Oryza rufipogon Griff., Dongxiang wild rice (DX), at its two-leaf stage. The correlation coefficients between RC and the plant survival rate (PSR) at the seedling and maturity stages were –0.85 and –0.9 (P = 0.01), respectively, indicating that RC is a reliable index for evaluating cold tolerance of rice. A preliminary mapping group was constructed from 151 BC2F1 plants using DX as a cold-tolerant donor and the indica variety Nanjing 11 (NJ) as a recurrent parent. A total of 113 codominant simple-sequence repeat (SSR) markers were developed, with a parental polymorphism of 17.3%. Two cold-tolerant QTLs, named qRC10-1 and qRC10-2 were detected on chromosome 10 by composite interval mapping. qRC10-1  was mapped at 148.3 cM, and qRC10-2  was mapped at 163.3 cM, which accounted for 9.4% and 32.1% of phenotypic variances, respectively. To fine map the major locus qRC10-2, NJ was crossed with a BC4F2 plant (L188-3), which only carried the QTL qRC10-2, to construct a large BC5F2 fine-mapping population with 13,324 progenies. Forty-five molecular markers were designed to evenly cover qRC10-2, and 10 markers showed polymorphisms between DX and NJ. As a result, qRC10-2 was delimited to a 48.5-kb region between markers qc45 and qc48. In this region, Os10g0489500 and Os10g0490100 exhibited different expression patterns between DX and NJ. Our results provide a basis for identifying the gene(s) underlying qRC10-2, and the markers developed here may be used to improve low-temperature tolerance of rice seedling and maturity stages via marker-assisted selection (MAS).Cold stress causes various injuries to rice seedlings in low-temperature and high-altitude areas and is therefore an important factor affecting rice production in such areas. In this study, root conductivity (RC) was used as an indicator to map quantitative trait loci (QTLs) of cold tolerance in qRC10-2 was fine mapped to a 48.5-kb candidate region, and Os10g0489500 and Os10g0490100 were identified as differently expressed genes for qRC10-2.With root electrical conductivity used as a cold-tolerance index, the quantitative trait locus  Oryza sativa L.) is one of the three major food crops in the world and feeds about 21% of the global population in terms of per capita energy Ctb-1, Ctb-2, qCTB3, qCTB7, and qCTB8) related to cold-induced rice damages to aboveground organs–including seedling mortality and growth, leaf wilting, and necrotic yellowing–were mapped Ctb-1, which improves the seed setting rate by enhancing spikelet fertility, has been isolated and cloned and has been proven to be involved primarily in the ubiquitin-proteasome pathway Rice  grown in Jiangxi Province , is the northernmost wild rice known in the world qRC10-2, related to root cold tolerance using DX as a cold-tolerant donor and the cold-sensitive indica variety Nanjing 11 (NJ) as a recurrent parent. We also evaluated the effects of qRC10-2 at the seedling and maturity stages. The analysis of the QTL derived from the wild rice will provide novel alleles for improving rice cold tolerance by MAS and also genetic materials for understanding the molecular mechanism of cold tolerance in rice.Dongxiang wild rice (DX), a Chinese common wild rice . Later, 150 simple-sequence repeat (SSR) markers covering the whole genome were used to determine the genetic background recovery rates, which varied from 89.33% to 94.67%  at the seedling and maturity stages. All plants were grown in the Yangzhou Wantou experimental fields at the Lixiahe Agricultural Research Institute of Jiangsu Province  in summer and in Sanya of Hainan Province  in winter.To eliminate the effect of o 94.67% . Among t2F1 and BC5F2 plants were measured as the average RC of the derived BC2F2 and BC5F3 plants across three replicates, respectively. Twenty seeds were randomly selected and placed in an illuminated incubator for growth to day 9. The seedlings were then given simulated diurnal alternating illumination treatment: 12 h dark at 4±1°C versus 12 h with 25,000 Lx of illumination at 4±1°C with relative humidity of 75–85%. After 48 h treatment, the 2-cm taproot section was immediately cut from each plant, and placed into an Eppendorf centrifuge tube with 2 mL deionized H2O (conductivity of 1.5 µs/cm). After soaking for 1 h, the RC of each plant was measured with a conductivity meter (Mettler Toledo 326) and data were adjusted for RC of deionized H2O.The RC phenotypes of the BC5F4 seeds were selected from each BC5F3 line with high or low RC to evaluate the PSR using the criteria of RC<10 and RC≥10 for cold tolerance and cold sensitivity, respectively. At day 9, the seedlings were cold-treated as described above and then returned to constant 28°C for recovery under illumination at 25,000 Lx for 72 h. The plants were divided into a sensitive group (with completely-withered leaves) and a tolerant group . PSR was estimated as the proportion of the plants showing a tolerant phenotype to all the plants by percentage (0–100%). The PSR of each BC5F3 line was measured in three replicates.Approximately 100 BC5F4 seedlings obtained from the BC5F3 lines were planted in the field on May 10, 2013. Uniform seedlings were then transplanted into a hydroponic greenhouse on June 10 at Yangzhou University. The hydroponic pool was 880 cm long, 130 cm wide, and 50 cm deep. The cement anchoring the rice plants was 135 cm long, 16.7 cm wide, and 2.5 cm thick, and each had a plate of 14 holes that were 4.0 cm in diameter and away from each other by 10 cm. A single seedling was planted in each hole, which was fixed by cork and sponges. Each group (low RC vs. high RC) contained 126 plants in three replicates. The hydroponic solution (pH 5.8) contained 10 p.p.m. of nitrogen (NH4NO3), phosphorus (NaH2PO4), potassium (K2SO4), calcium (CaCl2), and magnesium (MgSO4) each, 2 p.p.m. of iron (Fe(III)-EDTA), and 0.5 p.p.m. of manganese (MnCl2), and the solution was changed every other day. An oxygen pump was used to provide a steady flow of hydroponic nutrients, which maintained the nutrient concentrations and pH conditions in the pool during plant growth from seedling to maturity. These concentrations were elevated to twice the starting concentrations 60 days later. After 15-day post-heading, the cold hydroponic solution at 5±1°C was circulated around the roots for 24 h. After recovery at 28°C for two days, the PSR was measured.Root cold tolerance at maturity was evaluated as follows: The BCA genetic linkage map was constructed with Mapmaker/Exp 3.0 RM25570 and RM304 were chosen from the Gramene database (http://www.gramene.org). The sequences of indica cv. 93-11 and japonica cv. Nipponbare were downloaded from IRGSP1.0 (http://rapdb.dna.affrc.go.jp/) and used to design insertion/deletion (indel) markers and real-time polymerase chain reaction (PCR) markers using Premier 5.0 (DNA was extracted from leaves following the cetyltrimethylammonium bromide (CTAB) method mier 5.0  and S3.Taq DNA polymerase, 2 µL of 10×PCR buffer , and 1.5 µL of 2 µM forward and reverse primers each. The cycling conditions were 5 min at 94°C, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min. The PCR products were subjected to electrophoresis on 6% denaturing polyacrylamide gels, which were then stained with silver PCR and electrophoresis were performed following the method of Panaud et al. t-test using SPSS 11.0 for Windows . The correlation coefficient between RC and the PSR was calculated using the SAS software .Differences between parents and BILs for cold-related measurement were compared via DNaseI treatment. Total RNA (3 µg) was used as a template for cDNA synthesis using M-MLV transcriptase  with oligo (dT)18 primer. On an ABI PRISM 7500 system , real-time PCR was performed in a 20 µL volume containing 2 µL of first-strand cDNA, 10 µL of 2×SYBR Premix Ex Taq , 0.4 µL of 50×ROX reference dye, and 2 µL of each primer (2.5 µM). The used amplification protocol was 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Rice Actin 1 was used as an internal control, and each experiment was repeated at least three times.At the two-leaf stage after cold treatment for 0, 6, 12, or 24 h, the total RNA was extracted from 100 mg roots and 100 mg leaves using the RNeasy extraction kit , and any remaining genomic DNA was removed by P = 0.01, RC was significantly different for DX and NJ at day 8  was mapped at 148.3 cM between RM171 and RM1108, and qRC10-2 (LOD = 6.1) was mapped at 163.3 cM between RM25570 and RM304, which accounted for 9.4% and 32.1% of phenotypic variances, respectively  and 8 indel markers . The arrangement of these markers is shown in To obtain a fine map of RM25570 and RM304, a total of 211 BC5F2 BIL recombinants were identified. Among them, 64 showed the homozygous genotype for DX or NJ alleles and were divided into six groups  based on the genotypes of qc8, qc18, qc19, qc25, qc27, RM25681 and qc55  had DX alleles to the right of RM25681, indicating that qRC10-2 was located upstream of qc55. Groups R1 and R2 had recombinant loci between RM25681 and qc55, and there was a large range in RC for these groups. Based on this, we deduced that qRC10-2 was located between RM25681 and qc55  heterozygous in the qc48 allele. Therefore, qRC10-2 between qc45 and qc48 was restricted to 48.5 kb based on the Nipponbare sequence, in which a bacterial artificial chromosome clone (AC146481.2) contained six candidate genes, as shown in Based on the genotypes of the three polymorphic markers  RM25628 , groups P = 0.01), respectively  was selected as the reference genome to screen candidate genes. http://rapdb.dna.affrc.go.jp/).Os10g0489448, Os10g0489650, Os10g0489800, and Os10g0489900 shared a similar gene expression profile in DX, L188 and NJ. Os10g0489500 was induced under cold stress and showed high levels of inducible expression in the leaves of NJ at 6 h and 12 h, whereas no significant variation was observed in DX. This pattern was different from that in the roots, as it was highly expressed in the roots of DX at 6 h but not up-regulated in NJ until 24 h. Os10g0490100 in the roots of NJ showed higher expression than in DX before and after cold treatment. Therefore, Os10g0489500 and Os10g0490100 were considered as the genes associated with cold tolerance in the qRC10-2 region.To further identify genes related to cold tolerance, real-time PCR was used to assess gene expression in the leaf and root tissues after 0, 6, 12, and 24 h of cold treatment . The resindica and japonica showed cold injury symptoms below 18°C and 15°C, respectively qRC10-2. Moreover, the correlation coefficients (–0.85 and –0.9) between RC and the PSR were significant in seedlings and mature plants (P = 0.01), indicating that RC could be used as a reliable index for evaluating cold tolerance in rice.Cold stress is one of the major environmental concerns in rice cultivation, especially in low-temperature and high-altitude areas indica/japonica cultivars qCtss10 (LOD = 3.0) from W1943 (O. rufipogon) on chromosome 10, which accounted for 9.8% of phenotypic variations qRC10-1 and qRC10-2 were identified on the same chromosome in DX. A QTL for cold tolerance (qCTSS-10) was also identified in seedlings by Yang et al. qCTSS-10 for 7°C cold treatment was mapped by high-throughput sequencing in the F3 mapping population from a cross between a cold-tolerant japonica rice variety (Nipponbare) and a cold-sensitive indica variety. The interval of qCTSS-10 (15.48–21.06 Mb) overlapped that of qRC10-1 and qRC10-2, indicating that these QTLs may be controlled by the same cold-tolerant gene. Liu et al. also identified the same QTL  with 12% of the phenotypic contribution from the cold-tolerant line IL112 qCST10 was OSR33, which was the same marker as RM171 linked to qRC10-1 and qRC10-2 according to the Gramene marker data (http://www.gramene.org/). As described above, the same cold-tolerant QTL was identified on chromosome 10 among different parents and cold-treatment conditions based on a different cold tolerance index, suggesting that qRC10-2 was a stable and reliable cold-tolerant QTL for the rice seedling stage. Further studies on the candidate gene(s) in this region will aid in elucidating the molecular mechanism underlying rice cold tolerance.In previous studies, most cold-tolerant QTLs at the seedling stage were detected using recombinant inbred lines derived from Os10g0489500 and Os10g0490100 were differentially expressed between cold-tolerant and -sensitive plants. Os10g0489500 encoded a terpene synthase that was localized to the cytoplasm. In our study, Os10g0489500 was up-regulated in response to cold stress in the leaves of NJ. In the roots, however, its expression in DX and L188 was ∼18-fold higher than that in NJ, suggesting that the root may be a key organ for the Os10g0489500 activity. Yang et al. also identified Os10g0489500 as a gene up-regulated by low temperatures in a cold-tolerant variety indica (K354) and japonica (C418) under controlled conditions (25°C) and cold stress (4°C) for 48 h, Zhang et al. identified two terpene synthase genes (Os02g36140 and Os08g07100) that were associated with signal transduction cascades from K354 during cold-stress responses in seedlings Os10g0490100, which encoded virulence factors that degraded the pectin components of the plant cell wall QUASIMODO1, a gene encoding the glycosyl transferase required for pectin synthesis, caused reduced pectin content, leading to less cell adhesion and faster water loss during cold stress in common rice plants compared with the wild type Os10g0490100, a pectin lyase fold family protein that participated in pectin degradation, was more highly expressed in NJ roots than in DX and L188 roots before and after cold treatment, indicating that fewer pectin was maintained in cold-sensitive plants.In the candidate region, qRC10-2 based on other QTLs and also offered a molecular basis for rice cold tolerance mechanisms at the seedling and maturity stages.In conclusion, our data provided useful information for cultivating cold-tolerant rice varieties by pyramiding Figure S14F2 plants.Genetic background recovery rates determined among the BC(JPG)Click here for additional data file.Table S1Primer sequences for SSR markers.(DOCX)Click here for additional data file.Table S2Real-time PCR primer sequences for candidate genes.(DOCX)Click here for additional data file.Table S3qRC10-2.Primer sequences and sizes of polymorphic markers developed for fine mapping locus (DOCX)Click here for additional data file.


