified QTLs based on the bin map and subsequently mined their potential causal genes. The six QTLs accounted for 48.6 % of the starch variation in the CI7/K22 RIL population, with only one QTL explaining >10 % of the phenotypic variation. These findings indicate that large-effect QTLs, as well as minor-effect QTLs, contribute to the phenotypic variation in starch content in the CI7/K22 RIL population. Results from this study improve our understanding of the genetic variants that give rise to variation in kernel starch content, as well as of the possible mechanisms that underlie each QTL, and will provide guidance in manipulating starch quantity and quality by molecular breeding or biotechnology-assisted improvement.7 RILs, along with both parents, were grown in a randomized complete block design with one replication in Beijing in 2013, Hainan in 2014 and Neimeng in 2014. Each genotype was grown in a single-row plot having 1 m rows with 0.67 m between rows. In each row, all five ears were self-pollinated and harvested after maturity. Three hundred kernels were bulked for each row, with equal amounts from each harvested ear. Then, 20 representative kernels from each plot were selected from the 300 bulked kernels to measure the starch content.A RIL population consisting of 210 lines was derived from the cross between inbred lines CI7 and K22. CI7 is a high carotenoid and late maturing line introduced from America, which is developed by the USDA-ARS and derived from a backcross of (L317 x 33–16) L317, and K22 is a Chinese elite inbred line derived from a cross between two Chinese inbred lines LK11 and Ye478 . Accordi2) and heat (ethanol). All of the samples were measured with two sub-samples analyzed in parallel, and the average was used for subsequent analyses.The starch content in maize kernels was determined using a fermentable carbohydrate assay as described by Zhou and Bao . In briewww.R-project.org). The linear mixed effect function lmer in the lme4 package of R Version 3.1.1 was fitted to each RIL to obtain the BLUP value for starch content: yi = μ + fi + ei + εi, where yi is the phenotypic value of individual i, μ is the grand mean for all environments, fi is the genetic effect, ei is the effect of different environments and εi is the random error. The grand mean was fitted as a fixed effect, and genotype and environment were considered as random effects. The aov function in R version 3.1.1 was used to estimate the variances of the starch content. The model for the variance analysis was y = μ + αg + βe + ε, where αg was the effect of the gth line, βe was the effect of the eth environment and ε is the error. All of the effects were considered to be random. These variance components were used to calculate the broad-sense heritability as h2 = σ2g/(σ2g + σe2/e) [2g is the genetic variance, σe2 is the residual error and e is the number of environments.All of the statistics were performed using R Version 3.1.1 (+ σe2/e) , where σ6 lines in the CI7/K22 RIL population, together with their parents, were genotyped using the Illumina MaizeSNP50 BeadChip, which contains 56,110 SNPs and covers 19,540 genes [www.maizego.org/Resources.html). Completely co-segregating markers were assigned to a chromosomal bin, and each bin was considered as one marker.All 210 F40 genes . The lea40 genes  was used40 genes  in a Linp < 0.05 [QTL mapping of the starch content was performed using composite interval mapping  implemenp < 0.05 . The conp < 0.05 . To furtp < 0.05 .Arabidopsis or rice linked in the MaizeGDB database (www.maizeGDB.org). Additional protein prediction information was obtained from the InterPro module in the European Bioinformatics Institute database (www.ebi.ac.uk/interpro/).Based on the information available in the Gramene BioMart database (ensembl.gramene.org/biomart), the genes within the refined QTL interval and their functional descriptions were extracted. The function of each gene was further confirmed from orthologs in All supporting data can be found within the manuscript and its additional files.


Agrobacteriumtumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone “Nanlin895” (Populus deltoides × P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis.Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use  Agrobacterium tumefaciens have focused on a variety of parameters, including different Agrobacterium strains and culture densities, various acetosyringone (AS) concentrations and other factors[Poplar is a versatile tree species that is highly amenable to vegetative propagation, has a rapid growth rate and is a good model system for the transformation of woody plant species. These characteristics have led to its widespread use in the pulp, paper and cosmetics industries and in both highland and lowland reforestation ,2,3,4. Fr factors,9,10,11.et al. [Agrobacterium, with a high copy of transgenes, is quite good for molecular analysis and genetic engineering. The benefits of improved transformation methods include the insertion of intact full-length cDNAs into the plant genome, a reduction in the number of undesirable mutations and an increased transgene copy number [Agrobacterium-mediated transformation efficiency [CarNAC6 gene from chickpea encodes a 308-amino-acid, plant-specific regulatory transcriptional factor belonging to the NAC  domain protein family that increases plant response reactions to biotic and abiotic stresses and plays an important role in plant development. Homologues of the CarNAC6 gene have been identified in Arabidopsis and rice [Populus deltoides × P. euramericana “Nanlin895”) with the PBI121:CarNAC6 binary vector to improve Agrobacterium-mediated transformation efficiency. In addition, we attempted to optimize MS culture medium supplemented with N-6-benzyladenine (6-BA) and thidiazuron (TDZ) hormones to minimize lateral shoot development. Other factors affecting transformation efficiency evaluated in this study included the preculture of wounded explants, Agrobacterium infective suspension concentration, Agrobacterium infection duration, Agrobacterium infective suspension pH, acetosyringone (AS) concentration, cold treatment of an Agrobacterium infective suspension, cocultivation incubation temperature and duration and sucrose concentration in cocultivation medium. All of these factors were treated independently in related experiments independent of the age of the explants. In this study, the transformation efficiency is expressed as the percentage of independently transformed explants relative to the total number of explants.The expression of high-copy number transgenes in transgenic plants may be more or less than only a single copy of transgene, and also, sometimes, this is good for molecular analysis and genetic engineering. Furthermore, Husaini  reportedet al.  reportedy number . For theficiency ,15. The and rice ,18,19,20Agrobacterium-mediated transformation of Populus species is presented in Populus deltoides × P. euramericana “Nanlin895”) was cultured in medium containing various concentrations of 6-BA and TDZ  terminator. Southern blot analysis showed that 1–2 transgene copies were present in each of the three lines generated in procedures that did not show improved transformation efficiency  and 6-BA concentrations. Various concentrations of indoleacetic acid (IAA) were also shown to affect the frequency of regeneration from poplar explants [et al. [P. nigra. Various media are required for shoot and root regeneration of different genotypes and are supplemented with various levels of indole-3-butyric acid (IBA) to induce rooting from aspen leaves and with zeatin to induce shooting [Agrobacterium-mediated transformation of various plant species has increased markedly [Agrobacterium strains [Agrobacterium has been the subject of many studies. The use of appropriate AS concentrations during cocultivation has been shown to increase transformation efficiencies. Moreover, the effects of the Agrobacterium concentration and infection time on transformation efficiency have been demonstrated by Han et al. [The detection of mRNAs of 0.5–0.8 kbp by northern blot analysis confirmed that the ficiency a and in ficiency b. The tr  poplar leaves were collected from young y, China . The pBI2 discs and cultured on MS medium supplemented with the two hormones. The medium was refreshed at 2-week intervals. Regenerated shoots of 2 cm in length were cut and rooted on sterile half-strength MS medium containing 25 g/L sucrose and 6 g/L agar adjusted to pH 5.8 with no added hormones. After 6–8 weeks, the roots were examined, and leaves were collected from well-developed plantlets for gene transformation.Murashige and Skoog (MS) medium  was suppAgrobacterium containing the binary vector, PBI121:CARNAC6, was inoculated into Luria–Bertani (LB) medium supplemented with 50 mg/L rifampicin (Rif) and 50 mg/L kanamycin (Kan) and cultured in an incubator without light at 28 °C for 72 h [Young leaves were cut into 0.5 × 0.5-cm sections and precultured on half-strength MS medium supplemented with 6-BA (0.5 mg/L) and TDZ (0.004 