Later, three independent studies have confirmed the presence of a major QTL for spikelet number by three different names  in the same chromosomal region, and have implicated the overexpression of Nal1 gene as the causal factor for high spikelet number. However, the effect of qGN4.1 in different rice genetic backgrounds and expression levels of the underlying candidate genes is not known.Rice is a major source of food, particularly for the growing Asian population; hence, the utilization of genes for enhancing its yield potential is important for ensuring food security. Earlier, we have mapped a major quantitative trait loci (QTL) for the grain number per panicle, qGN4.1 QTL in the genetic backgrounds of 12 different high-yielding mega varieties of rice, introgressed by marker assisted-backcross breeding (MABB) using two QTL positive markers for foreground selection and two QTL negative flanking markers for recombinant selection together with phenotypic selection for the recovery of recipient parent genetic background. Analysis of the performance of BC2F3 plants showed a significant increase in the average number of well-filled grains per panicle in all the backgrounds, ranging from 21.6 in CSR 30-GN4.1 to 147.6 in Samba Mahsuri-GN4.1. Furthermore, qGN4.1 caused a significant increase in flag leaf width and panicle branching in most backgrounds. We identified BC3F3qGN4.1 near-isogenic lines (NILs) with 92.0–98.0% similarity to the respective recipient parent by background analysis using a 50 K rice SNP genotyping chip. Three of the NILs, namely Pusa Basmati 1121-GN4.1, Samba Mahsuri-GN4.1 and Swarna-GN4.1, showed a significant yield superiority to their recipient parents. Analysis of differential gene expression revealed that high grain number in these QTL-NILs was unlikely due to the overexpression of Nal1 gene (LOC_Os04g52479). Instead, another tightly linked gene (LOC_Os04g52590) coding for a protein kinase domain-containing protein was consistently overexpressed in the high grain number NILs.Here, we report the effect of qGN4.1 QTL for high grain number per panicle into 12 different mega varieties of rice using marker-assisted backcross breeding. The advanced near-isogenic lines are promising for the development of even higher yielding versions of these high-yielding mega varieties of rice.We have successfully introgressed the The online version of this article (10.1186/s12284-017-0195-9) contains supplementary material, which is available to authorized users. Food shortage is becoming a serious global problem as the rate of increase in world population exceeds the rate of increase in food production. In this regard, rice, which is one of the most important staple food crops feeding more than half of the human population worldwide, grown on the most productive irrigated land has achieved nearly maximum production with the current varieties. There is immense dependence on rice to satiate the food need of a large population and decrease the hunger index. With the completion of rice genome project, efforts are on to characterize the genes responsible for yield component traits such as the number of panicle-bearing tillers per plants, number of well-filled grains (spikelets) per panicle, and thousand grain weight with profound implications on yield improvement. Although the mechanisms that regulate each component trait are not yet fully understood, the genes and the available knowledge on the associated molecular markers offer a set of tools that can be combined to achieve higher grain-yielding varieties.sd1 gene showing a consistent effect on grain yield enhancement of Indica rice varieties, leading to green revolution , only a few of them have been evaluated for their impact on grain yield. Most of the QTLs for grain yield have been detected in crosses between Japonica and Indica cultivar groups because inter-cultivar group diversity is significantly higher than intra-cultivar group diversity. QTL-based marker-assisted breeding has been advocated for the transfer of beneficial genes into elite cultivars for food security , most of which have minor effects but some also have a major effect. Due to its complexity, different allelic combinations can give rise to similar phenotypes. It is still unclear whether a single QTL has the potential to enhance grain yield in diverse genetic backgrounds of elite rice varieties, though there is an example of Gn1a and APO1 for number of grains per panicle  for plant architecture and photosynthesis rate  populations (Pusa 1266/Pusa Basmati 1 and Pusa 1266/Jaya) derived from a new plant type (NPT) Indica rice genotype Pusa 1266  recovery. The effect of qGN4.1 QTL on grain yield in diverse genetic backgrounds was evaluated in field trials.In the present study, we used marker-assisted backcross breeding (MABB) approach to introgress qGN4.1 QTL-positive RILs derived from the cross between an NPT Indica rice genotype Pusa 1266 and Pusa Basmati 1  used in this study, namely HG28 and HG67, were The genomic DNA was isolated from the leaves of field-grown plants three to 4 weeks after transplanting. The leaf tissues from individual plants were frozen in liquid nitrogen, and DNA was extracted using the method of Murray and Thompson . The quaqGN4.1 QTL was transferred into 12 different mega varieties of rice using the previously described MABB strategy , which reduced the number of BCnF1 plants by 50%. In the second step, two QTL-negative recombinant selection markers (RM2441 and HVSSR 4-49) flanking the QTL-positive markers on both sides were used to identify lines with minimum linkage drag of the DP  selected for maximum phenotypic similarity with the RP. For genotypic background selection, the short-listed QTL-NILs were compared with respective RPs using the Affymetrix 50 K Axiom® 2.0 SNP chip ‘OsSNPnks’ on GeneTitan® instrument . SNP genotypes were called using the Affymetrix Genotyping Console™ v4.1 (AGC) software package. SNPs with low call rates across all samples were removed from the dataset and high-performing SNPs with a development quality check (DQC) score of >0.85, and call rates of >95.0% were used for further analyses . The RPG similarity based on SNP markers was calculated using the formula, RPG (%) = (R + 1/2H) × 100/P, where R = number of markers homozygous for RP allele, H = number of heterozygous markers, P = total number of SNP markers used for background selection  present in the primary panicle. All the NILs and their respective RP varieties were planted in two-meter rows with row-to-row spacing of 30 cm and plant-to-plant spacing of 20 cm. The best five BC2F3 plants showing the maximum phenotypic similarity with the RP were used for measuring plant height, flag leaf length, flag leaf width, tiller number and number of grain per panicle. Pollen from one of the plants most similar to RP was used for backcrossing to produce BC3 seeds.Phenotypic traits were evaluated on BCP = 0.05) for each of the four comparisons, namely between checks, between NILs and checks, between NILs of the same block, and between NILs of different blocks  and Indian Agricultural Research Institute, New Delhi. Evaluation of yield-associated traits was done on NILs, their RPs and four checks using an augmented randomized complete block design (RCBD) with five blocks, each having a maximum of 21 entries including four checks. The data for each character were analyzed as per the procedure of augmented RCBD using the Statistical Package for Augmented Designs. The treatment sum of squares was partitioned into the sum of squares among NILs, among checks and between NILs and checks. The adjusted means of NILs and checks were obtained. For making all possible pair-wise treatment comparisons, critical difference was calculated at 5% level of significance , followed by 40 cycles of denaturation at 95 °C (30 s), 60 °C (20 s), and 72 °C (20 s), interrupted by the dissociation curve with denaturation at 95 °C (1 min), cooling at 55 °C (30 s) and gradually heating up to 95 °C (30 s) in 96-well optical reaction plate. The amplicon purity was determined when a single melting peak was reached. Two biological replicates for each sample and three technical replicates for each biological replicate were used for RT-PCR analysis. The rice eEF1a gene was used as internal control (primer pair 5′TTTCACTCTTGGAGTGAAGCAGAT3′ and 5′GACTTCCTTCACGATTTCATCGTAA3′). Gene-specific primers were designed using the online PrimerQuest tool of IDT-Integrated DNA Technologies (https://eu.idtdna.com/PrimerQuest/Home/Index) using full-length cDNA sequences of the three candidate genes in the QTL interval: (i) Nal1 (LOC_Os04g52479); (ii) Retrotransposon gene (LOC_Os04g52540), and (iii) Protein kinase domain containing protein gene (LOC_Os04g52590), (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/#search). Primers for qRT-PCR expression analysis were: 5′GGTGTTTGGTGTGATGTTGATG3′ and 5′CCTGAGAGCCTGAACCAATAC3′ for LOC_Os04g52479 (product size 136 bp); 5′TCAGGTCTTCGCATTGGCACAC3′ and 5′GTACCGTTGAGCGCATTGCTTC3’ for LOC_Os04g52540 (product size 105 bp); and 5′GCAGCTCTCTCAGTCATCTAATC3′ and 5′TCTCAAGGCAGCAACCATAC3′ for LOC_Os04g52590 (product size 113 bp). Relative expression levels of the three genes were assayed on a real-time PCR system . The relative gene expression of the target gene was calculated using the equation: Exp = 2-ΔCt, where ΔCt = Cttargetgene − CteEF1a . Only the RNA samples with 260/280 ratio between 1.9 and 2.1 and 260/230 ratio greater than 2.0 were used for the analysis. First-strand cDNA was synthesized from 6 μg of total RNA using oligo dT as a primer and reverse transcriptase mix  in a 20 μl reaction volume. The PCR mixture contained 2 μl of template cDNA, 5 μl of 2× SYBR Green qPCR Master Mix (Agilent Technologies), 0.4 μl of gene-specific forward and reverse primers, 0.15 μl of ROX reference dye1, and 2.05 μl of nuclease-free HqGN4.1, a major QTL for grain number per panicle, into 12 different mega varieties of rice by marker-assisted backcross breeding  and the number of well-filled grains per panicle both increased significantly in the QTL-NILs of all 12 RP genetic backgrounds. Panicle length, number of primary and secondary branches per panicle, total number of grains per panicle and spikelet fertility directly influence the number of well-filled grains per panicle, which has a direct relevance to enhancing of yield potential  lines with the highest yielding Indica rice cultivars has shown that the original NPT lines had significantly lower tillering capacity , Samba Mahsuri (96.5%), Swarna (93.8%), IR 64 (98.0%), MTU 1010 (93.8%), HUR 105 (96.5%), Sarjoo 52 (92.0%), Pusa 44 (94.3%), CSR 30 (97.4%), Ranjit (97.7%), CR 1009 (96.0%), and Pusa Basmati 1 (93.4%). RPG recoveries for QTL qGN4.1 career chromosome 4 in these lines were: 94.6%, 93.1%, 89.6%, 95.4%, 90.6%, 93.3%, 78.6%, 91.3%, 85.8%, 91.3%, 90.3% and 91.5%, respectively , Samba Mahsuri (2.7 Mb), Swarna (4.1 Mb), IR 64 (2.4 Mb), MTU 1010 (3.4 Mb), HUR 105 (4.2 Mb), Sarjoo 52 (1.2 Mb), Pusa 44 (2.5 Mb), CSR 30 (2.8 Mb), Ranjit (4.3 Mb), CR 1009 (2.3 Mb), and Pusa Basmati 1 (2.8 Mb) despite conscious efforts to reduce the linkage drag by recombinant selection using QTL-negative flanking markers , 2 (1.8 Mb), 7 (0.6 Mb), 8 (3.7 Mb) and 12 (2.2 Mb); MTU 1010-qGN4.1 has 163 DP segments of >0.2 Mb through out the genome except chromosomes 7; HUR 105-qGN4.1 has 43 DP segments of >0.2 Mb on chromosome 1, 2, 11 and 12; Sarjoo 52-qGN4.1 has a large 8.7 Mb DP segment on chromosome 4 and 50 smaller segments of >0.2 Mb on chromosomes 2, 9, 11 and 12; Pusa 44-qGN4.1 has 104 DP segments on chromosomes 1, 2, 3, 5, 11 and 12; CSR 30-qGN4.1 has three large DP segments on chromosomes 3 (1.4 Mb), 5 (0.3 Mb) and 8 (2.4 Mb); Ranjit-qGN4.1 has a large DP segment on chromosome 1(1.1 Mb), four separate large segments of >0.2 Mb on chromosome 3, and 0.2 Mb on chromosome 8; CR 1009-qGN4.1 has two large DP segments 1.1 Mb and 1.9 Mb on chromosome 1, 0.9 Mb on chromosome 3; 1.0 Mb and 0.9 Mb on chromosome 6, 1.4 Mb on chromosome 9 and 1.1 Mb on chromosome 12; and Pusa Basmati 1-qGN4.1 has 85 DP segments of 0.2 Mb on chromosomes 2, 3, 6, 8, 9,10, 11 and 12. Thus, the high-resolution background analysis using 50 K SNP chip showed high RPG recovery in Pusa Basmati 1121, IR 64, CSR 30, Ranjit and CR 1009, and need for additional back crossing in Samba Mahsuri, Swarna, MTU 1010, HUR 105, Sarjoo 51, Pusa 44 and Pusa Basmati 1 for the elimination of the remaining large DP chromosome segments to develop QTL-NILs with uniform high RPG recovery.The RPG recovery was variable, but DP alleles were commonly present in the qGN4.1 QTL in PB 1121, Samba Mahsuri and Swarna backgrounds as compared to their respective RPs, but the maximum gain was observed in Samba Mahsuri genetic background where the yield was enhanced from 6690 kg/ha in the RP Samba Mahsuri to 8667 kg/ha in Samba Mahsuri-qGN4.1. Significant yield increases were also observed with Pusa Basmati 