We isolated an avian influenza virus H9N2 strain from a wild bird in the Guangxi Province of southern China in 2013 named A/turtledove/Guangxi/49B6/2013(H9N2) (GX49B6). We aimed to understand the genetic characters of the GX49B6 strain by analyzing the complete genome sequence. The results showed that our isolated strain has features of low pathogenic avian influenza viruses and viruses that infect humans. The discovery of the complete genome sequence of the GX49B6 strain may be helpful to further the understanding of the epidemiology and surveillance of avian influenza viruses in the field. Orthomyxoviridae. It causes a variety of infections in avians and mammals. H9N2 subtype AIVs are widespread in the world and are the most prevalent subtype of avian influenza viruses reported in China over the last decade (2Avian influenza A virus (AIV) is a single-strained negative-sense RNA virus belonging to the family t decade . Althoug decade 25 have ca decade 2 and they decade 2. GuangxiIn this study, we isolated an H9N2 strain named A/turtledove/Guangxi/49B6/2013(H9N2) (GX49B6) from a wild bird in the Guangxi province. We amplified eight genes of the GX49B6 strain by RT-PCR using the universal primers of the influenza A virus , 9.The complete genome of GX49B6 strain consists of PB2, PB1, PA, HA, NP, NA, M, and NS segments. The full lengths of the segments are 2,341 nucleotides (nt), 2,341 nt, 2,233 nt, 1,742 nt, 1,565 nt, 1,459 nt, 1,027 nt, and 890 nt, respectively. The amino acid lengths of the proteins encoded by the eight genes as follows: PB2, 759 aa; PB1, 757 aa; PA, 716 aa; HA, 560 aa; NP, 498 aa; NA, 466 aa; M2, 97 aa; M1, 252 aa; NS2, 121 aa; NS1, 217 aa. The amino acid residue at the cleavage site (335-341) of the HA molecule is RSSR↓GLF, without multiple consecutive basic amino acids, which is characteristic of low pathogenic AIVs. The presence of 158 Glu, 627 Glu, 701Asp in the amino acid sequence of the PB2 protein, 436 Tyr in the PB1 protein, and 515 Thr in the PA protein, respectively, also provides evidence of low pathogenicity , 11. TheSequence analysis revealed that the nucleotide sequences of the HA and NA genes of the GX49B6 strain both belong to the Eurasian lineage. The HA gene shared 96.3% nucleotide homology with the isolate A/Chicken/Guangxi/55/2005(H9N2) that is thought to be a representative strain in China since 2007 . The PB2The data demonstrate that wild birds are involved in the transmission and evolution of AIVs. Our results may be helpful in epidemiological studies on H9N2 subtype AIVs.KJ725009 through KJ725016.The virus genome sequence of the A/turtledove/Guangxi/49B6/2013(H9N2) strain was deposited in the DDBJ/EMBL/GenBank database under accession numbers


TCF7L2) and type 2 diabetes mellitus (T2DM) in a Cameroonian population.To study the relationship between the rs12255372 (G/T) polymorphism of the transcription factor 7-like 2 . The Restriction Fragment Length Polymorphism - Polymerase Chain Reaction (RFLP-PCR) was used for genotyping.The T allele frequency was significantly higher in the diabetic group (0.44) than in the control group (0.17). This allele was significantly associated to a greater risk of developing T2DM as compared to the G allele . The codominant (additive) model explained best the risk of developing the disease, as the TT genotype was significantly associated to T2DM when compared to the GG genotype . By logistic regression adjusted for age, this OR was 4.33 .TCF7L2 gene is an important risk factor for T2DM in the Cameroonian population.Our findings suggest that the rs12255372 (G/T) polymorphism of the  Diabetes is a non-communicable disease characterized by chronic hyperglycemia and disturbances in carbohydrates, lipids and proteins metabolism due to defects in insulin secretion and its action, which results in severe acute and chronic complications . DiabeteTCF7L2 is involved in insulin secretion [TCF7L2 gene and T2DM [TCF7L2 gene associated to T2DM, amongst which rs7903146 (C/T), rs7901695 (T/C), rs12255372 (G/T) and rs11196205 (G/C) [TCF7L2 rs7903146 (C/T) variants with T2DM, some few have also shown a strong association with the rs12255372 (G/T) variants [T2DM is the most heterogeneous form of diabetes which is due by interactions between genetic and environmental factors. Genome-wide association studies have led to the identification of several susceptibility genes for T2DM , includiecretion ,6. It inand T2DM . Other s05 (G/C) . Even thvariants . On the variants , Arabs [variants .TCF7L2 variants gene and T2DM has never been studied in Central African populations where T2DM is very prevalent, with high morbidity and mortality rates [TCF7L2 rs12255372 (G/T) polymorphism and T2DM in a Cameroonian population.The relationship between the This is a case–control study involving 60 T2DM patients and 60 non-diabetic controls of Cameroonian ethnicity aged over 40 years. T2DM patients, diagnosed according to the IDF criteria , were co2 in m2, and the waist-to-hip ratio were calculated. The resting blood pressures were measured using standardized procedures with an automatic sphygmomanometer Omron HEM-705 CP .For all participants, we collected data on the sex and age; height, waist and hip circumference to the nearest 0.5 cm, and weight in light clothes to the nearest 0.1 kg were measured. The body mass index (BMI) as weight in kg/heightBlood samples were collected for biochemical and molecular assays. Fasting plasma glucose (glucose oxidase–peroxidase method), serum triglycerides (glycerol phosphatase oxidase-phenol4-amino antipyrene peroxidase method), serum cholesterol and high-density lipoprotein (HDL)-cholesterol (cholesterol oxidase phenol4-amino antipyrene peroxidase method) were determined by spectrophotometer (UV Mini 1240) using Chronolab kits . Low-density lipoprotein (LDL)-cholesterol was calculated using the Friedwald’s formula .TCF7L2 was genotyped by Restriction Fragment Length Polymorphism – Polymerase Chain Reaction (RFLP-PCR) using the following primers: Forward 5′-CTG GAA ACT AAG GCG TGA GG-3′, Reverse 5′-GGG TCG ATG TTG TTG AGC TT-3 . A final reaction volume of 20 μL for the Polymerase Chain Reaction (PCR) was constituted, which contained 100 ng of genomic DNA, 0.25 μM of each primer, 1.5 mM of MgCl2, 0.2 mM of each deoxynucleotide triphosphate (dNTP), 0.5 U of Go Green Taq DNA polymerase (PROMEGA), 1× Go Green Flexi buffer and 11.3 μl of nuclease free water. The PCR was carried out on a BIOMETRA T3 Thermal Cycler under the following conditions: 95°C for 2 minutes, followed by 35 cycles of 95°C for 30 seconds, 54°C for 30 seconds, 72°C for 30 seconds, and a final extension of 72°C for 5 minutes. The amplicons (346 bp) were then digested with Thermus species (Tsp509I) restriction enzyme at 65°C for 3 hours. The reaction volume was set to 15 μl, containing 7 μl of amplicons, 1× NEB buffer1 (New England Biolabs), 1U of Tsp509I, and 6.3 μl of nuclease free water. The digested products were separated by electrophoresis on a 3.5% agarose gel in presence of ethidium bromide (10 mg/mL) and visualized under a UV transilluminator.Genomic DNA was extracted from whole blood on filter paper by the Chelex method  and storThe study was approved by the National Ethical Review Board of the Cameroon Ministry of Public Health. Written informed consent was obtained from all the participants. The study was conducted in accordance with the Helsinki Declaration.2 statistics or the fisher’s exact test. Continuous variables were compared using non parametric tests . The Hardy Weinberg equilibrium was tested using the goodness-of-fit chi-square. Odd ratios were calculated by logistic regression adjusting for age. A p value less than 0.05 was considered statistically significant.Data was analyzed with STATA 11.0 . Genotype and allele frequencies were compared using the χp < 0.0001), waist-to-hip ratio , fasting plasma glucose , total cholesterol  and LDL-cholesterol  , versus 5.21% (6/115) and 27.82% (32/115) for the GT and the TT genotypes respectively. Genotype frequencies violated the Hardy-Weinberg equilibrium in the general population. The G allele was major with a frequency of 70%, as compared to the minor T allele which showed a frequency of 30%. The T allele frequency was 43.96% in diabetic group against 16.7% in nondiabetic and was found to significantly increase the risk of T2DM with an odds ratio of 3.92  (Table The frequency of the TT genotype was significantly higher in diabetics than in controls (41.37% vs. 14.03%) and was found to be significantly associated to T2DM with an OR of 4.45  (Table TCF7L2 rs12255372 (G/T) polymorphism on T2DM. Assuming the dominant model (GG vs. GT + TT = XT), the T allele carriers (XT genotype) had a significantly higher risk of T2DM than those with the GG genotype with an OR of 3.64 . When assuming the recessive model (GX = GG + GT vs. TT), a significant association was also found with an OR of 4.32 . Thus, the highest risk was observed with the codominant model with an OR of 4.45 /4.33 , after adjusting for age  genotypes, the clinical  and biochemical  characteristics of the T2DM patients were stratified, and no association was found. Comparative analyses of normoglycemic controls and rs12255372 (G/T) genotypes also revealed no association with anthropometrical and biochemical characteristics (data not shown).TCF7L2 gene with T2DM in a Cameroonian population. The frequency of the minor T allele was found to be 30%, and was comparable to those observed in the Czech population (30.15%) [TCF7L2 rs12255372 (G/T) variant and T2D was found in Chinese [Although the burden of T2DM is huge in sub-Saharan populations, epidemiological data on the disease are limited, especially on the genetic determinants of the disease . This pi(30.15%) , the Ira(30.15%)  and the (30.15%) . The var(30.15%) . This al(30.15%) ,16,17, w(30.15%)  and in A(30.15%) . However Chinese , Arab [1 Chinese , Pima In Chinese , and Sou Chinese  populatiTCF7L2 gene and T2D, the codominant model best fitted the association with an OR of 4.45 /OR (adjusted for age) of 4.33 , thus a 4-fold risk increase. This finding is in accord with that of Faranak et al. who demonstrated that the codominant model best fitted the effect of these gene variant on the risk of T2DM in the Iranian population [TCF7L2 gene and the risk of T2DM is still unclear, it can be speculated that the TCF7L2 gene has a role in insulin secretion and possibly adipose tissue development. Furthermore, the genetic variants that have so far been studied are present in the introns rather than in the coding regions. However, this may still lead to functional consequences in terms of protein stability and/or expression of alternatively spliced variants [The frequency of the TT genotype was significantly higher in diabetic patients than normoglycemic individuals . The GT genotype frequency was similar between the 2 groups and no association was found with of T2D. When assuming 3 models  to explain the association between the rs12255372 (G/T) polymorphism of the pulation . A meta-pulation . Our valTCF7L2 gene and type 2 Diabetes in a Cameroonian population. The Hardy-Weinberg equilibrium was violated in the general population, and this could have been due to genotyping errors, as RFLP-PCR data is sometimes difficult to interpret. For this reason, we excluded all doubtful genotypes from analysis. Deviation from Hardy-Weinberg equilibrium in this study was most probably due to our small sample size [TCF7L2 and T2DM in our population.This study, with a caution on sample size showed an association between the rs12255372 (G/T) polymorphism of the ple size .Thus, ouTCF7L2 gene is probably associated with T2DM in this population. This variant could help to predict the occurrence of T2DM in the Cameroonian population and possibly other sub-Saharan populations. Our findings should be confirmed by larger study with more accurate genotyping tools.The rs12255372 (G/T) polymorphism of the