mg/L) for 1, 2 or 3 days. This medium was tested on 37–40 lines for each variable, with four replicates. Agrobacterium colony was inoculated into 5 mL of LB medium containing 50 mg/L Rif and 50 mg/L Kan and incubated at 28 °C for 24 h with constant agitation (220 rpm). An additional 50 mL of LB were added and growth was continued overnight under the same conditions until an OD600 of 2.0 was reached. The culture was centrifuged at 3000 rpm for 10 min. The pellet was diluted in liquid MS medium with 5% sucrose to prepare an Agrobacterium-infective suspension. All Agrobacterium experiments were carried out independently using 20–25 precultured explants in triplicate.A single Agrobacterium-infective suspensions . The precultured explants were also cold treated independently at 0, 4 or 10 °C. Other parameters tested independently for their effects on transformation efficiency included the duration of the cocultivation of the Agrobacterium-infective suspension with explants , the AS concentration  and the pH of the Agrobacterium infection suspension . Wounded leaf discs were dried on sterile paper to remove surface water. The infected explants were co-cultivated in a dark incubator at various temperatures  and for different durations  on semi-liquid MS induction medium containing 0.5 mg/L 6-BA, 0.004 mg/L TDZ, 3 g/L agar, various sucrose concentrations  and 200 µM AS to induce the expression of the AgrobacteriumVir genes [Agrobacterium culture OD600 of 0.5, 10 °C cold pre-treatment, 30 min of Agrobacterium cocultivation, 100 µM AS, an Agrobacterium-infective suspension at pH 5.8, cocultivation in darkness at 28 °C for 48 h and 25 g/L sucrose in MS induction medium. Putative transformed explants were transferred to MS selection medium supplemented with 0.5 mg/L 6-BA, 0.004 mg/L TDZ, 6 g/L agar, 25 g/L sucrose, 400 mg/L cefotaxime (Cef) and 50 mg/L kanamycin (Kan) at pH 5.8 and cultured under 16-h/8-h light/dark conditions at 23 ± 1 °C in a phytotron. Selected regenerated shoots were separated and transferred to MS shoot elongation medium supplemented with 0.25 mg/L 6-BA, 0.002 mg/L TDZ, 6 mg/L agar, 25 mg/L sucrose and 300 mg/L Cef. Shoots of approximately 2 cm in length were then transferred onto half-strength MS rooting medium supplemented with 6 mg/L agar, 25 mg/L sucrose and 300 mg/L Cef with no added hormones. Plants that were well developed were transferred to a greenhouse for experiments. Transformation efficiencies were calculated using the following equation:  × 100.The precultured explants were immersed in various concentrations of ir genes . The parSalI and EcoRI at 37 °C for 4 h and separated overnight on a 0.8% agarose gel at 15 V. The digested genomic DNA was transferred to a Hybond N+ nylon membrane  and detected using DIG (digoxigenin) reagent, according to the manufacturer’s instructions . The primers, F5'-TACCGAGGATATTACACTACCAGGA-3' and R 5'-AGTCCAGTTTTGCAGCCAAG-3', were used to generate an 868-bp PCR product to use as a probe on the southern blot. Northern blot analysis was performed using 7 µg of total RNA from young leaves of WT plants and three transgenic poplar lines extracted using TRIzol reagent , according to the manufacturer’s instructions. Total RNA was separated on a 1.2% agarose formaldehyde gel, transferred to a Hybond N+ nylon membrane (Amersham Biosciences BV), and processed for northern hybridization. Random primers labelled with digoxigenin-11-dUTP using DIG-High Prime (Roche) were used to generate probes from the CarNAC6 cDNA (GenBank Accession Number FJ477887.1). The primers, F 5'-CTGGCAAAGGTTGGAGAAAG-3' and R 5'-CGTGTTGGTCTTGTTGTTGT-3', were used to generate a 433-bp PCR product to use as a probe for northern blotting.For southern blot analysis, 10 µg of genomic DNA were extracted using the CTAB (cetyl trimethylammonium bromide) method ,31 from All data analyses were performed using ANOVA  with a mean separation calculated using Duncan’s test with SPSS version 16  and Excel 2013 software . Confidence intervals showing no overlap of the mean values with an error value of 0.05 were considered to indicate statistically significant differences.Populus deltoides × P. euramericana “Nanlin895”) could be optimized to yield an 85%–90% regeneration rate. We successfully transformed poplar with the CarNAC6 biotic and abiotic stress resistance gene using Agrobacterium-mediated transformation. We showed that the transformation efficiency was increased by using a two-day preculture of wounded regeneration explants, an Agrobacterium-infective suspension with an OD600 of 0.7, cocultivation of the Agrobacterium infection suspension with explants for 120 min, an Agrobacterium-infective suspension pH of 5.0, an AS concentration of 200 µM, a 4 °C cold treatment of the Agrobacterium-infective suspension, a cocultivation incubation temperature of 28 °C, a cocultivation duration of 72 h and a cocultivation medium sucrose concentration of 30 g/L.In this study, we demonstrated that the regeneration efficiency of hybrid poplar “Nanlin895” (