1121-qGN4.1 from 5808 kg/ha for RP to 6833 kg/ha for QTL-NIL, and with Swarna-qGN4.1 from 3667 kg/ha for RP to 5250 kg/ha for QTL-NIL. Other eight varieties also showed the numerical superiority of yield for qGN4.1 QTL-NILs as compared to the RP control, but the gain was not statistically significant  was identified as the most important region controlling the grain number per panicle in the NPT genotype Pusa 1266 . This list was too large for identification of causal gene(s) underlying qGN4.1 QTL. Therefore, QTL mapping was complemented with genome-wide transcriptome profiling to identify differentially expressed genes at early panicle development stage  that codes for a serine and cysteine protease. Of the eight differentially genes, only one coding for protein kinase domain-containing protein (LOC_Os04g52590) showed overexpression in the high grain number genotype; Nal1 gene was actually downregulated by −4.0 and −1.1 folds in microarray and qRT-PCR studies, respectively  and retrotransposon (LOC_Os04g52540) genes actually showed down regulation in the qGN4.1 QTL-NILs (Table SPIKE, GPS and LSCHL4 QTLs where overexpression of Nal1 gene is shown to be the causal factor for high spikelet number (Fujita et al. qGN4.1 QTL-NILs is being further investigated by genetic recombination and transformation studies.The Ls Table . This isqGN4.1 QTL for high grain number per panicle in the backgrounds of 12 different mega varieties of rice. Introgression of qGN4.1 resulted in a significant increase in the number of grains per panicle in all the 12 backgrounds. The associated morphological traits such as flag leaf width and the number of primary and secondary branches in the panicle also increased significantly, but the total number of tillers was reduced. However, the number of productive tillers remained unchanged in all backgrounds except Pusa Basmati 1121 and Sarjoo 52, which showed a significant reduction in the number of productive tillers per plant. Even with the normal planting density, the QTL-NILs showed significantly higher yield as compared to the respective RPs in the field experiments for three varieties, namely Pusa Basmati 1121, Samba Mahsuri and Swarna. Our results showed that the qGN4.1 QTL can be effectively deployed to further enhance the yield potential of popular mega varieties of rice.This study describes the development and evaluation of NILs with


In the fall of 2014, highly pathogenic avian influenza (HPAI) subtype H5N8 clade 2.3.4.4 was introduced into North America by migrating waterfowl from Asia where, through reassortment, novel HPAI H5N2 and H5N1 viruses emerged.Assess the susceptibility of pigs to HPAI H5N1, H5N2, and H5N8 clade 2.3.3.3 from North America.Pigs and trachea explants were inoculated with a representative panel of H5NX clade 2.3.4.4 HPAI viruses from North America. Nasal swabs, BALF, and sera were collected to assess replication and transmission in challenged and direct contact pigs by RRT‐PCR, virus isolation, hemagglutination inhibition, and ELISA.Limited virus replication was restricted to the lower respiratory tract of challenged pigs, though absent in the nasal passages and trachea cultures, as determined by RRT‐PCR in all samples. Seroconversion of inoculated pigs was detected by NP ELISA but was not reliably detected by antigen‐specific hemagglutination inhibition. Boost with adjuvanted virus was required for the production of neutralizing antibodies to assess cross‐reactivity between wild‐type avian strains. All RRT‐PCR and serology tests were negative for contact animals indicating a failure of transmission from primary inoculated pigs.H5NX clade 2.3.4.4 strains can replicate in the lower respiratory tract of swine upon high titer inoculation, though appear to be incapable of replication in swine nasal epithelium in vivo or ex vivo in trachea explants in culture. Infected pigs did not produce high levels of serum antibodies following infection. Collectively, our data show HPAI H5NX clade 2.3.4.4 viruses to be poorly adapted for replication and transmission in swine. In North America, the circulating IAV subtypes H1N1, H1N2, and H3N2 are genetically diverse products of multiple reassortments, combining the viral gene segments of avian, human, and swine influenza lineage IAV.To assess the ability of H5NX clade 2.3.4.4 viruses to replicate and transmit in swine, pigs were experimentally infected with representative strains from the 2014‐2015 wild bird and poultry outbreak in North America. Cohorts of naïve animals were later co‐housed with challenged pigs to assess pig‐to‐pig transmission. Additionally, to better understand the tissue tropism of clade 2.3.4.4 in swine, porcine trachea explants were infected with an expanded panel of North American H5NX viruses. Replication of IAV in the upper and lower respiratory tracts was evaluated by titration of nasal swabs and BALF and pathological analysis of lung tissue at 3 and 5 days post‐infection (dpi). Seroconversion of pigs challenged with H5NX was evaluated using the hemagglutination inhibition assay and nucleoprotein (NP) enzyme‐linked immunosorbent assay (ELISA). These data provide a comprehensive understanding of the replication, pathology, and transmission of H5NX clade 2.3.4.4 from North America in swine.22.150) titers were determined as previously described.H5NX clade 2.3.4.4 viruses, A/American green‐winged teal/Washington/195750/2014 (H5N1), A/northern pintail/Washington/40964/2014 (H5N2), A/turkey/Minnesota/7172‐1/2015 (H5N2), and A/gyrfalcon/Washington/41088‐6/2014 (H5N8), highly pathogenic avian influenza virus from human A/Vietnam/1203/2004 (H5N1), human pandemic virus A/CA/04/2009 (H1N1), and North American low pathogenic avian influenza (LPAI) virus A/quail/CA/K1400794/2014 (H5N8) were propagated in the allantoic cavity of 10‐day‐old embryonated chicken eggs. The allantoic fluid of inoculated eggs was collected, and 50% egg infective dose . Following the incubation period, explants were washed three times with PBS. At 8, 24, 48, 72 hours post‐infection (hpi), 300 μL of infection media was added to the apical chamber of trachea explant containing transwell inserts and incubated for 30 minutes at 37°C followed by removal and storage at −80°C. Supernatant from each time point was titrated on MDCK cells, and 50% tissue culture infectious dose (TCID50) was calculated as previously described.Swine trachea explants were derived using methods of Jones et al2.3Mycoplasma hyopneumonia. Prior to inoculation, pigs were confirmed to be serologically negative for influenza A virus by ELISA for antibodies against IAV nucleoprotein (NP). Fifteen pigs per virus were inoculated intranasally with 106 EID50 of virus in 2‐mL Eagle's minimal essential media , and 5 naïve animals were housed separately to serve as non‐challenged controls. Two days post‐infection (dpi), 5 naïve pigs were introduced into the same pen, one for each virus infection group, to serve as targets for direct contact transmission. Nasal swabs from inoculated and contact pigs were collected on 1, 3, 5, and 7 dpi. Five animals from each challenge group were humanely euthanized on 3 and 5 dpi, and three pigs from each group were euthanized on 21 dpi, non‐challenged control animals at 5 dpi, and all direct contact animals at 21 dpi. Blood and whole lungs were collected during necropsy. Two animals from each group were boosted intramuscularly with live virus plus adjuvant (128 HA with Emulsigen D) at 27 dpi and again at 35 dpi with adjuvanted whole inactivated virus preparations (128 HA units with Emulsigen D). All remaining animals were euthanized and blood collected at 42 dpi.A total of 85 three‐week‐old weaned pigs were used in this study. Weaned piglets were purchased from a high‐health status herd, free of IAV, porcine reproductive and respiratory syndrome virus, and 2.4Nasal swabs were collected at 1, 3, and 7 dpi from donor, contact, and control animals and stored in 2 mL of minimum essential medium (MEM). Whole lungs were taken from five necropsied pigs at 3 and 5 dpi and from three primary inoculated pigs and all five contact animals at 21 dpi. Lavages were performed on excised lungs using 50 mL of MEM. Broncho‐alveolar lavage fluid (BALF) was collected as previously described.2.5g for 10 minutes. Swine serum was then diluted 1:4 with receptor destroying enzyme (RDE)  and incubated for 18 hours at 37°C. Next, RDE was heat inactivated by incubation at 56°C for 30 minutes. HI assays were performed in U bottom 96‐well plates  by diluting 50 μL RDE‐treated sera 1:2 in PBS, incubating with 25 μL 4 hemagglutinating units (HAU) at room temperature for 1 hour, followed by a 30‐min incubation with 0.5% turkey red blood cells at room temperature. ELISA for IAV nucleoprotein (NP) was performed using FlockChek AI MultiS‐Screen Antibody Test kit following manufacturer's instructions .Seroconversion of challenged and contact animals was assessed using a commercial enzyme‐linked immunosorbent assay (ELISA) and the hemagglutination inhibition (HI) assay. Whole blood was collected, and the serum was separated by centrifugation at 290 2.6P ≤ .05 was considered to be statistically significant.GraphPad Prism 7  was used for statistical analysis of RRT‐PCR data. Two‐way analysis of variance was used to compare groups. 33.1Nasal swabs were collected from all experimentally infected pigs on 1, 3, 5 dpi, and BALF was collected from 5 animals at 3 and 5 dpi , LPAI A/quail/CA/1400794/2014 (H5N8), and A/CA/04/2009 (pH1N1) were included as control viruses. While all H5N8 and most H5N2 viruses failed to replicate ex vivo, including low pathogenic avian influenza virus A/quail/CA/K1400794/2014, the A/northern pintail/WA/40964/2014 H5N2 did replicate, though only to low titers (Table 3.2Serum was collected on 21 dpi in primary pigs and 19 dpc in direct contact pigs and assayed for antibodies against IAV hemagglutinin (HA) and nucleoprotein (NP). Serum antibodies, as assessed via HI assays, were not detected in serum from pigs infected with A/gyrfalcon/WA/41088‐6/2014 H5N8 and A/American green‐winged teal/WA/195750/2014 H5N1 and in only 2  animals from A/turkey/MN/7172‐1/2015 H5N2 and A/northern pintail/WA/40964/2014 H5N2 challenge groups. Antibodies against IAV NP were, however, detected by ELISA at an increased frequency, but not from all infected animals  with the exception of A/northern pintail/WA/40964/2014 H5N2, which did not demonstrate an increase in HI titers. Heterologous HI antibody reciprocal titers were generally within twofold to fourfold of the homologous virus titers  and low mortality (80%). Similar to mice, HPAI H5NX clade 2.3.4.4 viruses exhibit low virulence in inoculated ferrets characterized by moderate infectious titers detected in nasal wash samples and are incapable of direct contact transmission to co‐housed, naïve ferrets.Following the emergence of HPAI H5NX clade 2.3.4.4, multiple studies have shown these viruses to be poorly adapted for replication in mammals. Kim et alortality 0%. Simil50) recovered from nasal swab samples collected 2‐4 dpi, although no evidence of contact transmission was observed.Our study is the first to describe the experimental inoculation of pigs with HPAI H5NX clade 2.3.4.4 viruses. Previous studies have detailed porcine infections with viruses of A/goose/Guangdong/1/1996 clade 0, 1, 2.1, 2.2, and 2.3 H5N1 HPAI. Experimental inoculation of pigs with the first human (A/Hong Kong/156/1997) and related chicken (A/chicken/Hong Kong/258/1997) HPAI H5N1 viruses resulted in high infectious virus titers (3.6‐4.5 log EIDSwine are susceptible to infection with low pathogenic avian influenza viruses (LPAI) of multiple subtypes. Multiple studies have shown pigs inoculated with LPAI exhibit little to no clinical disease and no contact transmission.Ex vivo inoculation of swine trachea explants was conducted to further assess the replication kinetics of HPAI H5NX in porcine respiratory tissue. Trachea explants maintain the normal in vivo tissue architecture and sialic acid receptor distribution, abundant alpha‐2,6‐linked (human) and a complete absence of alpha‐2,3‐linked (avian) receptors.The data presented here demonstrate that the North American wild bird lineage H5N1, H5N2, and H5N8 clade 2.3.4.4 viruses are capable of infecting pigs by direct inoculation. Despite this, replication is restricted to the lower lung and none of the isolates were transmitted by direct contact to naïve pigs. The absence of a robust humoral immune response following challenge, as determined by HI, highlights the need for improved serological assays to ensure accurate detection of porcine infection with novel HPAI viruses should these events occur. In their current state, these data indicate that HPAI H5NX clade 2.3.4.4 viruses in North America pose little risk to the health of pigs though continued surveillance, especially in areas of domestic poultry outbreaks, is warranted.Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.