TaHKT2;1 multigene family was previously identified as a Na+ transporter with a possible role in root Na+ uptake. In the present study, the existing full-length cDNA of this member was used as a basis to query the International Wheat Genome Survey Sequence to identify all members of the TaHKT2;1 family. Individual TaHKT2;1 genes were subsequently studied for gene and predicted protein structures, promoter variability, tissue expression and their role in Na+ and K+ status of wheat.A member of the TaHKT2;1 genes were characterized which included four functional genes  and two pseudogenes , on chromosomes 7A, 7B and 7D of hexaploid wheat. Variability in protein domains for cation specificity and in cis-regulatory elements for salt response in gene promoters, were identified amongst the functional TaHKT2;1 members. The functional genes were expressed under low and high NaCl conditions in roots and leaf sheaths, but were down regulated in leaf blades. Alternative splicing events were evident in TaHKT2;1 7AL-1. Aneuploid lines null for each functional gene were grown in high NaCl nutrient solution culture to identify potential role of each TaHKT2;1 member. Aneuploid lines null for TaHKT2;1 7AL-1, TaHKT2;1 7BL-1 and TaHKT2;1 7BL-2 showed no difference in Na+ concentration between Chinese Spring except for higher Na+ in sheaths. The same aneuploid lines had lower K+ in roots, sheath and youngest fully expanded leaf but only under high (200 mM) NaCl in the external solution. There was no difference in Na+ or K+ concentration for any treatment between aneuploid line null for the TaHKT2;1 7DL-1 gene and Chinese Spring.Six TaHKT2;1 is a complex family consisting of pseudogenes and functional members. TaHKT2;1 genes do not have an apparent role in controlling root Na+ uptake in bread wheat seedlings under experimental conditions in this study, contrary to existing hypotheses. However, TaHKT2;1 genes or, indeed other genes in the same chromosome region on 7AL, are candidates that may control Na+ transport from root to sheath and regulate K+ levels in different plant tissues. Alignment with genomic DNA identified a splice site within exon 1 resulting in the deleting of exon 2 and exon 3 and intervening introns relative to its larger cDNA counterpart [Genbank: KJ540616]. The alternatively spliced TaHKT2;1 7AL-1 sequence had the dinucleotide motif 5′-AT and AC-3′ at the exon-intron splice junction different to the conserved 5′GT and AG-3′ splice junction motifs in all other transcripts.FL-cDNA transcripts of functional TaHKT2;1 genes was conducted by evaluating tissue Na+ and K+ concentrations in wheat aneuploid lines null for each member of the TaHKT2;1 gene family, when exposed to NaCl and different K+ supply in the external solution. The absence of DNA amplicons from aneuploid lines using gene specific PCR primers  or high (4 mM) K+ status when genotypes were treated with an external solution containing 200 mM NaCl . Since this study cloned FL-cDNAs of each member of the gene family, further investigations can be pursued using heterologous systems to evaluate the functional significance of the protein sequence variability for ion transport in the TaHKT2;1 gene family.Although all members of the monocots , DNA seqnsidered ,35. In t systems ,23,36,37TaHKT2;1 7AL-2 and TaHKT2;1 7AL-3 shared typical characteristics of unprocessed pseudogenes, such as in-frame stop codons, location within close proximity to the functional genes on the same chromosome, and the absence of transcription [HKT super-family but their functional relevance is unknown [TaHKT2;1 7AL-2 and TaHKT2;1 7AL-3 was an ancient event created during evolution of the A genome progenitor species prior to, and maintained during, polyploidization of the tetraploid and hexaploid genomes. The Ka/Ks between the functional genes was close to the average Ka/Ks ratio of 0.2 previously estimated for functional wheat genes [TaHKT2;1 gene family was subjected to resilient purifying selection. Although TaHKT2;1 7AL-2 and TaHKT2;1 7AL-3 had characteristics of pseudogenes the Ka/Ks value between these genes and TaHKT2;1 7AL-1 was less than the theoretical value of 1.0 for a bona-fide pseudogene [cription . Pseudog unknown . Pseudog unknown  and may  unknown . Conservat genes , providieudogene , indicatTaHKT2;1 gene family was variable for presence and frequency of salt induced CREs. Plant response to salinity is mediated by immediate and extensive reprogramming of the spatial and temporal gene transcription, instigated by the interaction of transcription factors with four major groups of CREs including ABA-independent CREs  and ABA-dependent CREs  [TaHKT2;1 gene family. Interestingly, promoter diversity in AtHKT1;1 genes resulted in variable salt response in natural populations of Arabidopsis[TaHKT1;5 in bread wheat and wild relatives under salt stress [HKT genes are regulated by CREs mediated mechanisms. The presence of these elements with varying copies and combinations predict salt response with variable expression patterns of individual TaHKT2;1 members.Each member of the MYC/MYB) ,43, all abidopsis. Similart stress , supportTaHKT2;1 gene would show diverse transcriptional regulation, yet, no obvious transcript differences were detected between low and high NaCl treated plants. Similar observations were reported [+ deficient conditions did not induce differences in TaHKT2;1 gene expression in two week old wheat seedlings. However, expression of TaHKT2;1 7BL-1/-2 was up regulated by imposing K+ starved conditions in six day old wheat [+ in younger seedlings. In the present study, there was an obvious down regulation of TaHKT2;1 transcripts in leaf blades regardless of the NaCl concentration of the external solution, confirming adjustment of gene expression in this tissue. The extent of gene expression was further explored by mining wheat EST databases including NCBI, Transcriptome Shotgun Assembly and the TIGR Plant Transcript Assemblies, identifying a number of ESTs derived from pre-anthesis spikes and kernel tissue with 98-100% identity to members of the TaHKT2;1 gene family (data not shown). Further studies are needed to understand the expression of TaHKT2;1 gene family and how the various members influence ion homeostasis during different stages of plant development, various environmental conditions and variable salt levels.Given the CREs identified in this study, it was expected that each reported  where sald wheat  indicatiTaHKT2;1 gene expression was the level of complexity caused by alternative splicing of a 571 bp transcript unique to TaHKT2;1 7AL-1. Alternative splicing is evident in 25 to 42% of bread wheat genes whereby some forms are thought to play a role in regulating functional transcript levels [OsHKT1;5 resulting in a premature stop codon and non-functional transcripts as a possible means of regulating mRNA degradation [. Similarly, alternative splicing event in TaHKT2;1 7AL-1 indicates a rare unstable splice-junction motif that may act as an embedded molecular switch for post-transcriptional regulation and mRNA turn-over processes, which presumably may then influence levels of protein production.An interesting feature of t levels . Alternaradation . SimilarTaHKT2;1 family provided a system to analyse phenotypic effects of the genes on tissue ion concentrations. This approach provided evidence that a gene does not have a specific role when there was no significant difference between its corresponding aneuploid line and Chinese Spring (containing the full chromosome complement) whereas differences indicated that the gene would be a candidate controlling tissue Na+ and/or K+ status. The results, therefore, corroborate that TaHKT2;1 genes neither regulate root Na+ uptake nor Na+ accumulation in bulk leaf blades and youngest fully expanded leaf blade. Nevertheless, presence of transcripts for all TaHKT2;1 gene family members in these tissues under low and high NaCl conditions indicate alternative roles if they are translated into proteins. The higher sheath Na+ concentration in 7AL-1 and Dt7BS is likely from increased Na+ concentration in the xylem flow and being preferentially stored in sheaths. Wheat and other grasses do have mechanisms for withdrawal of Na+ from xylem in roots and sheath to reduce delivery of Na+ into the leaf blade [TaHKT2;1 7AL-1 and TaHKT2;1 7BL-1/-2 genes are candidates regulating Na+ accumulation in sheaths, perhaps by modifying Na+ in the root xylem that affects the flow of Na+ into sheaths. The possibility of other genes in the same chromosomal region contributing to this phenotypic effect cannot be excluded and further studies are needed to confirm the function of TaHKT2;1 genes on 7AL and 7BL in controlling Na+ accumulation in the sheaths.The use of wheat aneuploid lines null for specific members of the af blade . TherefoTaHKT2;1 7AL-1 gene may also regulate K+ accumulation in the sheath but only under high NaCl conditions, whereas TaHKT2;1 7BL-1/-2 may serve a similar role in roots and the youngest fully expanded leaf blade, evident by the lower K+ in these tissues in the Dt7BS line. Since TaHKT2;1 genes are not involved in K+ uptake by roots under saline conditions [+ concentration observed for Dt7BS in this study would be due to a greater net K+ translocation from roots to shoots. Therefore, it is reasonable to assume that TaHKT2;1 7BL-1/ TaHKT2;1 7BL-2 genes control K+ translocation in specific tissues of salt stressed wheat. Extensive functional redundancy of K+ transport systems [TaHKT2;1 7BL-1/TaHKT2;1 7BL-2 and TaHKT2;1 7AL-1 genes in controlling K+ homeostasis.The nditions ,49, the  systems  can be aTaHKT2;1 genes has been of interest because the encoded proteins implement pathways for Na+ entry into wheat in saline conditions [TaHKT2;1 gene family in controlling Na+ or indeed, of K+, during plant growth and development. The present study has highlighted diversity amongst the TaHKT2;1 gene family and predicted proteins, as well as promoter variability in salt signalling and presence of TaHKT2;1 transcripts in different tissues, under low and high levels of external Na+ and K+, gaining knowledge that TaHKT2;1 genes could assume multiple roles in bread wheat. The phenotypic effects when individual genes were deleted in bread wheat showed that TaHKT2;1 gene family are not involved in regulating root or leaf blade Na+ concentration but TaHKT2;1 7AL-1 and TaHKT2;1 7BL-1/-2 may be candidate genes controlling sheath Na+ concentration and K+ status in different tissues and experimental conditions. TaHKT2;1 7DL-1 had no apparent role in regulating Na+ and K+ status in different tissue and treatments in this study. In summary, the present findings from combined molecular and physiological studies provided a broader perspective of the role of the TaHKT2;1 family of genes in bread wheat.nditions . HoweverTriticum aestivum L. cv. Chinese Spring, six nullisomic-tetrasomic lines , eight deletion lines  and ditelosomic lines (Dt7BL and Dt7BS) were kindly provided by Dr John Raupp, Wheat Genetic and Genomic Resources Centre, Kansas State University, USA. Six hexaploid cultivated bread wheat varieties Calingiri, Ducula, Krichauff, Westonia, SARC-1 and a tetraploid variety (T. turgidum L.) Tamaroi were obtained from the germplasm collection at the Department of Agriculture and Food, Western Australia. DNA