The higher resolution obtained reveals previously unresolved spectral features of W(CO)6. The spectrum shows two higher-energy bands (in the energy ranges of 7.22–8.12 eV and 8.15–9.05 eV), one of them with clear vibrational structure, and a few lower-energy shoulders in addition to a couple of lower-energy metal-to-ligand charge-transfer (MLCT) bands reported in the literature before. Absolute photoabsorption cross sections are reported and, where possible, compared to previously published results. On the basis of this combined experimental/theoretical study the absorption spectrum of the complex has been totally re-assigned between 3.9 and 10.8 eV under the light of spin–orbit coupling (SOC) effects. The present comprehensive knowledge of the nature of the electronically excited states may be of relevance to estimate neutral dissociation cross sections of W(CO)6, a precursor molecule in focused electron beam induced deposition (FEBID) processes, from electron scattering measurements.High-resolution vacuum ultraviolet photoabsorption measurements in the wavelength range of 115–320 nm (10.8–3.9 eV) have been performed together with comprehensive relativistic time-dependent density functional calculations (TDDFT) on the low-lying excited sates of tungsten hexacarbonyl, W(CO) W(CO)6 is a precursor molecule used in electron beam induced deposition (EBID) to produce well-defined tungsten-containing nanostructures [6 irradiated at 500 eV electron impact energy were analysed in situ by X-ray photoelectron spectroscopy (XPS), mass spectrometry and reflective absorption infrared spectroscopy (RAIRS) measurements [6 molecules have shown contaminations by C and O due to incomplete ligand desorption yielding tungsten oxide and an enhanced degree of tungsten oxidation from the presence of co-adsorbed water. These contaminations are then incorporated into the carbonaceous matrix. Recently, we note ab initio molecular dynamics simulations of focused electron beam induced deposition (FEBID) precursor molecules adsorbed on fully and partially hydroxylated SiO2 surfaces [6, with no formation of bare W− metal anions. Negative ion states of transition-metal hexacarbonyls have been obtained by electron transmission spectroscopy (ETS) with W(CO)6 attachment energies of 1.53, 2.46 and 4.26 eV [6 clusters [6 dimer [6 at 248 nm (ca. 5 eV) assigning the first CO elimination to a translational energy release process. As part of an ongoing effort to fully characterize the electron-induced fragmentation channels of W(CO)6, as needed for FEBID simulations, the purpose of the present work is to provide a high-resolution vacuum ultraviolet (VUV) absorption spectrum, representative of transition-metal carbonyl complexes for which unresolved spectral features remain to be solved, with a description as complete as possible of the electronic states.The electronic structure of tungsten hexacarbonyl, W(CO)5–350 nm , photoel5–350 nm –10, phot5–350 nm  and elec5–350 nm  spectros5–350 nm , molecul5–350 nm , and ele5–350 nm  have beetroscopy , as welltroscopy  and infrtroscopy –20 spectructures –22. Nanourements . These ssurfaces . Electrosurfaces  revealed 4.26 eV . We note 4.26 eV , on the clusters  and on t[6 dimer . As far [6 dimer , making [6 dimer . Another[6 dimer  explored6 followed by the results together with a discussion and comparison with previous data where possible. Finally, some concluding remarks that can be drawn from this study are given.In the next section we provide details on the experimental and theoretical methods used in this study for W(CO)6 in the photon energy range of 3.9–10.8 eV . For measurements above 220 nm, air is allowed into this gap to let oxygen absorb higher harmonics of light  that may be passing through the cell.The high-resolution VUV photoabsorption studies of W(CO)–10.8 eV  were per−18 cm2) are obtained using the Beer–Lambert attenuation law It = I0·exp(−nσx), where It is the radiation intensity transmitted through the gas sample, I0 is the radiation intensity transmitted through the evacuated cell, n is the molecular number density of the sample gas, σ is the absolute photoabsorption cross section, and x is the absorption path length (here 15.5 cm). ASTRID2 is operated in a “top-up” mode, keeping the stored electron beam current (and thus the intensity for a given wavelength) quasi-constant, which is achieved by adding small amounts of current to the storage ring to make up for the constant beam decay. This procedure causes the beam current to vary by about 3% during a scan, with this effect being compensated for by a normalization of the data to an accurately determined beam current. The accuracy of the cross section is estimated to be better than ±5%. Only when the sample absorbs very weakly (I0 ≈ It), does the error increase as a percentage of the measured cross section.Absolute photoabsorption cross sections  level with B3LYP functional [The structure of W(CO)nctional  with allnctional , leadingnctional ), respecnctional . Howevernctional . The vernctional –40. Spinnctional . The comnctional –48.The sample used in the photoabsorption measurements was purchased from Sigma-Aldrich, with a stated purity of ≥99%. The sample was used as delivered.6 belongs to the Oh point group with the calculated electronic configuration of the outermost valence orbitals for the ground state 1A1g being (7eg)4 (3t2g)6 (1t1g)6 (11t1u)6 (4t2g)6, and only optically allowed 1A1g→1T1u transitions. The analysis of the ground-state Kohn–Sham (KS) orbitals , 11t1u, has σCO character, the third-highest (HOMO−2), 1t1g, and the fourth-highest occupied molecular orbitals (HOMO−3), 3t2g, have CO character. The lowest unoccupied molecular orbital (LUMO), 12t1u, and (LUMO+1), 3t2u, have mainly π*CO antibonding character, while (LUMO+2), 13t1u, and (LUMO+3), 2t1g, have π*CO and σ*CO character, respectively.W(CO)orbitals  shows th6 without SOC is depicted in Figure S2 of Interpretation of the experimental data and results from quantum chemical calculations of the lowest electronic states with spin–orbit coupling (SOC) are summarised in The measured high-resolution VUV photoabsorption spectrum is presented in 6 in a solvent report a weak band at 3.794 eV, which was assigned to a spin-forbidden d→d transition. In contrast, our calculations do not provide evidence for the presence of low-lying metal-centred (MC) states in this period 6 transition-metal carbonyl complex, as it is the case in the period 4 analogue Cr(CO)6 [1A1g→a,b3T1u transitions occur. The present calculations including SOC effects show that three “spin–orbit” states, namely 1T1u, 2T1u and 3T1u of metal-to-ligand charge transfer (MLCT) character, which are composed essentially of 13T1u, 13Eu and 13T2u states, contribute to this region (The temperature-dependent absorption studies of Beach and Gray  of W(CO) Cr(CO)6 . The ele Cr(CO)6  have pros region .The photoabsorption spectrum in the energy region of 3.9–4.8 eV is shown in 1u state composed mainly of the 11T1u←11A1g transition involving the contribution from 12t1u←4t2g (0.66) and 3t2u←4t2g (0.33) of MLCT character and calculated to be at 4.119 eV  origin band is not pronounced in the spectrum, although tentatively estimated to be at 4.04(6) eV , accordi1A1g→a1T1g and 1A1g→a1T2g symmetry-forbidden charge-transfer transitions. However, on the basis of the present calculations it seems that these symmetry-forbidden transitions do not participate in this band. In contrast, the mixed MLCT/intra-ligand (IL) 6T1u “spin–orbit” state calculated at 4.309 eV, with a modest oscillator strength (0.002) and composed essentially of 23T1u, 13A1u and 23Eu  in the ground state [The calculations of Rosa et al.  assign tand 23Eu , should nd state , althoug1T1u←11A1g MLCT transition involving the contribution from (3t2u←4t2g) (0.64) and (12t1u←4t2g) (0.31)  from Band I and the ν2-mode has an average excitation energy of 0.047 eV, which may be compared to 0.052 eV (392 cm-1) in the ground state [2g(π)→t2g(π*) transitions, whereas Koerting et al. [1A1g→1A1g symmetry forbidden transitions.The photoabsorption spectrum of band II is shown in ) 0.31)  calculat  calculan from 3tu←4t2g (0←4t2g) 0.4 and (12 mode ν2 . The ν1-nd state . Gray an←4t2g) 0.4 and  but at lower energies , exploring the velocity distributions of the nascent photofragment yields [The photodissociation mechanisms of W(CO)t yields . A detait yields . The sin3T1u←11A1g transition involving the contribution from 12t1u←1t1g (0.27), 3t2u←1t1g (0.16), (12t1u←3t2g (0.16) and 3t2u←3t2g (0.11) calculated at 7.965 eV  33E/(0.20) 33T2u from the electronic ground state (13t1u←4t2g) (0.92) , and WC stretching mode ν2 . The lowest-lying vertical ionisation energy of tungsten hexacarbonyl has been reported by Lloyd and Schlag [The photoabsorption spectrum of band IV is presented in ) (0.92)  calculatnd 33T1u  may contansition –7, in cod Schlag  at 8.38 1T1u states of IL or sigma-bond-to-ligand charge transfer (SBLCT) character, whereas 3T1u and 3Eu states may also contribute significantly. In some states the singlet/triplet mixing may be rather important such as in 27T1u and 32T1u for instance.Tungsten hexacarbonyl displays a broad and structureless feature in this energy region . The mai6. Our high-resolution synchrotron photoabsorption measurements allowed for the identification of previously unresolved experimental features for the first time. The level of accuracy of our relativistic calculations has allowed the reassessment and reassignment of some states, particularly those previously explored below 8 eV. However, despite a reasonable agreement between the TDDFT and VUV absorption spectra we should be aware that the description of double excited states or diffuse Rydberg states is not accessible at the present level of calculation due to the limitation of TDDFT to single excitations. Unfortunately, the use of large diffuse basis sets necessary to correctly describe Rydberg states is beyond the current computational capabilities because of numerical problems due to near-linear dependencies in the basis sets. The combined experimental/theoretical investigation appears relevant to assess the role of W(CO)6 low-lying electronic states that can lead to dissociation. Thus, a reasonable assumption is given that the electronic excitation of tungsten hexacarbonyl leads to direct and statistical bond breaking, the former reminiscent of a repulsive dissociation character by the translational energy distribution of the first CO ligand, the latter correctly modelled by statistical product energy distributions [We have presented a comprehensive investigation of the experimental and theoretical electronic transitions in tungsten hexacarbonyl, W(CO)ibutions .File 1Additional computational data.


The DNA binding sites in the promoters of phosphate-responsive genes have not been defined, nor have any structure-function relationships been established for the Pho7 protein. Here we narrow this knowledge gap by (i) delineating an autonomous DNA-binding domain (DBD) within Pho7 that includes the Zn2Cys6 module, (ii) deploying recombinant Pho7 DBD in DNase I footprinting and electrophoretic mobility shift assays (EMSAs) to map the Pho7 recognition sites in the promoters of the phosphate-regulated pho1 and tgp1 genes to a 12-nucleotide sequence motif [5′-TCG(G/C)(A/T)xxTTxAA], (iii) independently identifying the same motif as a Pho7 recognition element via in silico analysis of available genome-wide ChIP-seq data, (iv) affirming that mutations in the two Pho7 recognition sites in the pho1 promoter efface pho1 expression in vivo, and (v) establishing that the zinc-binding cysteines and a pair of conserved arginines in the DBD are essential for Pho7 activity in vivo.Fission yeast phosphate homeostasis entails transcriptional induction of genes encoding phosphate-mobilizing proteins under conditions of phosphate starvation. Transcription factor Pho7, a member of the Zn 2Cys6 family of fungal transcription regulators, is the central player in the phosphate starvation response in fission yeast. The present study identifies a 12-nucleotide Pho7 DNA binding motif [5′-TCG(G/C)(A/T)xxTTxAA] in the promoters of phosphate-regulated genes, pinpoints DNA and protein features important for Pho7 binding to DNA, and correlates them with Pho7-dependent gene expression in vivo. The results highlight distinctive properties of Pho7 vis-a-vis other fungal zinc binuclear cluster transcription factors as well as the divergent cast of transcription factors deployed for phosphate homeostasis in fission yeast versus budding yeast.Fungi respond to phosphate starvation by inducing the transcription of a set of phosphate acquisition genes that comprise a phosphate regulon. Pho7, a member of the Zn Schizosaccharomyces pombe is achieved by regulating the transcription of genes encoding three proteins involved in extracellular phosphate mobilization and uptake: a cell surface acid phosphatase, Pho1; an inorganic phosphate transporter, Pho84; and a glycerophosphate transporter, Tgp1  analyses of DNA occupancy by Pho7-TAP in phosphate-replete and phosphate-starved cells affirmed the role of Pho7 in the phosphate starvation response, while highlighting an additional function in driving expression of multiple stress response genes independent of phosphate status  (pho1 transcription start site (We have shown that a 283-nucleotide (nt) segment of DNA 5′ of the in silico analysis of the Pho7 ChIP-seq data and (ii) biochemical footprinting of the Pho7 DNA-binding domain (DBD) on the pho1 and tgp1 gene promoters. We identify DNA features important for Pho7 binding and correlate them with Pho7-dependent gene expression. Our results provide new insights into the fission yeast phosphate response and illuminate distinctive properties of Pho7 vis-a-vis other fungal zinc binuclear cluster transcription factors.In the present study, we define the DNA site recognized by Pho7, via two independent approaches: (i) a revisited S. pombe Pho7 protein is most closely related to its 683-aa homolog from Schizosaccharomyces octosporus. Alignment of the two polypeptides highlights 291 positions of side-chain identity/similarity across their full lengths, with the highest conservation being in the central Zn2Cys6 domain  in which pho7 was either wild type (WT) or had double-alanine mutations of three serial cysteine pairs . Spot testing for growth on YES (yeast extract with supplements) agar medium showed that the pho7Δ strain grew slower than wild type at 20, 25, and 30°C (as gauged by colony size) and was extremely sick at higher (34 and 37°C) and lower (18°C) temperatures  eratures . All thr2Cys6 domain of Pho7 is homologous to the N-terminal Zn2Cys6 DNA-binding domain (DBD) of Saccharomyces cerevisiae Gal4, a prototypal fungal zinc cluster transcription factor. The DBD consists of a folded zinc finger module of two α-helices that recognizes the target DNA sequence, a linker that contacts the DNA phosphate backbone, and a helical coiled-coil module that mediates dimerization of the DBD -TAP and pho7(272–738)-TAP alleles. Both truncation strains grew like the wild type at 18 to 30°C; the truncation strains grew better than the pho7Δ mutant at 34 and 37°C, albeit with colony sizes slightly smaller than wild type  and pho7(272–738) truncation strains were 22% and 11% of that of the wild type, respectively  were processed for Pho7-TAP peak localization above background, and the peak sequences were screened for enriched motifs in MEME  on separate pages for phosphate-starved (−Pi) and phosphate-replete (+Pi) conditions. The motifs that correlate with the Pho7 DBD DNase I footprints in the pho1 and tgp1 promoters are highlighted in yellow. Download TABLE S1, XLSX file, 0.1 MB.Individual motif occurrences within the sequences of the Pho7 ChIP peaks, identified by FIMO , are lisCopyright © 2017 Schwer et al.2017Schwer et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01218-17.2TABLE S2 i) and phosphate-replete (+Pi) conditions. Download TABLE S2, XLSX file, 0.1 MB.Annotated genes located within 1 kb downstream of the Pho7 ChIP peaks (group 1) and those that have a Pho7 ChIP peak within the annotated gene (group 2) are listed on separate pages for phosphate-starved  protein (henceforth referred to as Pho7 DBD) was separated from the tag by a second round of nickel affinity chromatography and purified further by gel filtration. SDS-PAGE analysis revealed a predominant polypeptide that migrated at ~17 kDa relative to size standards , which spans the conserved Zntandards , althougg ladder  demarcatart site . We procg ladder  assignedectively . Both ofectively , that reanalysis .pho1 promoter by electrophoretic mobility shift assay (EMSA) using the 160-bp 5′ 32P-labeled DNA probe comprising nt −249 to −90 upstream of the pho1 transcription start site. Pho7 DBD formed a single discrete DNA-protein complex at lower Pho7 DBD concentrations that was converted to a more slowly migrating complex as the Pho7 DBD concentration was increased , between the two Pho7 sites (mutant 3), or between the Pho7 sites and a putative TATA box element, −34TATTTAA−28, preceding the transcription start site (mutants 6 and 7). The deleted dinucleotides are indicated by brackets above the pho1 promoter sequence in pho1Δ cells carrying the wild-type and promoter mutant plasmids were assayed for acid phosphatase activity. Mutation of the TATA box sequence effaced Pho1 expression, as did the dinucleotide deletions 1, 2, and 4 within the Pho7 binding sites  was annealed to RNA isolated from phosphate-starved yeast cells and then subjected to reverse transcription (RT). The RT primer extension product was analyzed in parallel with a chain-terminated sequencing ladder generated by DNA polymerase extension of the same 32P-labeled primer annealed to a DNA template containing the tgp1 locus (not shown). A single 5′ end was thereby located 42 nt upstream of the start codon of the tgp1 open reading frame (ORF)  .tgp1 promoter, we constructed a plasmid reporter in which a genomic DNA segment containing nt −871 to +42 of the tgp1 transcription unit (with +1 being the mRNA start site) was fused to the pho1 ORF  reduced acid phosphatase expression by 85%, mimicking the effects of pho7Δ and the promoter truncation AxxTTxAA site identified in silico. To understand the role of nucleobase sequence in Pho7 recognition, we prepared a series of 5′ 32P-labeled 24-bp DNA duplexes based on the footprinted Pho7 site in the tgp1 promoter that corresponded to the native wild-type dodecamer DNA sequence 5′-TCGGACATTCAA or had 2-base substitutions (underlined) as follows: 5′-TGCGACATTCAA (Mut1), 5′-TCGGACAAACAA (Mut2), or 5′-TCGGACATTCTT (Mut3). The 24-mers were tested by EMSA for binding to Pho7 DBD  or the GG (site 2) dinucleotide is altered in the site 1 or site 2 dodecamers of the pho1 promoter (A/T)xxTTxAA. We independently identified the same motif as a Pho7 recognition element via in silico analysis of available genome-wide ChIP-seq data  In contrast, the tgp1 promoter appears to contain only a single Pho7 binding site that is important for Pho7-dependent tgp1 promoter activity.The 738-aa Pho7 protein is the key transcriptional activator underlying fission yeast phosphate homeostasis. Here, we purified a recombinant Pho7 DNA-binding domain (DBD) that includes the Znin vivo, as gauged by two criteria: complementation of the conditional growth defects of a pho7Δ strain and the ability to mount a Pho1 inductive response to phosphate starvation. We surmise that DNA binding by Pho7 is necessary for its biological activity. The Pho7 DBD also has a pair of arginines that are conserved in Gal4, where they contact the phosphate backbone of the Gal4 DNA recognition site. Mutation of this arginine pair in Pho7 eliminated Pho1 expression during phosphate starvation, again consistent with DNA binding being crucial for Pho7 activity during the starvation response.The Pho7 DBD resembles the DBDs of other zinc cluster fungal transcription factors with respect to the presence and spacing of its six conserved cysteines. We find that the three pairs of zinc-coordinating cysteines are essential for Pho7 function S. octosporus  growth phenotype. With respect to phosphate homeostasis, deletion of the N domain sharply reduced Pho1 production in response to phosphate starvation. It is noteworthy that eight sites of Pho7 phosphorylation have been annotated within the N domain: S230, S244, S247, S249, S253, S257, T260, and T261 (http://www.pombase.org). Five of these phosphorylation sites are conserved in the S. octosporus N domain.In many of the zinc cluster fungal transcription factors, the DBD is located at the very N terminus of the polypeptide . This istosporus . A trunc2Cys6 proteins , which typically recognize pairs of CGG triplets that are arranged as inverted, direct, or everted repeats  it might have a unique binding mode in which the component promoters of a homodimer recognize different nucleotide sequence motifs or (ii) it might bind to DNA as a monomer, a la the Aspergillus transcription factor AlcR (The Pho7 DNA recognition site 5′-TCG(G/C)(A/T)xxTTxAA differs from those of other well-studied fungal Zn repeats –13. The tor AlcR . FurtherS. pombe is a member of the fungal Zn2Cys6 