samples of the diploid species, T. monococcum (accession 4044) was provided by Dr Evans Lagudah, CSIRO Plant Industry, Canberra, Australia.Bread wheat variety TaHKT2;1 FL-cDNA, U16709, was used as the query sequence in BLASTN to search IWGSS database (http://www.wheatgenome.org) and retrieve coding and promoter regions of the individual members of TaHKT2;1 gene family. In-silico gene structure predictions for individual members of the TaHKT2;1 family were made using Spidey (http://www.ncbi.nlm.nih.gov/spidey/), GeneSeqer (http://www.plantgdb.org/cgi-bin/GeneSeqer/index.cg) and SIM4 (http://pbil.univ-lyon1.fr/sim4.php) software. Intron-exon structures for each member were confirmed by aligning retrieved genomic sequences with the FL-cDNA, U16709. DNA and protein sequence analysis and structure predictions were made using GENEIOUS 6.0.3 [.The US 6.0.3  and phylUS 6.0.3 .http://www.cbs.dtu.dk/services/TMHMM/) and MPEx 3.2 [http://blanco.biomol.uci.edu/mpex/) software suites. Three dimensional structures of the proteins were predicted by PHYRE 2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index). Protein sequence alignments, hydrophobicity plots and the 3-D structures were used to elucidate structural domains of the putative proteins.The predicted proteins were analysed for structural and functional elements including hydrophobicity and membrane topology using TMHMM Server 2.0 (MPEx 3.2  (http://cis-acting regulatory elements (CREs) associated with salt stress response in PlantCARE [DNA sequence 2000 bp upstream of predicted initiation site were analysed for lantCARE  and PLAClantCARE  databaseTaHKT2;1 family. Gene specific primer pairs were designed by targeting insertions and/or deletions (INDELs) and single nucleotide polymorphisms (SNPs) based on 3′ terminus mismatch described in [http://www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html) was used to confirm primer compatibility. Primer information is summarized in Table 2, 0.2 mM of each deoxyribonucleotides, 10 μM of each primer, 1X PCR buffer, 0.1 U taq DNA polymerase  and 50 ng of template DNA in a 20 μl reaction volume. Cycle conditions for standard PCR were 35 cycles of 94°C 30 sec, primer annealing temperature 30 sec, 72°C 30 sec and the final extension 7 min at 72°C; and for touchdown PCR were five cycles of 94°C 30 sec, 55-50°C 30 sec, 72°C 30 sec; and then 35 cycles of 94°C 30 sec, 50°C 30 sec, 72°C 30 sec and the final extension 7 min at 72°C. PCR products were separated on 1.5% agarose gel in 0.5X Tris-acetate EDTA at constant voltage (90 V) for 30 min and visualized under UV light using Gel Doc System  after staining with ethidium bromide.Plant genomic DNA was extracted from leaves using a phenol-chloroform-based method as described in  and was ribed in . NetPrim+, 3.95; Ca2+, 4; Mg2+, 0.4; NH4+, 0.625; NO3−, 4.375; SO42−, 1.9; HPO42−, 0.2; Fe-EDTA, 0.05; MES, 1.0; and micronutrients of one-quarter-concentration in Hoagland solution (pH was adjusted to 6.5 using Ca(OH)2). Four day old seedlings were then transferred to full strength nutrient solution in aerated, foil-covered, 4.5 L pots for 12 days. Each pot carried one individual of each of all six genotypes arranged randomly in each pot. There were four replicates of each genotype for each treatment in the experiment and the pots were arranged in a completely randomised design. Seedlings were held upright by a foam holder at the stem base inserted into individual holes in the pot lids. The experiment was carried out in a temperature controlled phytotron (20 ± 3°C/15 ± 2°C day/night) and the photosynthetically active radiation (PAR) recorded at midday was at ca. 1,400 - 1,500 μmol m−2 s−1. Solution levels in pots were maintained by topping up with deionized water.Seeds of six selected wheat deletion and ditelosomic lines were washed in 0.04% sodium hypochlorite (42 g/L) for 30 sec, rinsed in deionized water, and placed on a mesh float on aerated 0.1 strength nutrient solution in the dark for three days. Seedlings were then transferred to 0.25 strength nutrient solution for one day. Nutrient composition at full strength was: (mM) KTaHKT2;1 gene family, the plants were subjected to low K+ conditions prior to imposing salt treatments [TaHKT2;1 transporters might be involved with either Na+ and/or K+ transport [+ levels: low (0.2 mM) K+, where high affinity transport systems for K+ dominate and high (4 mM) K+, where K+ uptake kinetics was determined by low affinity systems [+ conditions for four days in a modified nutrient solution without added K+. The media contained (mM) Ca2+, 4; Mg2+, 0.4; NH4+, 0.825; NO3−, 4.375; SO42- , 0.4; HPO42-, 0.2; Fe-EDTA, 0.05; MES, 1.0; and micronutrients at one-quarter-concentrations in Hoagland solution 2) at a background Na+ and K+ concentrations of 0. 3 mM and 0.1 mM, respectively. After 4 days of low K+ level pre-treatment, 200 mM NaCl was applied to designated pots, so as to obtain four different Na+/K+ combinations: no added Na+/0.2 mM K+, no added Na+/4 mM K+, 200 mM Na+/0.2 mM K+ and 200 mM Na+/4 mM K+. The NaCl treatment was applied in 50 mM increments at 12 hour intervals and plants were maintained at the final 200 mM concentration for a full 3 days. The youngest fully expanded leaf blade, bulk leaf blades , sheaths and roots of each plant were sampled. Prior to excision, roots were washed three times for 10 secs each in a solution containing 4 mM CaSO4 and 368 mM mannitol (200 mM treated plants) or in 4 mM CaSO4 (plants subjected to no added NaCl). Leaves and sheath samples were rinsed in deionized water. All tissue samples for ion analyses were oven-dried at 70°C, weighed and then ground to a powder. Samples of dried powdered tissue (100 mg) were extracted in 5 ml of 0.5 M HNO3 for 3 days on a mechanical shaker. Na+ and K+ were measured in technical triplicate using a flame photometer  and mean values represented each treatment replicate tissue sample. The reliability of the methods was confirmed by analyses of a reference tissue  taken through the same procedures. Mean values from 4 treatment replicates of each genotype were analysed by ANOVA using SAS 9.4  software.In order to induce expression and study phenotypic effects of the eatments ,46. As kransport ,21,22 Na systems . 12 day T. aestivum var. Chinese Spring was grown in hydroponics and treated with NaCl as described above. Leaf blades (including youngest fully expanded leaf blade), sheaths and roots of plants treated with 4 mM K+ with no added Na+ and 4 mM K+ with 200 mM NaCl were harvested after treating the plants for three days, snap frozen in liquid nitrogen for RNA extraction and RT-PCR. Duplicate tissue samples were harvested for Na+ analysis (described above). Total RNA was isolated using RNeasy Plant Mini Kit . First strand cDNA was synthesised using RT Omniscript Kit . Integrity of synthesised cDNA was verified by RT-PCR using primers  designed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene [TaHKT2;1 gene family were amplified from cDNA preparations using primer pairs FL:A1 F and FL:A1 R, FL:A2/3 F and FL:A2/3R, FL:B1/2 F and FL:B1/2 R and FL:D1 F and FL:D1 R (Table DH) gene . FL- cDNTaHKT2;1 FL-cDNA were selected and DNA templates were purified using Wizard® Plus SV Minipreps . The cloned fragments were sequenced using BigDye™ sequencing chemistry  and analysed using GENEIOUS 6.0.3 [RT-PCR amplicons were separated and excised from 1% agarose gels, purified using Wizard® SV Gel and PCR Clean-Up System , and cloned into pGEM®-T Easy Vector Systems . Three bacterial colonies containing cloned US 6.0.3 .The authors declare they have no competing interests.HACKA conducted the bioinformatic work, designed the experiments, generated and analysed data, interpreted results and wrote the manuscript. TUH contributed to designing and conducting experiments for transcript analysis. TDC contributed to designing phenotyping experiments, data interpretation and edited the manuscript. MGF contributed to design of all experiments, data interpretation and edited the manuscript. All authors read, revised and approved the final manuscript.TaHKT2;1 gene family.Hydrophobicity of predicted proteins from the functional members of the Click here for file


Nanoparticle-like nucleation centers are formed at lower temperatures (220 °C), mixtures of morphologies  are seen at 230 and 240 °C, in which the nanodisks are predominant, while big hexagonal/prismatic crystals are obtained at 260 °C according to TEM results. A mixture of chalcocite and digenite phases was found at 230 and 240 °C, while a clear transition to a pure digenite phase was seen at 260 °C. The evolution of morphology and transition of phases is consistent to the electrical, optical, and morphological properties of the copper sulfide. In fact, digenite Cu1.8S is less resistive (346 Ω/sq) and has a lower energy band gap (1.6 eV) than chalcocite Cu2S . Low resistivity was also obtained in CuxS synthesized in aqueous solution, despite its amorphous structure. All CuxS products could be promising for optoelectronic applications.Copper sulfide is a promising p-type inorganic semiconductor for optoelectronic devices such as solar cells, due its small band gap energy and its electrical properties. In this work nanocrystalline copper sulfide (Cu Copper-rich sulfides (Cu2S), CuxS with x = 0.03, 0.2, 0.25, and CuS are widely reported [1.97S), digenite (Cu1.8S) or analite (Cu1.75S) [xS, some of which have reflections with narrowly spaced positions  . The lasreported –27. The nite Cu1.S or analCu1.75S) . These cCu1.75S) –32. Howeions see . This prxS is an essential challenge nowadays, because these parameters are dependent on several factors [2S obtained from a simple one-pot route [On the other hand, the control of size, shape, distribution and stoichiometry of Cu factors 15,18,21.ot route ,20,23–26xS obtained from aqueous solution.In this work, the growth evolution and the phase transition of copper sulfide in the temperature range from 220 to 260 °C in an organic solvent is reported. The full electrical, morphological and optical properties of these crystalline samples synthesized in the organic solvent were compared with the amorphous CuxS) depend on the synthesis and the reaction temperature   phase and djurleite phase (JCPDS 20-0365). At 240 °C  appear at 28.26, 30.02, 32.66, 42.42, 46.62, 52.32, and 55.12 corresponding to the rhombohedral structure, which is consistent to the literature [xS synthesized at 260 °C presents sharp peaks at 2θ = 27.84, 32.22, 32.66, 46.24, 55.12, and 67° of the digenite phase.The structural properties of the copper sulfide samples (Cuperature . A fullyperature . However31-0482) . Above at 240 °C , well deterature . Small p2S chalcocite phase can be presented: monoclinic , hexagonal , and cubic  [2S) to hexagonal (β-Cu2S) occurs at 103.5 °C and 101.8 °C for bulk and nanostructure chalcocite, respectively [xS products maintained the crystalline phases after we stored them for one year at room temperature, which is indicative of a good stability of the Cu2S chalcocite and Cu1.8S digenite phases (results not shown here).Three shapes of unit cells of Culcocite) . It is wectively . Accordiectively  the monoectively –15. In fectively , while cectively . So, theThe grain size and stress of the crystalline copper sulfide samples from organic synthesis at 230–260 °C were obtained from the full widths at a half maximum (FWHM) of the diffraction peaks and the linear combination of the following equation :D is the effective crystallite size, and ε is the effective strain. A plot of β cos(θ)/λ versus sin(θ)/λ for all the samples gives the grain size and the strain, as