transcription factor family. In contrast, transcription of the phosphate regulon in phosphate-starved S. cerevisiae depends on cooperative action of two distinct transcription factors: Pho4 and Pho2 , they rely on quite different classes of DNA-binding transcription factors to achieve this response . As affiand Pho2 –17. Pho2nd Pho2 –. The crynd Pho2 –. There ioformans , 21. Thepho7-TAP integration cassettes marked with hygMX by PCR amplification using genomic DNA as a template and oligonucleotides that introduced restriction sites for cloning. The cassettes consisted of the following elements, proceeding from 5′ to 3′: (i) a 515-bp segment of genomic DNA 5′ of the pho7+ start codon; (ii) an open reading frame encoding wild-type Pho7, or mutated versions thereof, fused to a 560-bp ORF encoding the TAP tag  an hygMX gene (1.69 kbp) conferring resistance to hygromycin; and (v) a 728-bp segment of genomic DNA from nt +2517 to +3245 downstream of the pho7+ start codon. The integration cassettes were excised from the pKS plasmids and transformed into a haploid S. pombe strain. Correct insertions were verified by Southern blotting and sequencing of PCR-amplified DNA segments to ascertain the presence of the desired allele. To gauge the effect of these mutations on vegetative growth, cultures of S. pombe strains containing the indicated pho7-TAP allele were grown in liquid medium until the A600 reached 0.6 to 0.9. The cultures were adjusted to a final A600 of 0.1, and aliquots (3 µl) of serial 5-fold dilutions were spotted on YES agar plates, which were then incubated at 18, 20, 25, 30, 34, and 37°C.We constructed a series of pKS-based plasmids carrying  TAP tag ; (iii) aS. pombe pho7Δ and pho7-TAP strains were grown in YES medium at 30°C until the A600 reached 0.6 to 0.8. Aliquots (10 A600 units) of cells were collected by centrifugation and lysed in 20% trichloroacetic acid. Total acid-insoluble protein was recovered by centrifugation; the pellets were washed with ethanol and resuspended in 1 M Tris-HCl (pH 8.0). Aliquots of the samples, adjusted to contain the same total protein content based on the A280 of the extracts, were adjusted to 2% SDS and 0.1 M dithiothreitol (DTT) and then analyzed by electrophoresis through 8% polyacrylamide gels containing 0.1% SDS. The gel contents were then transferred to a 0.2-µm-pore polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membranes were probed by Western blotting with either rabbit polyclonal antibody recognizing the TAP tag (Thermo Fisher) or affinity-purified rabbit polyclonal anti-Spt5 antibody (antibody . Immune GSE39498) (S. pombe genome (ASM294v2.28) using Bowtie-2 2.2.9 . The motifs that correlate with the Pho7 DBD DNase I footprints in the pho1 and tgp1 promoters are highlighted in yellow. Annotated genes within 1 kb of the peaks were identified using a python script (Pho7 ChIP-seq data sets were accessed from GEO data sets (accession no. SE39498) . The FAS-2 2.2.9 . The res-2 2.2.9 . Pho7 pe-2 2.2.9 . For pea-2 2.2.9  and saven script  and are 10.1128/mBio.01218-17.3TABLE S3 i) and phosphate-replete (+Pi) conditions. Download TABLE S3, XLSX file, 0.1 MB.Position probability matrices for the Pho7 motifs predicted by MEME  under phCopyright © 2017 Schwer et al.2017Schwer et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01218-17.4TABLE S4 i) and phosphate-replete (+Pi) conditions. Download TABLE S4, XLSX file, 0.1 MB.The Pho7 ChIP peaks identified by HOMER  are listCopyright © 2017 Schwer et al.2017Schwer et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10-Smt3-Pho7(279–368) encodes the central Zn2Cys6 domain fused to an N-terminal His10-Smt3 module under the transcriptional control of a T7 RNA polymerase promoter. The plasmid was transfected into E. coli BL21(DE3) cells. Cultures (2 liters) amplified from single kanamycin-resistant transformants were grown at 37°C in Terrific Broth containing 50 µg/ml kanamycin until the A600 reached 0.8. The cultures were chilled on ice for 1 h and adjusted to 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and then incubated for 20 h at 18°C with constant shaking. All subsequent steps of purification were performed at 4°C. Cells were harvested by centrifugation and resuspended in 35 ml buffer A . The cells were lysed by sonication, and the insoluble material was removed by centrifugation at 18,000 rpm for 45 min. Supernatants were mixed for 1 h with 4 ml of Ni-nitrilotriacetic acid (NTA) resin (Qiagen) that had been equilibrated with buffer A. The resin was recovered by centrifugation and resuspended in 50 ml buffer A. The washed resin was centrifuged again, resuspended in 50 ml buffer A, and then poured into a column. The bound material was step eluted with buffer A containing 300 mM imidazole. The polypeptide compositions of the flowthrough and eluate fractions were monitored by SDS-PAGE. The 300 mM imidazole eluate fractions containing His10-Smt3-Pho7(279–368) were supplemented with Smt3-specific protease Ulp1 and then dialyzed overnight against 2 liters buffer B . The dialysates were mixed for 1 h with 2 ml of Ni-NTA resin that had been equilibrated with buffer B. Tag-free Pho7(279–368) was recovered in the flowthrough fractions and then subjected to gel filtration through a Superdex-200 column  equilibrated in a mixture of 50 mM Tris-HCl (pH 7.5), 200 to 250 mM NaCl, and 10% glycerol. Peak fractions were pooled, concentrated by centrifugal ultrafiltration, and then stored at −80°C at a concentration of 10 mg/ml. Protein concentration was determined as follows. Aliquots of Pho7 DBD and known amounts of bovine serum albumin (BSA) standard were serially diluted and adjusted to 2% SDS and 0.1 M DTT and then resolved by electrophoresis through a 15% polyacrylamide gel containing 0.1% SDS. The gel contents were stained with Coomassie brilliant blue R-250 followed by destaining in a solution containing 22.9% ethanol and 8.4% acetic acid. The gel was scanned, and the staining intensity of the Pho7 DBD and BSA polypeptides was quantified using ImageJ software. A BSA standard curve was generated by plotting intensity as a function of input BSA protein (micrograms); the concentration of Pho7 DBD was calculated by interpolation to the BSA standard curve.Plasmid pET28b-His32P-labeled DNA fragments were generated by PCR amplification of pho1 and tgp1 promoter segments using 5′ 32P-labeled forward primers (prepared with [γ-32P]ATP and T4 polynucleotide kinase) and nonlabeled reverse primers. The PCR fragments were purified by electrophoresis through a native 8% polyacrylamide gel in 1× TBE buffer , eluted from an excised gel slice, ethanol precipitated, and resuspended in 10 mM Tris-HCl (pH 7.4)–1 mM EDTA at a concentration of 0.6 to 1 µM. Alternatively, 5′ 32P-labeled oligonucleotides containing Pho7 binding sites were separated from [γ-32P]ATP by Sephadex G25 gel filtration and annealed to a 1.5-fold molar excess of a complementary nonlabeled DNA oligonucleotide in a mixture of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 250 mM NaCl by heating for 20 min at 65°C followed by slow cooling to room temperature. The labeled oligonucleotide duplexes were ethanol precipitated, resuspended in 10 mM Tris-HCl (pH 7.4)–1 mM EDTA–10% glycerol, and then purified by electrophoresis through a native 12% polyacrylamide gel. EMSA reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.4), 10% glycerol, 340 ng poly(dI-dC) (Sigma), 0.6 or 1 pmol 32P-labeled DNA (as specified in figure legends), and 2 µl Pho7 DBD  were incubated for 10 min at room temperature. The mixtures were analyzed by electrophoresis through a native 6% polyacrylamide gel containing 2.5% (vol/vol) glycerol in 0.25× TBE buffer. 32P-labeled DNAs (free and Pho7 bound) were visualized by autoradiography. Where indicated, the extent of DNA binding to Pho7  was quantified by scanning the gel with a phosphorimager.32P-labeled DNA fragments of the pho1 and tgp1 promoters were generated by PCR amplification as described above, using either a 5′ 32P-labeled forward primer and nonlabeled reverse primer (to label the top strand) or a 5′ 32P-labeled reverse primer and nonlabeled forward primer (to label the bottom strand). Footprinting reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.4), 10% glycerol, and 32P-labeled DNA and Pho7 DBD as specified were incubated for 10 min at room temperature. The mixtures were then adjusted to 2.5 mM MgCl2 and 0.5 mM CaCl and reacted with 0.04 U DNase I (New England Biolabs) for 90 s at room temperature. The DNase I reaction was quenched by adding 200 µl of stop solution . The mixture was phenol-chloroform extracted, ethanol precipitated, and resuspended in 90% formamide–50 mM EDTA. The samples were heated at 95°C and then analyzed by urea-PAGE. 32P-labeled DNAs were visualized by autoradiography.S. pombe cultures in YES (yeast extract with supplements) medium were harvested, the cells were washed in water and adjusted to A600 of ~0.3 in PMG (Pombe glutamate) medium without phosphate. After incubation for 5 h at 30°C, cells were harvested, washed and suspended in water. To quantify Pho1 acid phosphatase activity, reaction mixtures (200 µl) containing 100 mM sodium acetate (pH 4.2), 10 mM p-nitrophenylphosphate, and cells (0.02 or 0.1 A600 unit) were incubated for 5 min at 30°C. The reactions were quenched by adding 1 ml of 1 M sodium carbonate, the cells were removed by centrifugation, and the absorbance of the supernatant at 410 nm was measured. Acid phosphatase activity is expressed in A410 (p-nitrophenol production) to A600 (cells). Each data point is the average (±standard error of the mean [SEM]) from three phosphatase assays using cells from three independent cultures.Aliquots of exponentially growing pho1  were pooled, and cultures were grown in YES medium containing G418 (150 µg/ml). Aliquots of exponentially growing cultures were harvested, washed, and suspended in water. Pho1 acid phosphatase activity was quantified as described above. Each data point in the bar graphs in To measure acid phosphatase activity using the pho1  and tgp1gp1-pho1  plasmid  deleted , were tr