shown in Figure S2 in −5) and the highest was at 230 °C (−2.73 × 10−3).where β is the FWHM measured in radians, θ the Bragg angle of the peaks, λ the XRD wavelength, in our case in nanometers (λ = 0.154 nm), xS from aqueous solution is constituted of nanometric particles with undefined shape that are agglomerated into clusters  and width (w) of about 13.97 ± 2.7 × 5.86 ± 1.09 nm (from 260 particles), are seen in xS crystals at the higher temperature is not significantly different. However, the amount of crystals with a prism geometry is increased. These two types of morphology are consistent to the mixture of phases that were shown in the X-ray results. Big crystals with different sizes (25–80 nm) are observed for the samples of CuxS synthesized at 260 °C (l/w) of the CuxS nanorods are about 2.38 (230 °C), 2.29 (240 °C), and 2.63 (260 °C) taken from the data of size distribution  and irregularly shaped prisms (50–100 nm). The movement of the nanodisks results in the formation of the digenite phase through fusion of the nanodisks.xS samples show the following Cu/S ratios: 1.58 ± 0.02 for the sample at 220 °C, 1.92 ± 0.05, and 1.83 ± 0.08 for crystalline chalcocite/digenite at 230 and 240 °C, respectively, and 1.69 ± 0.05 for the digenite phase (at 260 °C). These values are similar to the chalcocite Cu2S and digenite Cu1.8S phases, respectively.The EDS patterns shows two peaks at 0.9 and 8.0 keV attributed to Cu Kα and Cu Lα emission, while a third peak at 2.3 keV is due to the S Kα emission. xS are shown in xS from organic synthesis at 220 °C, present a weak and broad absorption band at approximately 500 nm. However, crystalline CuxS samples show a well-defined absorbance band between 490 to 600 nm. In fact, a red shift of about 40 to 60 nm is presented from the chalcocite (Cu2S) to the digenite phases (Cu1.8S), which is in agreement to the increment of crystal size. This phenomenon is related to the free charges due to the copper deficiency in the samples. For example, the maximum absorbance band has been reported at 450 nm for Cu2S, while it is observed at longer wavelength (950 nm) for CuS [The optical absorbance spectra of the Cu for CuS . It is cxS sample prepared in aqueous solution shows an Eg about 2.2 eV for the direct and 2.0 eV for the indirect transition, respectively  [Eg values were obtained at high deposition times without modifying the chalcocite phase [On the other hand, the direct  2.0 eV) , so, it te phase .xS films prepared in aqueous solution are amorphous with undefined morphology. They exhibit a low square electrical resistivity (about 103 Ω/sq) as shown in xS from organic solution has a resistance of the order of 105–106 Ω/sq, while crystalline CuxS has a resistivity of about 107 Ω/sq at 240 °C and 102 Ω/sq at 260 °C, respectively. In fact, the samples obtained at 230 and 240 °C, which consist of a mixture of chalcocite and digenite phases, are more resistive than the digenite phase (sample at 260 °C). This means that the copper deficiency improves the conductivity of the CuxS, which is consistent to the reports in the literature [1.75S) have been grown onto the surface of CuS thin films, which improved their conductivity [The Cuterature . Deficieuctivity .xS is reported for the first time , tri-n-octylphosphine oxide (OP(C8H17)3, TOPO Sigma-Aldrich, ≥98%), 1-dodecanethiol , and dichlorobenzene  were used as received.For the organic reaction: copper(I) acetate , copper(II) sulfate pentahydrate , triethanolamine , and sodium acetate The aqueous reaction: deionized water (10 MΩ·cm), thiourea  during 120 min. The colloidal brown products were washed three times with dichlorobenzene by centrifugation  and were re-dispersed in dichlorobenzene. The organic products were cast on a Corning glass substrate and dried at 60 °C in an electric grill in order to form films.It consisted of a one-pot colloidal process previously reported by Wang et al.  with sli4·5H2O) were the sulfur and copper precursors, respectively, and the TEA ligand was an intermediary in the reaction. The synthesis proceeded as follows: A three-necked reactor containing 440 mL of deionized water was placed on a hot plate with magnetic stirring at 40 °C for 30 min. Clean Corning glass substrates were immersed inside the reactor in order to obtain the films by in situ deposition. Subsequently 1.3389 g of CuSO4·5H2O, previously dissolved in 20 mL of deionized water (1.3389 g/20 mL), 0.4354 g/14.5 mL of NaCOOCH3 and, 5.18 mL/20 mL of TEA. Finally, 0.2 g/31 mL of H2NCSNH2 was added in three aliquots each for 25 min. The substrates were withdrawn from the reactor and rinsed with deionized water. The precipitated products were washed with deionized water three times, immediately they were centrifuged and dried at room temperature. Both films and powder products, received a thermal treatment at 100 °C in air in a stove during 1 h.In this reaction thiourea and copper(II) sulfate pentahydrate , electrically by the four-points-probe technique, by UV–vis spectroscopy (Thermo Scientific Genesys 10S UV–vis spectrophotometer in the range of 200 to 1100 nm) in order to determine, the structural phase, the electrical resistance and optical absorbance spectra, respectively. The photoresponse measurements were made by applying a potential of 1 V at the sample: 20 s in darkness, 50 s under illumination and another 50 s in darkness. For this, two rectangular metallic contacts (0.5 × 0.2 cm) were painted on the surface of the films with silver paint in a square sample of 0.5 cm2.Powders of two syntheses, aqueous and organic, respectively, of CuxS powder to make the punctual analysis.Energy dispersive X-ray spectroscopy (EDS) was carried out in a JSM-6060LV SEM at 20 keV by using KBr pellets containing granules of CuFile 1Additional Figures.


Saccharomyces cerevisiae suggests the need of a proper redox balance. Nevertheless, there are no direct evidence of the links between NAD(P) levels and cold growth and how engineering of enzymatic reactions requiring NAD(P) may be used to modify the performance of industrial strains at low temperature.Cold stress reduces microbial growth and metabolism being relevant in industrial processes like wine making and brewing. Knowledge on the cold transcriptional response of S. cerevisiae modified for increased NADPH- and NADH-dependent Gdh1 and Gdh2 activity were tested for growth at low temperature. A high-copy number of the GDH2-encoded glutamate dehydrogenase gene stimulated growth at 15°C, while overexpression of GDH1 had detrimental effects, a difference likely caused by cofactor preferences. Indeed, neither the Trp− character of the tested strains, which could affect the synthesis of NAD(P), nor changes in oxidative stress susceptibility by overexpression of GDH1 and GDH2 account for the observed phenotypes. However, increased or reduced NADPH availability by knock-out or overexpression of GRE3, the NADPH-dependent aldose reductase gene, eliminated or exacerbated the cold-growth defect observed in YEpGDH1 cells. We also demonstrated that decreased capacity of glycerol production impairs growth at 15 but not at 30°C and that 15°C-grown baker’s yeast cells display higher fermentative capacity than those cultivated at 30°C. Thus, increasing NADH oxidation by overexpression of GDH2 would help to avoid perturbations in the redox metabolism induced by a higher fermentative/oxidative balance at low temperature. Finally, it is shown that overexpression of GDH2 increases notably the cold growth in the wine yeast strain QA23 in both standard growth medium and synthetic grape must.Recombinant strains of S. cerevisiae at temperatures below the optimal. An adequate supply of NAD(P) precursors as well as a proper level of reducing equivalents in the form of NADPH are required for cold growth. However, a major limitation is the increased need of oxidation of NADH to NAD+ at low temperature. In this scenario, our results identify the ammonium assimilation pathway as a target for the genetic improvement of cold growth in industrial strains.Redox constraints limit the growth of The online version of this article (doi:10.1186/s12934-015-0289-2) contains supplementary material, which is available to authorized users. Variations in ambient temperature are a common phenomenon in nature that influences the microbial growth and metabolism. Temperature drops modifies the molecular topology, the enzyme kinetics, and increases the molecular order of membrane lipids , 2, affeSaccharomyces cerevisiae . Plates were incubated for 22 or 48 h at 30 and 15°C, respectively. Yeast transformants carrying the geneticin (kanMX4) resistant module were selected on YPD agar plates containing 200 mg/l of G-418 (Sigma)  supplemented with the appropriate drop-out mixtures . Synthetic grape must was prepared as described by Riou et al. . For gas (Sigma) . EscheriSaccharomyces cerevisiae CEN.PK2-1C  [MATaade2-1 his3-11,15 leu-2-3,112 trp1-1 ura3-1 can1-100 GAL mal SUC2) [MATa trp1-1 leu2-3,112 ura3-52 his4 can1 FUS1::lacZ::LEU2) [gpd1::URA3) [houra3 mutant strain (MJH201), an Ura− haploid derivative of the wine yeast strain QA23 , was previously described [GDH1 and GDH2 gene, including its own promoter and terminator were obtained with specific synthetic oligonucleotides  obtaining the plasmids pGEM-GDH1 and pGEM-GDH2. Then, EcoRI/SSpI (pGEM-GDH1) and BglII/PvuII (pGEM-GDH2) fragments were released and ligated into the plasmid YEplac195   and W303al SUC2)  wild typZ::LEU2)  and PW40escribed . PCR-amp5  previour [LEU2, ) shuttler [LEU2, . The del600 = 0.25 from overnight seed cultures of YEplac195, YEpGDH1 and YEpGDH2 transformants of the CEN.PK2-1C strain, and cultivated at 30°C. When OD600 reached 0.5, aliquots were withdrawn for their immediate analysis (30°C-control), and cultures were transferred to 15°C for 18 h (15°C-samples). Cells were harvested by centrifugation, washed twice with 10 mM PBS (pH 7.2) and resuspended in the same buffer (0.25 units of OD600 per ml). Then, dihydrorhodamine 123  was added at 5 μg per ml of cell culture from a 2.5 mg/ml stock solution in ethanol and cells were incubated in the dark for 90 min at 28°C. Finally, cells were harvested, washed, resuspended in PBS buffer and analysed using the “Annexin V and Cell Death” channel of a flow cytometer Muse Cell Analyzer (Millipore). The settings were adjusted using negative (DHR 123-untreated cells) and positive (4 mM H2O2/60 min-stressed cells treated with DHR 123) controls. Data are expressed as the percentage of cells showing DHR 123-positive staining. The data represent the mean ± SE of four independent experiments.Five milliliters of SCD-Ura medium were inoculated at OD2 evolved recorded in a Fermograph II apparatus . Values are expressed as ml of CO2 and represent the mean ± SE of total volume of gas produced after 180 min of fermentation or are shown as the time–course graph of total gas. At least four independent experiments were conducted for each yeast strain.Molasses-grown cells (30 or 15°C) were collected, washed, resuspended and the final yeast concentration adjusted to 30 mg (dry weight) per ml as previously described . Fifteent test. The samples denoted with different letter were significantly different with a p < 0.05.Sample averages were compared using a Student’s