Uracil derivatives have a great attraction because they play an important role in pharmacological activities. Pyrimidodiazepines, lumazines, triazolopyrimidines and xanthines have significant wide spectrum activities including anticancer, antiviral as well as antimicrobial activities.b]diazepines 5a–e, 6a–d, lumazines 7a–d, triazolopyrimidine 8 and xanthines 9, 10 was prepared in a good yields. The antimicrobial and antioxidant activities of compounds 5a, 5b, 6a, 6d and 8 exhibited a wide range activity against the pathogenic tested microbes . Compound 8 showed activity against the fungus Aspergillus niger. The highest antioxidant activity was noticed for compound 5a.A newly synthesized compounds pyrimidodiazepines 5a–e, 6a–d, lumazines 7a–d, triazolopyrimidine 8 and xanthines 9, 10 was prepared from 5,6-diamino-1-(2-chlorobenzyl)uracil 3 in good yields. Compounds 5a–e, 6a–d were prepared by sequential manipulation of 3 with α,β-unsaturated ketones. Lumazines 7a–d were obtained from 3 by treatment with phenacyl bromides in the presence of TEA. Compound 8 was prepared by treatment of 3 with HNO2, while xanthines 9, 10 were obtained from 3 by consecutive acetylation then intramolecular cyclodehydration or heating with malononitrile under solvent-free condition. The antimicrobial and antioxidant activity of this series was evaluated in vitro and they showed either weak or moderate activities.A series of novel pyrimido[4,5- Uracil is a basic scaffold for design of significant pharmaceuticals –6. They To solve these problems, researchers are required to modify the structure of uracil and subsequently these problems can be overcome by innovation of new derivatives with beneficial pharmacological and pharmacokinetic effects. These new fused uracil derivatives as antibacterial agents can be obtained via replacement at N-1, N-3, C-5 and C-6 positions with different substituents on uracil ring. Seven-member heterocyclic compounds containing nitrogen atom, such as 1,4-diazepine derivatives, are considered as an important drug discovery because they have a wide range of antimicrobial activities .1H-NMR, mass spectral data, IR and elemental analysis.The purpose of this study is to evaluate the in vitro effect of antimicrobial fused uracil derivatives, pyrimidodiazepines, lumazines, triazolouracil and xanthines. Simultaneously, a MIC-kinetic curve for the inhibition activity of the new molecules was also obtained. The structure of newly synthesized uracil-based derivatives was proven on the basis of their 3 diazepine-7-carbonitrile. The IR spectra of these diazepines displayed the C≡N stretching band at 2222–2217 cm−1 confirming cyclization, the stretching band of the two C=O groups (Amide I) was red-shifted within the range 1690–1610 cm−1. Derivatives 5d, e displayed two separate bands for the two C=O groups. The imide linkages in this series displayed keto-iminol tautomerism, since they showed O–H stretching bands 3634–3617 cm−1 and additional O–H stretching bands in compound 5d at 3495 cm−1 and N–H stretching bands 3164–3141 cm−1. The nitro group in compound 5e showed strong asymmetric and symmetric NO2 stretching bands at 1518 and 1350 cm−1, respectively. The intrinsic significance of the IR spectra is that they exclude the possibility of the cyclization pass way that lead to compounds 4a–e due to absence of any blue-shifted C=O stretching bands.To our endeavor toward developing new uracil-based architectures of potential pharmacological significance, 5,6-diamino-1-(2-chlorobenzyl)uracil 3  was chos3 –33, nitr3 diazepine-7-carbonitriles (6a–d) in 53–69% yields after recrystallization from DMF/EtOH  biosynthesis and, consequently the production of nucleic acid bases necessary for its replication. Sulphonamides (sulpha drugs) are common inhibitors of FH4 biosynthesis and act as bacteriostatic. Therefore, substrate 3 was treated with different phenacyl bromides in refluxing DMF containing TEA to afford lumazines 7a–d in good yields as potential folate antagonists 2 under solvent-free condition. The IR spectrum displayed the C≡N stretching band at proper frequency 2200 cm−1, while the 1H-NMR disclosed two signals at δ 5.08 ppm for the NCH2 protons and at δ 4.10 ppm for the protons in the CH2CN group.Xanthine This series displayed, in their EI-MS spectra, molecular ions peaks corresponding to the mass of each formula and their elemental analyses agreed as well.6b exhibited low to moderate activity only against Pseudomonas aeruginosa. Compound 7a exhibited low to moderate activity only against Saccharomyces cerevisiae. Some other compounds  exhibited activities against wide range of pathogenic tested microbes. The minimal inhibitory concentrations (MIC) of these compounds had been measured  have been measured (Table 5a showed the highest activity (39.9%) followed by the compound 8. The lowest antioxidant activity recorded for the compound 6c is 1.9. Two compounds 7a and 7b showed no antioxidant activity.The percentages of antioxidant activity (AA%) of compounds ed Table  and the Materials and instruments1H-NMR) spectra were recorded on Bruker 400 MHz Spectrometer and 13C-NMR spectra were run at 125 MHz in dimethylsulfoxide (DMSO-d6) and TMS as an internal standard, Applied Nucleic Acid Research Center, Zagazig University, Egypt. All new compounds gave corresponding elemental analyses . All reactions were monitored by TLC using precoated plastic sheets silica gel (Merck 60 F254) and spots were visualized by irradiation with UV light (254 nm). The used solvent system was chloroform: methanol (9:1) and ethyl acetate: toluene (1:1).All melting points were determined by an Electrothermal Mel.-Temp. II apparatus and were uncorrected. Element analyses were performed at Regional Center for Mycology and Biotechnology at Al-Azhar University. The infrared (IR) spectra were recorded using potassium bromide disc technique on Nikolet IR 200 FT IR. Mass spectra were recorded on DI-50 unit of Shimadzu GC/MS-QP 5050A at the Regional Center for Mycology and Biotechnology at Al-Azhar University. The proton nuclear magnetic resonance  was added over 15 min to ammonium sulphide solution (36 ml) at 70–80 °C with stirring. The formed precipitate was collected by filtration, washed with ethanol and dried in vacuum desiccator to give 92% diazepines 5a–e, 6a–d, lumazines 7a–d, triazolopyrimidine 8 and xanthines 9, 10 were prepared by a simple method from 5,6-diamino-1-(2-chlorobenzyl)uracil 3. The novel compounds were screened for both antimicrobial and antioxidant activities. Compounds 5a, 5b, 6a, 6d and 8 showed a wide range activity against the pathogenic tested microbes  in comparison to the standard drug Benzylpenicillin. Compound 8 was the only novel synthesized compound exhibited activity against the fungus A. niger in comparison to the standard drug Nystatin. On the other hand, Compound 5a showed the highest antioxidant activity followed by compound 8. While, compounds 7a and 7b showed no antioxidant activity.A series of newly synthesized compounds of pyrimido[4,5-


Ceratogyrus darlingi. Cd1a reversibly paralysed sheep blowflies (PD50 of 1318 pmol/g) and inhibited human Cav2.2 (IC50 2.6 μM) but not Cav1.3 or Cav3.1 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp electrophysiological assays Cd1a inhibited rat Cav2.2 with similar potency (IC50 3 μM) without influencing the voltage dependence of Cav2.2 activation gating, suggesting that Cd1a doesn’t act on Cav2.2 as a classical gating modifier toxin. The Cd1a binding site on Cav2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Cav2.2-mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Nav1.1–1.2 and Nav1.7–1.8 (IC50 0.1–6.9 μM) but not Nav1.3–1.6 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp assays, Cd1a strongly inhibited human Nav1.7 (IC50 16 nM) and produced a 29 mV depolarising shift in Nav1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Nav1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Cav2.2 inactivation gating, associated with the Cav2.2 α-subunit pore, while altering the activation gating of Nav1.7. Cd1a was inactive at some of the Nav and Cav channels expressed in skeletal and cardiac muscles and nodes of Ranvier, apparently contributing to the lack of side effects at efficacious doses, and suggesting potential as a lead for development of peripheral pain treatments.Spider venoms are rich sources of peptidic ion channel modulators with important therapeutical potential. We screened a panel of 60 spider venoms to find modulators of ion channels involved in pain transmission. We isolated, synthesized and pharmacologically characterized Cd1a, a novel peptide from the venom of the spider  Cav and Nav are structurally similar, consisting of a pore-forming α subunit and 1–3 auxiliary subunits, including a number of Nav β isoforms and Cav β, α2δ, and γ auxiliary subunits ethanesulfonic acid; HEPES 10), plus 0.1% BSA and nifedipine (10 μM) to fully inhibit endogenous Cav1 channels. The crude venoms (at 40 μg/well) were tested for activity against human (h) Cav2.2 channels in SH-SY5Y cells using a FLIPRTETRA  Ca2+ imaging assay, as detailed below -GVIA; iodinated at Tyr22) was prepared using IODOGEN as previously described [125I]-GVIA was diluted to 20,000 cpm/50 μL ([30 pM]). For displacement studies, [125I]-GVIA was incubated with mouse brain membranes or SH-SY5Y membranes and varying concentrations of the competing ligand on 96-well plates. The plates were incubated with shaking for 1 h at room temperature and vacuum filtered through a glass-fibre filter pre-soaked in 0.6% polyethyleneimine (PEI) to reduce non-specific binding and washed with buffer containing 20 mM HEPES and 125 mM NaCl at pH 7.2 using a Tomtec harvester vacuum system . The filters were then dried at 37°C before being placed in sample bags and soaked in liquid scintillant. Retained radioactivity was then counted using a MicroBeta JET microplate liquid scintillation counter . Non-specific binding was determined in the presence of 50 μL of unlabelled peptides. Specific binding was calculated as the difference between total and non-specific binding. A one-site model was fitted to the data using GraphPad Prism v5.0. Results are presented as the mean ± SEM of 3–6 replicates, performed at minimum 3 independent experiments. Statistical significance was determined using analysis of variance (ANOVA) or a Student’s t-test, with statistical significance defined as p < 0.05.Radiolabelled conotoxin GVIA  Cav2.2 (Cav α1b co-expressed with Cav β1b and Cav α2δ subunits) or mutant rCav2.2-G1326P, as previously described /[1+exp(Va–Vm)/ka)], where Vm is the test potential, Va is the half-activation potential, Erev is the reversal potential, Gmax is the maximum slope conductance, and ka reflects the slope of the activation curve. The voltage dependence of steady-state inactivation was calculated by dividing the amplitude of the test current (I) by the