By expression of wild-type and mutant PIP5K2A in human embryonic kidney cells, we were able to provide evidence of impaired kinase activity of the mutant PIP5K2A. We defined the region K813–K823 of GluA1 as critical for the PIP2 effect by performing an alanine scan that suggested PIP2 binding to this area. A PIP strip assay revealed PIP2 binding to the C-terminal GluA1 peptide. The present observations disclose a novel mechanism in the regulation of GluA1.Ionotropic glutamate receptors are the most important excitatory receptors in the central nervous system, and their impairment can lead to multiple neuronal diseases. Here, we show that glutamate-induced currents in oocytes expressing GluA1 are increased by coexpression of the schizophrenia-associated phosphoinositide kinase PIP5K2A. This effect was due to enhanced membrane abundance and was blunted by a point mutation (N251S) in PIP5K2A. An increase in GluA1 currents was also observed upon acute injection of PIPThe online version of this article (doi:10.1007/s00424-013-1424-8) contains supplementary material, which is available to authorized users. Ionotropic glutamate receptors are the most important mediators of excitatory signal transduction in the central nervous system , 36. The3) has been shown to control synaptic function by maintaining AMPA receptors clustered at the postsynaptic membrane [2) might be important for the retention of NMDA receptors at the cell surface of cortical neurons [2, is a minor phospholipid component of cell membranes and enriched in the inner layer of the plasma membrane where it is a precursor of the second messengers IP3 and PIP3. Moreover, by regulating functions of numerous ion channels and transporters, it is considered a second messenger in its own right. We show a regulatory effect of PIP2 on GluA1 receptor function, prove direct binding of PIP2 to a defined amino acid stretch in the GluA1 C-terminus, and analyze the interaction between PIP2 and GluA1 by an alanine scan, PIP strip assay, and 3D modeling.Phosphoinositides are phosphorylated derivatives of phosphoinositol and serve as important second messengers in the cell. These phosphorylated lipids are produced at cell membranes during signaling events and contribute to the recruitment and activation of various signaling components as well as the trafficking of intracellular membranes , 6. Phosmembrane , and for neurons . Phospho neurons . Here, w neurons . The mai2 diC8  was used in the intracellular pipette solution in patch clamp experiments and injection into oocytes, respectively.The mutations for the alanine scan  were introduced using the QuikChange II Site-Directed Mutagenesis Kit  as described in the user manual. The primers used for mutagenesis were the following: ACGCATCCCGTAGCGAGTCGAAGCGG (K813A), TACAAAGCCCGTAGCGAGTCGAAGCGG (S814A), CTACAAATCCGCTAGCGAGTCGAAGCG (R815A), TCCCGTGCCGAGTCGAAGCGGATGAAG (S816A), TCCCGTAGCGCGTCGAAGCGGATGAAG (E817A), TCCCGTAGCGAGGCGAAGCGGATGAAG (S818A), TCCCGTAGCGAGTCGGCGCGGATGAAG (K819A), TCCCGTAGCGAGTCGAAGGCGATGAAG (R820A), TAGCGAGTCGAAGCGGGCGAAGGGT(M821A), AGCGAGTCGAAGCGGATGGCGGGTTTCTG (K822A), ATGAAGGCTTTCTGTTTGATCCCACAGC (G823A). Template complementary DNA (cDNA) was linearized with a suitable restriction enzyme. Complementary RNA (cRNA) was synthesized from 1 μg of linearized cDNA using an in vitro transcription kit . cRNA concentrations were evaluated by spectrophotometry, and transcript quality was checked by agarose gel electrophoresis. PIPXenopus laevis as described elsewhere [2, 2.0 mM KCl, 1.0 mM MgCl2, and 5 mM hydroxyethyl piperazineethanesulfonic acid (HEPES)–NaOH, pH 7.2 with NaOH, all from Sigma-Aldrich, Munich, Germany).Oocytes of stages V–VI were surgically removed from the ovaries of lsewhere . Oocytes2 was replaced by 1.8 mM MgCl2, 1.0 mM BaCl2, and 0.3 mM niflumic acid (pH 7.4 with NaOH) in the recording solution, and in addition, oocytes were injected with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid  60 min prior to recording. Current and voltage electrodes were filled with 3 M KCl and had resistances of 0.5–1.5 MΩ. Oocytes were held at −70 mV, and an agonist ) was applied by superfusion for 10 s at a flow rate of 10–14 ml/min. Half maximal effective concentration (EC50) values for glutamate were measured with nine different agonist concentrations. Data from each oocyte were fitted separately with the GraphPad Prism program to obtain an EC50 value. EC50 values of five to six oocytes were eventually averaged.To rule out calcium influences, 1.8 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES adjusted to pH 7.3 with NaOH. The pipette solution contained 130 mM CsF, 2 mM MgCl2, 1 mM CaCl2, 11 mM EGTA, and 10 mM HEPES adjusted to pH 7.3 with KOH; PIP2 diC8 was added to the pipette solution to a final concentration of 20 nM. Brief (10 ms), repetitive (10–100) pulses of glutamate (1 mM) were applied using a borosilicate theta capillary mounted to a piezo-driven actuator . Capacitances of the excised patches were determined using the time-domain technique [Human embryonic kidney (HEK) 293-T cells were transfected with GluA1(Q)flip-pIRES2-EGFP using Metafectene Pro . One day after transfection, cells were plated onto 30-mm glass bottom dishes  at a density of 60,000–80,000 cells per dish. Outside-out recordings were performed 2 days after plating using an EPC-9 amplifier . Currents were digitized with a sampling rate of 10 kHz and filtered at 3 kHz. Pipettes were pulled from borosilicate glass to resistances of 10–15 MΩ. The extracellular solution contained 140 mM NaCl, 4 mM KCl, 2 mM CaClechnique . For dat4 cells/3.5-cm dish. After another 24 h, the cells were fixed. For fixation, the 3.5-cm dish plate was washed with 1× phosphate-buffered saline (PBS) to remove media and treated with 4 % PFA /PBS for 15 min. Finally, slides were mounted with Fluoromount , and samples were examined by fluorescence microscopy  with the appropriate emission band-pass filters for detection of green fluorescent protein (GFP) and AxioVision 4.8 software. For analysis, ImageJ software was used.HEK293 cells were transfected with plasmid DNA /pcDNA3) using Metafectene Pro  and after 24 h seeded at a density of 4 × 105 cells/dish, grown for 48 h, and then transfected with 3 μg plasmid DNA (PIP5K2A/pcDNA3 or PIP5K2A(N251S)/pcDNA3) using Metafectene Pro . Control cells were treated with Metafectene Pro alone. For lysis of transfected HEK293 cells, the 3.5-cm dish plate was washed with 1× PBS to remove residual media. Then, 150 μl of 1× cell lysis buffer /3.5-cm dish was added and the plate incubated on ice for 5 min. The cells were scraped and sonicated briefly, followed by a centrifugation for 10 min at 14,000×g in a cold microfuge. The supernatant was removed and stored at −80 °C until needed. Prior to gel electrophoresis and Western blotting, a protein assay  was performed to determine the total protein concentrations. For Western blotting, 10 μg of total protein was applied per lane. PIP5K2A monoclonal purified rabbit antibody was used as the primary antibody . For the detection of α-calnexin, primary rabbit α-calnexin antibody  was used. The secondary antibody used was horseradish peroxidase-conjugated sheep anti-rabbit antibody .HEK293 cells were maintained in DMEM  supplemented with 10 % fetal bovine serum (Invitrogen) and 1× non-essential amino acids . To determine expression levels of wild-type and mutant PIP5K2A kinases, cells were plated onto 3.5-cm dishes at a density of 2.5 × 102 T75 flasks of HEK293-T cells ((1) without DNA, (2) GluA1/pIRES2/EGFP, (3) GluA1/pIRES2/EGFP + PIP5K2A/pcDNA3, or (4) GluA1/pIRES2/EGFP + PIP5K2A(N251S)/pcDNA3) were transfected with or without the corresponding DNA using the calcium phosphate transfection method. Confluent cells (90–95 %) were incubated with Sulfo-NHS-SS-Biotin for 30 min at 4 °C. Cells were scraped and transferred to a single 50-ml tube after adding a quenching solution to each flask. Cells were centrifuged  and transferred into a lysis solution. After sonication, cells were incubated for 30 min on ice, vortexing every 5 min. Cell lysate was centrifuged ; clarified supernatant was transferred to a prepared NeutrAvidin agarose column and incubated for 60 min at room temperature. After washing of the column, it was incubated with SDS-PAGE sample buffer containing DTT for 60 min at room temperature. Elution of the purified cell surface proteins was performed by centrifugation . Samples were analyzed by Western blot. Antibodies were obtained from Abcam, Cambridge, UK .Cell surface protein was isolated according to the manual of Pierce Cell Surface Protein Isolation Kit from Thermo Scientific, Bonn, Germany (Cat# 89881). Briefly, four 75-cm2 amounts was carried out using the PIP2 Mass ELISA Kit as specified by the user manual . Briefly, dried lipid extracts were dissolved in 200–600 μl of the sample buffer , loaded into a 96-well plate, and incubated with a PIP2 detector protein. After incubation, samples were transferred to a PIP2-coated 96-well plate for competitive binding. To determine the amount of PIP2 detector protein binding to the plate, a peroxidase-conjugated secondary detection reagent was added. For visualization, a colorimetric substrate was used, and the absorbance at 450 nm was measured with a microplate reader.Lipid extraction from transfected HEK293 cells was performed as previously described . BrieflyConsensus homology models of human PIP5K2A and GluA1 were generated using several individual modeling steps within YASARA Structure v10.1: The PIP5K2A consensus homology model is based on the amino acid sequence Lys21–Thr406, and the GluA1 homology model represents the amino acid sequence Leu13–Gly841. Subsequent refinements of high-resolution models using a CASP-approved protocol were achieved . The Prohttp://www.rcsb.org/pdb/explore.do?structureId=2YBX), but it lacks three highly relevant regions for our modeling . Two of these regions  are directly involved in the formation of the ligand-binding domains. Therefore, template structures 1bo1 and 2gk9 from the highly homologous PIP5K2β/PIP5K2γ represent far better templates to generate a consensus working model of PIP5K2A. However, comparison of the modeled structure to the solved parts of the structure of PIP5K2A over 284 analyzed residues revealed a relatively small root-mean-square deviation (RMSD) of 1.87 ± 1.13 Å, further indicating the relatively high quality of the PIP5K2A model.There is a solved crystal structure of PIP5K2A P were docked to PIP5K2A-ADP and PIP5K2A-ATP, respectively, resulting in the complexes PIP5K2A-ADP–PIP2 and PIP5K2A-ATP–PI(5)P. These models were energy-minimized again. In both model complexes, the residue Asn251 was mutated to serine to obtain PIP5K2A(N251S)-ADP–PIP2 and PIP5K2A(N251S)-ATP–PI(5)P. All four models were incorporated in simulation boxes filled with 0.9 % NaCl–H2O. MD simulations were performed for 2 ns to drive the models into stable conformations. The following settings were used: force field AMBER03, temperature at 298 K, pressure at 1 bar, pH 7.0, coulomb electrostatics at a cutoff of 7.86, 0.9 % NaCl, solvent density 0.997, 1-fs time steps. The average structures were determined from the 2,000-ps simulations and were used to calculate the root-mean-square fluctuations (RMSFs) for the respective PIP5K2A model  homology model in a similar position as described for PKA in a procedure described before . Both mo2A-ADP–PI,5P2 and 2 molecules were generated de novo using ACD/ChemSketch (version 12.01), manually docked in close proximity to the residues Ser813–Lys822, and the resultant GluA1–PIP2 complex was energy-minimized. The GluA1 model without PIP2 and the GluA1–PIP2 complex were both placed into a simulation box and inserted into a virtual membrane consisting of equal quantities of phosphatidylethanolamine (PEA), phosphatidylcholine (PCH), and phosphatidylserine . Membrane height was 43 Å (density ∼0.861 g/l), and the height of the hydrophobic membrane core was 28 Å. The GluA1 model