maximal current elicited (Io). Steady-state inactivation curves were fitted using the Boltzmann equation: I/Io = 1/(1+exp((Vm–Vh)/k)), where Vh is the half-inactivation potential, k is the slope factor and Vm is the holding voltage. Statistical significance was determined by paired or unpaired Student’s t-tests and one-way or repeated measures ANOVA for n ≥ 6 independent experiments, followed by Dunnett’s post-hoc test. Differences were considered significant if p < 0.05. Data are expressed as mean ± SEM.The Cahttp://www.iasp-pain.org), in agreement with the Animal Care and Protection Regulation Qld (2012), and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 8th edition (2013) (http://www.nhmrc.gov.au). Ethics approval was obtained from the University of Queensland Institutional Animal Ethics Committee. C57BL/6J, an inbred mouse strain widely used in biomedical research . Cm. Cmv chav1.3, hCav2.2 and hCav3.1 channels expressed in SH-SY5Y cells [v2.2 responses, in SH-SY5Y cells : Cd1a 2.6 ± 0.93 μM; Cm1a 0.4 ± 0.2; and Cm1b 1.1 ± 0.6; v1.3 and mibefradil/pimozide-sensitive Cav3.1 (Cd1a tested up to 30 μM and Cm1a and Cm1b 10 μM). Despite a tendency to some variability in potencies, the Cav2.2 activity of the three peptides were not statistically different (two-way Anova).We assessed Cd1a activity on native hCa5Y cells , and com125I-GVIA) in competitive binding studies to investigate the Cd1a mode of action. ω-Conotoxin GVIA is known to bind to Cav2.2 channel α-subunit pore, in a region localized within the external EF hand motif of domain III S5–S6 (see 125I-GVIA from both human SH-SY5Y (IC50 of 0.18 ± 0.01 nM) and mouse brain cell membranes (IC50 of 0.27 ± 0.01 nM)   with pot2+ currents measured using whole-cell voltage-clamp electrophysiology on rCav2.2  expressed in tsA cells. Cd1a (3 μM) inhibited the currents by ~50% (n = 9) and there was no recovery from block during a 5 min wash-off period  were not significantly different from untreated cells   . In cont–54.6 mV . The absnd ω-IVA .v2.2 carrying a G1326P mutation (Cav2.2-G1326P), which lies on the large extracellular loop region of the Cav2.2 α subunit. This mutation results in localized structural disruption within the domain III S5–H5 region associated with the external EF hand motif within domain III S5–S6 [v2.2 channels showed no recovery from Cd1a-evoked block, Cd1a inhibition of the mutant Cav2.2-G1326P channel was reversed following a 5 min washout  in contrast to its lack of effect at the wild-type channel. The voltage dependence of activation of the mutant, like the native rCav2.2, was unaffected , whereas the voltage dependence of inactivation underwent a small but significant shift , with each peptide inhibiting Nav1.1, Nav1.2 and Nav1.7 , but inhibited hNav1.3 and Nav1.6. Cd1a was the only peptide with activity at Nav1.8 , while depolarization to 0 mV following a conditioning pulse was used to assess its effects on the steady-state inactivation (Vh)  (h) of hNav1.7  (We assessed the Cd1a effect on hNaion (Vh) . Interes n = 11) . Further, n = 9) .v1.7 voltage sensor domains. Under control conditions there was no significant change between a first (P1) and third (P3) pulses separated by a strong positive pulse (P2)  Insert. Cd1a atv1.7 channels [50 = 0.36 ± 0.12 pmol) (V1.7 reversed spontaneous pain behaviours (40 pmol), TTX caused sedation, and reduced motor coordination  in mice , an effechannels . Intrapl12 pmol) . For comdination  in all m50 measured 1 hour after injection of 1318 ± 58 pmol/g , or Nav channels found in skeletal muscle (Nav1.4) or nodes of Ranvier in motor nerves (Nav1.6). The Cd1a selectivity profile is consistent with its analgesic efficacy and lack of side effects at maximal efficacious doses in a mouse model of peripheral pain shown in this work.We describe the discovery and characterization of Cd1a, the first Nafamily 1 , a classteolysis , 44, feav channels, likely reflecting its relatively low potency against Cav2.2 (IC50 of ~3 μM at rat and human Cav2.2). Like Cd1a, the related peptides Cm1a and Cm1b from C. marshalli [v inhibitors and have little sequence similarity to Cav inhibitors. However we found for the first time that these peptides inhibit hCav2.2 with moderate potency, but were inactive at hCav1.3 and hCav3.1. Our results contrasted with a previous report on patch clamp electrophysiology, which indicated that Cm1a and Cm1b were inactive at HVA calcium currents of sensory neurons [v channel gating modifier toxins, Cd1a didn’t affect activation gating of rCav2.2 but instead induced a small shift in the voltage-dependence of inactivation, suggesting that at this channel Cd1a doesn’t act as a typical gating modifier toxin. Cd1a activity was enhanced at the rCav2.2α1B-G1326P mutant channel while channel inhibition became partially reversible, indicating possible partial overlap with the binding site for ω-conotoxins. In addition, Cd1a caused a small change in reversal potential of the mutant channel but not the wild-type, while channel inhibition became partially reversible, indicating binding near the ion permeation/selectivity pathway. However, competitive binding studies revealed that the Cd1a binding does not completely overlap with the GVIA binding site on mutant Cav2.2. Thus, we suggest a possible allosteric coupling between the Cd1a and GVIA binding site that is introduced by the rCav2.2α1B-G1326P mutation. Residues in the outer vestibule of the channel pore may allosterically affect Cd1a binding and its ability to interact with the inactivated state of the channel. Precisely how Cd1a influences the voltage dependence of inactivation remains to be determined, but it is possible that the toxin interacts with one or more of the S6 regions, which have been linked to fast inactivation [Cd1a has little sequence similarity to other venom peptides that inhibit Caarshalli  are know neurons . However neurons , thus attivation .v inhibitor peptides Cm1a and Cm1b suggested that Nav channels may be the primary high-affinity target for Cd1a. Indeed, in electrophysiological studies Cd1a was ~200-fold more potent at recombinant hNav1.7 than rCav2.2, inhibiting hNav1.7 with an IC50 of ~15 nM. Whereas we have previously shown that species differences can lead to discrepancies in potencies [50 for Cd1a at human and rat Cav2.2 were similar in both in patch clamp and fluorescence assays (~3 μM), confirming the primary target for Cd1a is Nav1.7.The high level of homology between Cd1a and the Naotencies , the IC5v channels, including Nav1.1–1.2 and Nav1.7. However, Cd1a potency at Nav1.7 was lower using the FLIPR assays than when using patch clamp electrophysiology (~200-fold), consistent with previous reports [v1.8 activity for Cm1a and Cm1b, in contrast with previous report [v isoforms is typically conserved, differences in potencies between patch clamp electrophysiology (‘the gold standard method for assessing ion-channel function’) and fluorescence based assays have been described [Using a FLIPR membrane potential dye assay, we identified that Cd1a, Cm1a and Cm1b were similarly active across a range of hNa reports . In addis report . Althougescribed , 47, 48.v2.2, the human Nav1.7 inhibition was driven by a depolarising shift in Va. This effect on Nav1.7 is consistent with gating modifying activity through one or more of the voltage sensor domains (VSD) [v voltage sensor domains has been achieved with strong positive depolarizing pulses, releasing gating modifier toxins from the voltage sensor domains [Although Cd1a didn’t affect the steady-state voltage parameters of activation of rat Cans (VSD) , 39, 42. domains , 42 and  domains , 42.v channels, Cd1a was named β-theraphotoxin-Cd1a (or β-TRTX-Cd1a as abbreviation), based on the rational nomenclature devised for spider toxins [v channels remains to be elucidated, spider-venom peptides with similar modes of action have been found to bind to VSDs DI—DIII but not to DIV, which appears to influence inactivation rather than activation [Due to its pharmacology profiling and mode of action, with the highest activity at Nar toxins . While ttivation –52.v1.7 channel activator OD1 [v1.1–1.4 and/or Nav1.6 [v1.3, Cav2.1 Nav1.4, Nav1.5 and Nav1.6 (30 μM). Cd1a was potent at centrally expressed Nav1.1–1.2, however, it is unlikely to cross the blood-brain barrier and interact with these channels centrally [We assessed the analgesic potential of Cd1a by examining its ability to reverse spontaneous pain induced by intraplantar injection of the Naator OD1 , 30, 43.r Nav1.6 . Consistentrally .In future, Cd1a needs to be tested in other more conventional animal models of pain, such as the inflammation and neuropathic pain models, to confirm its usefulness as a peripheral analgesic lead, and to provide information tools to compare Cd1a with peptides from other animal venoms.Whereas the analgesic effect of Cd1a might be useful as a therapeutic lead, we wondered about the ecological significance of Cd1a. Pain-inducing venom components for defensive purposes have been previously reported from tarantula venoms , but thev2.2, Nav1.7 and Nav1.8. Cd1a additionally inhibits the central Nav1.1–1.2, but not peripheral off-target channels such as Cav1.3 and Cav2.1 or Nav1.4–1.6. Interestingly, Cd1a acts at Cav2.2 and Nav1.7 with distinct modes of action, inhibiting Cav2.2 activity affecting near the pore region, but not overlapping the ω-conotoxin binding site, interfering with inactivation with no apparent effects on the activation gating of the channel. Conversely, at Nav1.7 Cd1a acts as a typical gating modifier toxin, interacting with one or more of the voltage sensor domains and changing the gating properties of this channel. The primary structure of Cd1a strongly suggests that it will fold into an ICK motif that is expected to provide a high level of chemical, thermal and biological stability. Thus, Cd1a may be a useful lead for development of a peripherally acting analgesic. In future studies it will be important to examine Cd1a analgesic potential in a wider range of pain models to confirm its usefulness as an analgesic lead.In summary, we have isolated and pharmacologically characterized Cd1a, a novel peptide inhibitor that showed insecticidal effects and acts on the anti-nociceptive targets CaS1 Table(DOCX)Click here for additional data file.S1 FigProposed transmembrane topology of the calcium channel α1 subunit indicating the location of the putative EF hand motif in IIIS5-H5 where G1326P is located.(TIF)Click here for additional data file.