without PIP2 and the GluA1–PIP2 complex were shrunk to 50 % of their size, and lipids bumping into the shrunk protein were deleted. Stepwise, the size of the protein/PIP2 complexes was expanded to reach their regular size and to fill the membrane pore. Each expansion step was followed by energy minimizations. The simulation box was filled with water, and 0.9 % NaCl was added to the system. MD simulations (2 ns) were performed on GluA1 models in complex with or without PIP2. The following simulation settings were used for the molecular dynamics simulation: force field AMBER03, temperature at 298 K, pressure at 1 bar, pH 7.0, coulomb electrostatics at a cutoff of 7.86, 0.9 % NaCl, solvent density 0.997, pH 7.0, 1-fs time steps, periodic boundaries, all atoms mobile. The average structures were determined from the simulations and were used to calculate the RMSFs for the respective model. Further, RMSD and energies were computed. During a 25-ps initial equilibration period, the membrane was artificially stabilized so that it could repack and cover the solute, while solvent water molecules are kept outside the membrane core for the initial 250 ps of the simulation.The GluA1 model generated was elongated at the C-terminal ends to Gly841 assuming an α-helical structure of the residues as predicted  Tween 20) and gently agitated for 1 h at room temperature. The peptide was solved in TBS-T + 3 % fatty-acid-free BSA and applied to the membrane. The membrane was incubated with 50 μg/ml peptide for 1 h at room temperature by gentle agitation. The washing of the membrane was performed three times with TBS-T + 3 % fatty-acid-free BSA for 10 min each time. For detection of fluorescence signal, the Molecular ImagerTM GelDoc from BioRad  was used. All incubation steps were performed in the dark.PIP strip membranes facilitate the analysis of phosphoinositide protein interactions by protein–lipid overlay assay. PIP strip membranes from Life Technologies  were used (# P23750). GluA1 peptide was synthesized by Caslo , and a fluorescein isothiocyanate (FITC) fluorochrome was conjugated to the N-terminus via an aminohexanoic acid (Ahx) linker. The GluA1 peptide sequence is (FITC)-Ahx-GALIEFCYKSRSESKRMKG. The first glycine was introduced for more flexibility of the peptide. The assay was performed as follows: the strip membrane was blocked with TBS-T + 3 % fatty-acid-free bovine serum albumin (BSA) . The point mutation at position 251 in PIP5K2A(N251S) was identified by Schwab and colleagues in schizophrenia patients when performing a schizophrenia association screen [Xenopus oocytes overexpressing GluA1 together with PIP5K2A than in oocytes expressing GluA1 alone. The stimulating effect of PIP5K2A on GluA1 was significantly less pronounced when GluA1 was coexpressed with the PIP5K2A (N251S) mutant P2 or another effect of the enzyme, we injected a water-soluble form of PIP2 and determined GluA1 currents. As illustrated in Fig. 2 before recording caused a significant increase in GluA1 current amplitudes. This stimulation by PIP2 was not further increased by coexpression of the kinase PIP5K2A  for GluA1 (17.2 ± 4.5 μM) or for GluA1 with PIP5K2A (19.6 ± 2.3 μM) or PIP5K2A(N251S) (10.6 ± 3.6 μM) revealed no significant differences when tested in oocytes P2 diC8 caused similar albeit slightly smaller but still significant effects on GluA1 channel functions PK2A Fig. , suggesttes Fig. . In ordeons Fig. .2 diC8 via the recording pipette. Significant changes in current amplitude were found for GluA1-expressing HEK293 cells exposed to PIP2 diC8 compared to cells not exposed to the lipid Ppid Fig. . Single-2 cascade may be the reason for the increase in GluA1 function.To test for membrane surface GluA1 protein, the relative abundance of an EGFP-tagged GluA1 expressed alone or together with PIP5K2A variants was analyzed. GluA1–EGFP abundance was increased in HEK293 cells coexpressed with PIP5K2A wt compared to GluA1–EGFP expressed without additional kinase variants Fig. . Coexpre2. To test whether the mutation N251S affects PIP5K2A function, we performed a quantitative PIP2 assay. After the treatment of HEK293 cells with transfection reagent plus PIP5K2A or PIP5K2A(N251S) cDNA or with transfection reagent alone, we first verified the overexpression of both PIP5K2A variants and the comparability of their expression levels via Western blot P2 production by PIP5K2A or PIP5K2A(N251S). As shown in Fig. 2 amounts compared to mock-transfected cells. In contrast, the PIP2 amounts of HEK293 cells expressing the mutant kinase PIP5K2A(N251S) were twofold lower compared to cells expressing wild-type PIP5K2A and on the same level as mock-transfected cells, supporting the notion that the mutation N251S results in reduced kinase activity.The impact of the schizophrenia-associated mutation N251S on the function of PIP5K2A thus far has not been investigated. The main catalytic function of PIP5K2A is the phosphorylation of PI(5)P to produce PIPlot Fig. . Mock-tr2 were docked into the ATP binding site of the PIP5K2A wt homology model in a similar position as proposed for PIP5K2β [2 -ATP–PI(5)P. On all four models, 2-ns MD simulations were performed. Overlays of the final structures are shown in Fig. 2 and PIP5K2A wt ATP–PI(5)P but not for the PIP5K2A(N251S)-ADP–PIP2 and PIP5K2A(N251S)-ATP–PI(5)P complexes. The PIP5K2A(N251S)-ATP–PI(5)P complex has more contact points than the PIP5K2A(N251S)-ADP–PIP2 complex. The former complex can be assumed to be more stable, creating an energy barrier during the catalytic circle and reducing the catalytic activity of PIP5K2A(N251S).We constructed a PIP5K2A homology model to visualize the catalytic domain of PIP5K2A and the location of the N251S mutation in PIP5K2A Fig. . The mod PIP5K2β , resulti [2 Fig. . In both2 on GluA1. We reasoned that in the case of PIP2 directly binding to GluA1, the binding region should be located in close proximity to the inner leaflet of the plasma membrane. The C-terminal amino acids K813–G823 span the region closest to the inner leaflet of the membrane. Therefore, we performed an alanine scanning mutagenesis of this stretch of the GluA1 sequence. As shown in Fig. Xenopus oocytes.Our data point to an effect of PIP2 binding, we coexpressed the GluA1 mutants (K813A–G823A) with PIP5K2A in oocytes and determined maximal current amplitudes. Alanine mutations at positions S814, S816, and E817 showed increased current amplitudes upon coexpression with PIP5K2A. For the other eight mutations , the sensitivity of currents to coexpression with PIP5K2A was lost, indicating that those amino acids might be involved in PIP2 binding  compared to those unlikely to be involved (blue). By using PIP strip membranes, we analyzed the binding of a GluA1 peptide, including the above identified amino acids possibly involved in PIP2 binding, to phospholipids. The peptide was labeled at the N-terminal with FITC for easy fluorescence detection. The assay proved direct binding of the GluA1 peptide to PIP2 as well as to PIP, PIP2, PIP2, PIP3, and phosphatidic acid Ping Fig. . The modcid Fig. .2 to the C-terminus of GluA1, which is in close proximity of the inner plasma membrane.Therefore, these results of the alanine scan and the PIP strip assay clearly suggest the binding of PIP2 molecules P2 for each subunit) were manually docked in close proximity to the residues Ser813–Lys822 P2 complex were inserted into a PEA, PCH, or PSE membrane (33 %:33 %:33 %). MD simulations  were performed on both models, and mean RMSD calculations suggested that the models had reached a stable conformation during the simulation  are rather rigid, consistent with the idea that conformational space of residues in these regions is restricted by the surrounding membrane. As residues 813–823 are positioned outside the membrane core, stabilization may result from interaction with the PIP2 headgroups. Indeed, stabilizing electrostatic interactions of specifically Lys815, Lys819, Arg820, and Lys822 side chains with the negatively charged PIP2 head group occur during the simulation and can be observed in the averaged structural models Pophrenia . Interesophrenia , 24, 25.ophrenia . However2. Homology modeling and molecular dynamics simulation suggest that the mutation has a relatively global effect on PIP5K2A structure and dynamics. As the distances between residue N251 and the ligands are relatively large , the effects on protein structure and function must be of allosteric nature. Possibly, the relatively global effects on the protein structure by the mutation result from destabilization of a loop (residues 215–251) involved in the formation of both ligand domains. The structural and dynamic nature of this loop may determine access of ligands and stability of the ligand–protein complexes as suggested by the MD simulations. As a result of the mutation, an energetic barrier in the catalytic cycle may prevent high-throughput catalyses as suggested by our MD simulations  less effectively produces PIP2 is a quantitatively minor phospholipid component of cell membranes and enriched at the plasma membrane where it is an important substrate for several signaling proteins, including several ion channels [2 on GluA1 currents is caused by direct binding to the C-terminus of GluA1 rather than a consequence of activation of second messengers by PIP2. Our 3D molecular model suggests that PIP2 stimulates GluA1 by interaction with the plasma membrane-adjacent region of the GluA1 C-terminus. A similar effect has recently been shown for the transmembrane AMPA receptor regulatory protein (TARP) molecules [2 to the C-terminus of GluA1 could impair binding of regulatory molecules, thus modifying receptor activity by increasing GluA1 protein at the plasma membrane resulting in increased ion channel function by altered clustering. This interpretation is supported by our suggestive experimental data as well as the 3D modeling. Interestingly, GluA1 amino acids shown here to be crucial for PIP2/PIP3 binding have also been reported to be the sites for phosphorylation, palmitoylation, and Band 4.1 interaction, implying complex cross talk of regulating signals [3 as a lipid-controlling synaptic function by maintaining AMPA receptor clustering at the postsynaptic membrane [3 action was not examined in that publication. Theoretically, by increasing the pool of diphosphoinositol, the pool of triphosphoinositol could be increased secondarily to enhance GluA1 stimulation. It seems rather likely that both PIP2 and PIP3 are involved in clustering processes, especially since we show that the identified GluA1 sequence seems to be a promiscuous binding site of PIP, PIP2, and PIP3. In a previous study, we showed the regulation of GluA1 by PIKfyve, which produces PIP2 2 and PIP3 are most abundantly expressed. Additionally, the local concentration of the respective phospholipid species is possibly determined by the locally present kinases. It is reasonable to assume that a functionally impaired kinase like PI5K2A(N251S) may disturb local PIP compositions leading to altered cellular function and possibly diseases including schizophrenia.It is known that PIPchannels . Here, wolecules . The TARolecules . Our obs signals . The groP2 , the mo2 directly interacts with the C-terminus of GluA1. Finally, we propose the structure of this carboxyterminal region of GluA1 based on homology modeling.In conclusion, the present observations disclose a novel mechanism regulating GluA1 function that may contribute to the pathogenesis of schizophrenia. Furthermore, we show for the first time that PIPESM 1(PDF 280 kb)Below is the link to the electronic supplementary material.


Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs. Collagen XIX was isolated from human rhabdomyosarcoma cells in 1992 by Hidei Yoshioka, under the name of collagen Y . In 1993The type XIX collagen is a homotrimer of 400 kDa, composed of the combination of three 112 kDa α1(XIX) chains. Each chain is encoded by a 250 Kbp gene (encompassing 51 exons) located on chromosome 6 q12-q14 and consists of 1142 residues, with 5 collagenous domains (Col1 to Col5) interrupted by six non-collagenous domains (NC1 to NC6) , 8. NC1 of the NC1 (XIX) domain on melanoma cells and to identify the involved signaling pathway that could explain its anti-tumor effects in melanoma. We demonstrated the involvement of αvβ3 integrin and a decrease in the phosphorylation of the FAK/PI3K/Akt/mTOR signaling pathway.p < 0.05), 33% (p < 0.05) and 31% (p < 0.05) at 24, 48 and 72 h, respectively . These results were confirmed on SK-MEL-28 melanoma cell line. In scratch wound assay, migration of SK-MEL-28 melanoma cells was significantly decreased after NC1(XIX) treatment Figure . NC1 , whereas adhesion was similar to the positive control, fibronectin. Adhesion of SK-MEL-28 cells was significantly decreased in the presence of 5 mM EDTA (p < 0.001). Cell adhesion was significantly restored when Ca2+ and Mg2+ were added into the media in order to counteract EDTA effect (p < 0.001)   Figure . Likewis) Figure . Taken tp < 0.001)  coated wells for 2 h at 37°C. The addition of the αvβ3 integrin monoclonal blocking antibody significantly decreased cell adhesion compared to control  Figure , suggestSK-MEL-28 extracts were analyzed by affinity chromatography on a NC1(XIX)-Sepharose column. Proteins bound to the affinity column were eluted with increasing concentrations of NaCl . Eluted samples were submitted to SDS-PAGE and analyzed by western blot. The 0.6 M eluted samples revealed a band which matched the 105 kDa band of the denaturated recombinant αvβ3 integrin Figure . This reThe interaction was confirmed in liquid phase binding assay. Recombinant αvβ3 integrin was incubated with biotinylated NC1(XIX) peptide with or without excess of unlabeled NC1(XIX) peptide. Integrin-peptide complexes were precipitated using sepharose-streptavidin beads and revealed by western blot. As shown in Figure SK-MEL-28 melanoma cells were cultured on glass slides, fixed with paraformaldehyde and incubated with biotinylated NC1(XIX) peptide, unlabeled NC1(XIX) peptide. Immunofluorescence studies showed biotinylated NC1(XIX) peptide (20 μM) (green) fixation on SK-MEL-28 cell surface. Competition assay with 10 fold-concentrated NC1(XIX) peptide (200 μM) completely abolished the biotinylated NC1(XIX) peptide labeling on SK-MEL-28 Figure . These dMoreover, SK-MEL-28 melanoma cells were cultured on glass slides, fixed with paraformaldehyde and incubated with biotinylated NC1(XIX) peptide and anti-αvβ3 integrin antibody 23C6). Competition assay with anti-αvβ3 integrin antibody (23C6) abolished the biotinylated NC1(XIX) peptide labeling on SK-MEL-28  -coated wells for 2 h at 37°C. The addition of cRGDfV blocking peptide significantly decreased cell adhesion compared to control  Figure .We characterized the expression of integrins at the cell surface and demonstrated the expression of the integrin αvβ3 on the cells surface using flow cytometry  and by iY397 phosphorylation was not modified even after 60 min of incubation with NC1(XIX). Total FAK level and FAKY397 / total FAK ratio remained also unchanged , while total FAK level remained unchanged. The FAKY861 / total FAK ratio was decreased by 50% at 5 min and was progressively restored after 30 min. In the same conditions, Tyr458 phosphorylation of the PI3K p85 subunit decreased gradually from 1 min to 60 min of incubation, while total PI3K p85 level remained unchanged. The PI3K p85Y458 / total PI3K p85 ratio was decreased by 20% at 5 min and 70% at 60 min  for 1, 5, 15, 30 and 60 min. Expression of total protein and correspondent phosphorylated protein were evaluated by western blot. FAKd Figure . By contn Figure . In the n Figure .T308 phosphorylation from 1 to 30 min of incubation, while total Akt level remained unchanged. The AktT308 / total Akt ratio decreased of 20% at 1 min and 50% at 15 and 30 min , while total mTOR level remained unchanged. The mTORS2448 / total mTOR ratio was decreased by 25% at 30 min and 60% after 60 min of incubation. In the same way, NC1(XIX) induced a decreased in mTORS2481 phosphorylation after 15 min. The mTORS2481 / total mTOR ratio also decreased of 30% from 15 to 60 min of incubation  induced a decrease in Aktn Figure . In the n Figure . As shown Figure .S9 / total GSK3β ratio was gradually decreased by 30% at 30 min, 40% at 60 min and remained decreased by 30% at 360 min  inhibits the migration of SK-MEL-28 cells. These results confirm previous studies from our laboratory demonstrating anti-tumor activity of NC1(XIX) .We showed that the adhesion of NC1(XIX) on SK-MEL-28 melanoma cells was calcium/magnesium/manganese dependent, evoking the involvement of integrins. Integrins play key roles in malignant process as well in the early stages of tumor invasion as in the tumor angiogenesis or in the development of metastases . We idenFAK is a multifunctional protein , especiaY861 phosphorylation has been well studied in tumors and it is clear that this phosphorylation is essential for tumor cell migration [Y861 was inhibited by NC1(XIX) as soon as 1 min of incubation. Afterwards, this inhibition is the starter of a series of FAK partner phosphorylation inhibitions, which may explain the anti-tumor and anti-angiogenic effects of NC1(XIX).FAKigration . We demovia integrins, the PI3K/Akt/mTOR pathway is involved in the proliferation, migration, and angiogenesis of many cancers, including melanoma [Two main signaling pathways, the RAS /RAF/MEK/ERK pathway and the FAK/PI3K/Akt/mTOR pathway are activated in melanoma and may explain the progression of this cancer . After amelanoma , 40.Phosphorylation by the PI3K activating subunit is important, especially for the migration and growth of tumor cells , 42. OurAs mentioned above, these two Akt-activating protein kinases are also activated by phosphorylation, essential for their activity, on serine 241 for PDK1 and on seS473, AktT308, PDK1S241, mTORC2S2481) of the signaling pathway. In addition, NC1(XIX) also induced an inhibition of phosphorylation of the second mTORC1 complex on Serine 2448. This phosphorylation is involved in tumor invasion, particularly in melanoma progression [We demonstrated that NC1(XIX) inhibited the phosphorylation of many partners  induced an inhibition of GSK3β (Glycogen synthase kinase 3-β) by decreasing its phosphorylation degree. GSK3β plays an important role in the activity of mTOR complex . It is aGSK3β implication in the mechanism of action of NC1 (XIX) suggests the involvement of the Wnt / β-catenin signaling pathway. This pathway is frequently deregulated in carcinogenesis and is most often of poor prognosis, in particular in colon cancer . HoweverIn summary, we have identified for the first time the αvβ3 integrin as a receptor of the NC1 domain of the collagen XIX. We have demonstrated that NC1(XIX) controls the majority of the partners of the FAK/PI3K/Akt/mTOR pathway by inducing the inhibition of their phosphorylations, essential for their biological activity Figure . Inhibit® . It was obtained by solid-phase synthesis using a FMOC (N-(9-fluorenyl)methoxy-carbonyl) derivative procedure. It was then purified by reverse phase high performance liquid chromatography using a C18 column, eluted by a gradient of acetonitrile in trifluoroacetic acid, and finally lyophilized [® and had the following sequence NPEDCLYPVSHAHQRTGGN-K-Biotin.NC1(XIX) peptide was purchased from Proteogenixphilized . The NC1® 25 cm2, 75 cm2 or 150 cm2 flasks  at 37°C in a humid atmosphere . All culture media and reagents were purchased from Life Technologies .SK-MEL-28 melanoma cell line was purchased from the American Type Culture Collection . Cell were grown in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) in Nunclon2 and 0.5 mM MgCl2 in DMEM. After counting, cells were seeded at a density of 60,000 cells in 100 μL/well and incubated for 2 h at 37°C in 5% CO2. Unattached cells were removed by washing with PBS. Attached cells were fixed with 1.1% glutaraldehyde in PBS for 20 min. After washing, cells were stained with 0.1% crystal violet for 20 min. After extensive washing with distilled water and air-drying, crystal violet was eluted in 100 μL/well of 10% acetic acid. Absorbance was then measured at 560 nm. To investigate the effect of divalent cations or EDTA on adhesion, cells were incubated for 2 hours at 37°C in 5% CO2 with or without 5 mM EDTA and/or 5 mM CaCl2 and 2.5 mM MgCl2 or 0.1 mM Mn2+. For MnCl2 adhesion assay, cells were centrifuged at 800 g for 5 min at room temperature and then incubated in HEPES buffer . For adhesion blocking assay, cells were preincubated with 20 μg/mL of anti-αvβ3 integrin antibody  diluted in 2 mM CaCl2 and 0.5 mM MgCl2 supplemented DMEM for 30 min on a carrousel at 37°C before adhesion assay. In complementary experiments, cells were incubated with 0.1 to 10 μg of cRGDfV  (blocking αvβ3 integrin peptide).For cell adhesion assays, 96-well microtiter plates were purchased from Dutscher . Plates were coated with the different substrates described below, diluted in carbonate buffer  overnight at 4°C. Bovine serum albumin (BSA)  at 0.1 μg/well was used as negative control whereas fibronectin  at 0.1 μg/well was used as positive control. NC1(XIX) was coated in the same carbonate buffer at 1 μg/well. Cells were detached with a Versene buffer  and centrifuged at 800 g for 5 min at room temperature. Cells were then incubated with 2 mM CaCl2. At confluence, cells were incubated with fresh DMEM without FBS overnight at 37°C. A homogenous wound was then created in each well with a sterile 200 μL pipet tip. After washing with DMEM, cells were incubated in DMEM with or without 20 μM NC1(XIX) for 24, 48 or 72 h at 37°C. Wounds were microphotographed at 0, 24, 48 or 72 h of incubation and wound closure was measured.SK-MEL-28 cells were seeded in 12-well plates and cultivated to confluence in DMEM supplemented with 10% FBS at 37°C in 5% CO2. Cells were fixed with 4% paraformaldehyde in PBS (w/v) and incubated for 10 min at room temperature. After washing with PBS, cells were then incubated with biotinylated NC1(XIX), NC1(XIX) and/or anti-αvβ3 integrin antibody  diluted 1/200 in 1% BSA-supplemented PBS for 1 h at room temperature. After washing with PBS, cells were incubated respectively with a 1/1000 diluted Alexa fluor 488- or 568-conjugated secondary antibody  in PBS supplemented with 1% BSA for 30 min in a dark chamber at room temperature. Coverslips were then mounted with aqueous mounting medium  and examined using a confocal laser scanning microscope .30,000 SK-MEL-28 melanoma cells were seeded on coverslip placed into 24-well culture plate and then incubated for 24 h in DMEM supplemented with 10% FBS at 37°C in 5% CO2, 1 mM MgCl2, pH 7.6, 1/100 PIC  (w/v) and 0.1% octylglucoside). Proteins bound to the affinity column were then eluted with elution buffer  and 0.1% octylglucoside) supplemented with increasing concentrations of NaCl . Eluted samples were then solubilized in SDS sample buffer with 10 mM DTT, denatured at 95°C for 5 min and submitted to western blotting using an β3 integrin antibody  previously functionalized with NC1(XIX) peptide, according to the manufacturer instructions. Unbound material was removed with 30 mL of washing buffer . This mixture was incubated with or without 7 μg of unbiotinylated NC1(XIX) peptide with gentle shaking at 4°C for 30 min. 10 μL of streptavidin sepharose beads  were added to the samples for a second incubation period of 30 min at 4°C. Samples were washed twice with 1 mL PBS and centrifuged at 500 g for 5 min at room temperature. 20 μL of loading buffer , 20% saccharose (w/v), pH 6.8, 75 mg bromophenol blue, 5% 2-mercaptoethanol (v/v)) were added to each pellet. Samples were denatured at 95°C for 5 min, centrifuged and supernatants were submitted to SDS-PAGE.One μg of recombinant αvβ3 integrin (3050-AV-0505 R&D Systems) was combined with 1 μg of biotinylated NC1(XIX) peptide in 500 μL of Dulbecco's PBS without divalent cations  diluted in DMEM without FBS for 0, 1, 5, 15, 30 or 60 min. All flasks were frozen at −80°C until protein extraction. Cells were scrapped with 200 μL of protein lysis buffer , supplemented with 1% PIC, 2 mM sodium orthovanadate and 50 mM NaF  and cell lysate were incubated for 30 min at 4°C. Cellular debris was pelleted by centrifugation of lysates at 10000 g for 10 min at 4°C. Protein concentration of the supernatant was quantified using Biorad Protein Assay  according to the manufacturer's instructions.SK-MEL-28 melanoma cells were cultured as previously described in Nunclon 25 cmFor western blot analysis, samples were reduced by 10 mM dithiothreitol and denaturated 5 min at 95°C. Samples were submitted to SDS-PAGE   and then transferred onto membrane using Transblot Turbo . Membranes were blocked with 5% BSA in TBS-T  for 2 h at room temperature. All antibodies were incubated in TBS-T supplemented with 1% BSA overnight at 4°C. Antibody references and dilutions are summarized in Table t test.Results were expressed as means +/− standard deviation or SEM. Statistical significance between groups was assessed by unpaired Student's