Improving the hydrolytic performance of hemicellulases to degrade lignocellulosic biomass is of considerable importance for second-generation biorefinery. Xylanase, as the crucial hemicellulase, must be thermostable and have high activity for its potential use in the bioethanol industry. To obtain excellent xylanase candidates, it is necessary to understand the structure–function relationships to provide a meaningful reference to improve the enzyme properties. This study aimed to investigate the catalytic mechanism of a highly active hyperthermophilic xylanase variant, XYL10C-ΔN, for hemicellulose degradation.By removing the N-terminal 66 amino acids, the variant XYL10C-ΔN showed a 1.8-fold improvement in catalytic efficiency and could hydrolyze corn stover more efficiently in hydrolysis of corn stover; however, it retained similar thermostability to the wild-type XYL10C. Based on the crystal structures of XYL10C-ΔN and its complex with xylobiose, Glu175 located on loop 3 was found to be specific to GH10 xylanases and probably accounts for the excellent enzyme properties by interacting with Lys135 and Met137 on loop 2. Site-saturation mutagenesis confirmed that XYL10C-ΔN with glutamate acid at position 175 had the highest catalytic efficiency, specific activity, and the broadest pH-activity profile. The functional roles of Glu175 were also verified in the mutants of another two GH10 xylanases, XylE and XynE2, which showed increased catalytic efficiencies and wider pH-activity profiles.XYL10C-ΔN, with excellent thermostability, high catalytic efficiency, and great lignocellulose-degrading capability, is a valuable candidate xylanase for the biofuel industry. The mechanism underlying improved activity of XYN10C-ΔN was thus investigated through structural analysis and functional verification, and Glu175 was identified to play the key role in the improved catalytic efficiency. This study revealed the importance of a key residue (Glu175) in XYN10C-ΔN and provides a reference to modify GH10 xylanases for improved catalytic performance.The online version of this article (10.1186/s13068-018-1150-8) contains supplementary material, which is available to authorized users. The rising global demand for renewable fuels and the climate change associated with the consumption of fossil fuels are considered as the biggest social challenges over the next few decades , 2. Bioed-xylanase, EC 3.2.1.8) is a key enzyme that randomly cleaves the internal β-1,4-d-xylosidic linkages of xylan to yield different chain lengths of xylo-oligosaccharides [8 barrel fold structure  with catalytic residues at the end of β-sheets 4 and 7 [Hemicellulose is the second most abundant polysaccharide on earth. Efficient conversion of hemicellulose to other value-added fermentation products has both environmental and economic benefits . Xylan, charides . Based ocharides . GH10 xy 4 and 7 , while G 4 and 7 , 13.π), fold on triggered trimerization, which affects the catalytic process of proteins, making them potential foci for protein engineering [Currently, the commercial uses of xylanases are mainly in the paper, food, and animal feed industries , 14, howineering –27. Amonineering , 26. Theineering , 29.Bispora sp. MEY-1 is an attractive candidate industrial enzyme that has remarkable activity and favorable thermostability [To meet the rigorous requirements of industrial processes, and to decrease the production-cost, highly active xylanases with excellent thermostability are of great interest. The GH10 xylanase, XYL10C, from acidophilic tability . The prePichia pastoris and purified . The enzymes had similar pH stability , and specific activity values  from piformis  as a seaTo fully understand the substrate binding and catalysis mechanisms, co-crystallization of XYL10C-ΔN and xylobiose was also completed at a resolution of 1.7 Å. As shown in Fig. As shown in the structure of the XYL10C-ΔN-xylobiose complex Fig. . Thus, rKm values (1.1–4.8-fold) and decreased kcat/Km values (1.0–37.3 times), as well as decreased specific activities (0.8–32.5 times) . The Km values of XYL10C-ΔN and XYL10C-ΔN-E175N were very similar, but were significantly different from those of other variants. The results indicated that residue substitution at position 175 probably altered the intramolecular interaction, the protein structure, or the substrate conformation, which consequently changed the enzyme’s properties.XYL10C-ΔN mutants with Glu175 replacement were successfully constructed, expressed, and purified . These results confirmed the functional roles of Glu175 in the catalytic performance and pH-activity profile of GH10 xylanases. However, the importance of Glu175 in the catalytic efficiency of other GH10 xylanases remains to be determined.Two xylanases, XylE and XynE2, were selected to verify the roles of Glu175 in the catalytic performance and pH adaptability of GH10 xylanases. Compared with the wild-type proteins, residue substitution caused no changes in the temperature and pH optima of most mutants , the reducing sugar amounts released were 9.1 and 9.4 µmol, and during the first 2 h of reaction, the sugar production rates increased by 2 and 1.6 times, respectively. The results indicated that T. reesei cellulase and XYL10C or XYL10C-ΔN have a synergistic activity to degrade steam pretreated corn stover, and that XYL10C-ΔN is more efficient than the wild-type for the hydrolysis of corn stover.Steam-exploded corn stover mainly comprises cellulose and hemicellulose. When using steam-exploded corn stover as the substrate, the hydrolytic capabilities of commercial cellulase from Bispora sp. MEY-1 showed great application potential in the bioethanol industry [Talaromyces leycettanus xylanase TlXyn10A (54%) [Penicillium canescens xylanase XylE (53%) [TlXyn10A (no enzyme activity loss after 1 h-incubation at 80 °C), which is better than XylE (completely inactive after 30 min incubation at 70 °C). Compared with other GH10 xylanases, such as reAuXyn10A from Aspergillus usamii (5448 mg/mL) [Geobacillus stearothermophilus [MpXyn10A from Malbranchea pulchella [GtXyn10 from Gloeophyllum trabeum [The study of the relationship between enzyme structure and function provides an important reference for efficient protein engineering . Artificindustry . Multiplindustry –39. In t0A (54%)  and PenilE (53%) , but has8 mg/mL) , the H17mophilus , MpXyn10ulchella , and GtX usamii 58 mg/mL [Cellvibrio japonicus xylanase 10C, having an insertion of Tyr and a substitution of Glu/Gly at subsites − 3 and − 2, showed very low activity on less polymerized substrates [8 enzymes [GH10 xylanases have conserved amino acids located in the shallow slot around the top face of the protein molecule, including two catalytic glutamates and five to seven substrate binding subsites –48. Amonbstrates . XYL10C-bstrates , 31. As  enzymes , 50. TheXylanases with high specific activity, good catalytic efficiency, and excellent stability are required for biorefinery uses. Recent studies have shown that the longer the xylo-oligosaccharides, the greater the extent of cellulase inhibition . In addiIn the present study, we obtained a xylanase variant XYL10C-ΔN with excellent thermostability, high catalytic efficiency, and synergistic action with cellulase in the degradation of straw. A key residue on loop 3, Glu175, was found to interact with Lys135 and Met137 of loop 2, and could account for the improved catalytic efficiency and broadened pH-activity profile. This study not only provides an excellent candidate xylanase for potential industrial use, but also provides the theoretical and practical basis for the modification of GH10 xylanases.xyl10c-ΔN, was amplified by PCR with a primer set . The gene fragment was then cloned into the expression vector pPIC9 at the EcoRI and NotI restriction sites using the T4 ligase. The recombinant plasmid pPIC9-xyl10c-ΔN was linearized by BglII and transformed into P. pastoris GS115 competent cells using a Gene Pulser X cell Electroporation System (Bio-Rad). Recombinant expression and fermentation were conducted following the method previously described [The gene fragment coding for the truncated XYL10C (GenBank Accession Number: FJ492963) without the N-terminal 66 amino acid residues, designated escribed .g, 4 °C, and 10 min), followed by concentration through a Vivaflow ultrafiltration membrane (Vivascience) with a molecular weight cut-off of 10 kDa. The crude enzymes were loaded onto a FPLC HiTrap Q Sepharose XL 5 mL column  that was equilibrated with 20 mM McIlvaine buffer (pH 6.5). Enzymes were eluted utilizing a linear gradient of NaCl (1.0 M) in the same buffer at a flow rate of 4.0 mL/min. Fractions exhibiting xylanase activities were collected and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined using the Bradford method with albumin from bovine serum as a standard. To remove N-glycans, approximately 600 mg of purified recombinant XYL10C-ΔN was treated by endo-β-N-acetylglucosaminidase H  in 50 mM sodium citrate (pH 5.6) at 300 K for 1 day. The reaction mixture was then dialyzed twice against 50 mM sodium citrate (pH 6.0) containing 20 mM MES, followed by elution through a DEAE column in 200 mM NaCl. The eluted XYL10C-ΔN was re-dialyzed in 50 mM sodium citrate (pH 7.5) containing 25 mM Tris–HCl, 150 mM NaCl, and concentrated to 66 mg/mL using Amicon Ultra-15 Centrifugal Filter Units (Millipore). The protein purity was higher than 95% as shown by SDS-PAGE.The culture supernatants were collected by centrifugation . The crystallization conditions were then optimized to 0.4 M MgCl2·6H2O, 0.1 M Bis–Tris (pH 6.5), and 19% (w/v) PEG 3350. At day 5, the crystals reached to a size of approximately 1.0 mm × 0.4 mm × 0.3 mm. The crystals that diffracted to 1.5 Å resolution were cryo-protected with a solution containing 0.5 M MgCl2·6H2O, 0.15 M Bis–Tris (pH 5.5), 25% (w/v) polyethylene glycol 3350 and 10% (w/v) glycerol. Complete X-ray intensity data were collected from a single crystal at beam line BL15A1 of the National Synchrotron Radiation Research Center of China. A 0.5° oscillation angle was used, the exposure time was 3 s, and the distance between crystal and detector was 200 mm. The diffraction images were processed using the program HKL-2000 [Phaser program [Phialocephala scopiformis (53% sequence identity) as a search model (PDB code: 4F8X). Initial structure refinement using REFMAC5 resulted in a model with the Rwork and Rfree values of 0.125 and 0.175, respectively.The initial crystallization screen was performed manually using the 768 different reservoir conditions from Hampton Research kits (Laguna Niguel) and sitting-drop vapor-diffusion method. Briefly, 2 μL of XYL10C-ΔN solution  was mixed with 2 μL of reservoir solution in 24-well Cryschem Plates (Hampton Research), and equilibrated against 300 μL of reservoir solution at 298.15 K. The initial crystals of XYL10C-ΔN were obtained within 5 days using the Index condition No. 83  of XYL10C and XYL10C-ΔN were determined on a MicroCal™ VP-Capillary DSC . Each protein sample, 200 μg, were dissolved in 0.5 mL of 20-mM McIlvaine buffer (pH 6.5). The degassed protein samples and controls were treated with a heating rate of 120 °C/h over the temperature range of 20–110 °C. Each experiment was repeated at least three times.Beechwood xylan from Sigma was used as the substrate. The pH-activity profiles of the wild-type and variant enzymes were determined by measuring the xylanase activity after incubation at 80°C in McIlvaine buffer (pH 2.5–8.5) and glycine–NaOH (9.0–11.0) containing 1.0% beechwood xylan for 10 min. To determine the optimal temperature, the enzyme activities were measured at temperatures ranging from 30 to 95 °C in McIlvaine buffer of each optimal pH for 10 min. The amounts of reducing sugar released were determined using the 3,5-dinitrosalicylic acid (DNS) method . Thermal2HPO4-citric acid (pH 4.5) containing 2 μg/mL of xylobiose to xylohexaose or 1% (w/v) beechwood xylan, and incubated at 70 °C for 16 h. The hydrolysis products were resolved in a mobile phase of 100 mM NaOH using xylose to xylohexaose as standards.The hydrolysis products of xylo-oligosaccharides and beechwood xylan by XYL10C and XYL10C-ΔN were determined using the high-performance anion-exchange chromatography  equipped with a CarboPac PA200 guard column (3 mm × 250 mm). Each enzyme (0.2 U) was added into the 0.4 mL 50 mM Na2HPO4-citric acid buffer was used as the substrate for determination of the Km, Vmax and kcat values. The enzymatic activities of purified recombinant XYL10C-ΔN and its mutants were assayed under standard conditions (80 °C and pH 4.0) for 5 min. The data were plotted and calculated according to the Lineweaver–Burk method. The catalytic efficiency (kcat/Km) of each enzyme was calculated for comparison. Significant differences of the Km and kcat/Km values were tested using the ANOVA of SPSS 15.0 at the P < 0.05 level.Beechwood xylan of 1–10 mg/mL in 0.1 M NaP. scopiformis and XynE2 from Anoxybacillus sp. E2 were replaced with glutamic acid or other amino acid residues. The gene fragments of xylE and xynE2 (GenBank Accession Number: FJ860894 and GQ240232) without the signal peptide-coding sequence were synthesized by Biomed . The gene fragments harboring mutations were obtained by overlap PCR with specific primers  (Novagen) were digested by NcoI and HindШ, ligated by T4 ligase, and transformed into E. coli BL21 (DE3) competent cells for expression. The standard assay conditions for XynE2 and its mutant were 65 °C and pH 7.5.To verify the functional roles of Glu175 in other GH10 xylanases, the corresponding residue of XylE from w/v), followed by the addition of 100 U of T. reesei cellulase or 30 U of XYL10C or XYL10C-ΔN and 70 U of T. reesei cellulase. The reactions were incubated at 50 °C with an agitation rate of 165 rpm for 24 h. The amounts of reducing sugars released were determined at 1, 2, 4, 8, 12, 18, and 24 h using the DNS method. The experiment was performed in triplicate.Corn stover was treated with 15% (w/w) ammonia solution for 24 h as previously reported . PretreaAdditional file 1: Table S1. X-Ray data collection and structure refinement statistics. Table S2. Temperature and pH optima of XYL10C-ΔN and its mutants with beechwood xylan as the substrate. Table S3. Specific activity of XYL10C-ΔN and its mutants with beechwood xylan as the substrate under standard conditions (80 °C and pH 4.0). Table S4. Temperature and pH optima of XylE and its mutants with beechwood xylan as the substrate. Table S5. Specific activity of XylE and its mutants with beechwood xylan as the substrate under standard conditions (70 °C and pH 5.0). Table S6. Primers used in this study. Figure S1. Multiple sequence alignments of XYL10C-ΔN and other GH10 xylanases using the ClustalW and ESPript. Strictly conserved residues are marked in red. Similar residues are shown in black bold characters and marked in yellow. The catalytic residues are indicated by red diamonds. Some important amino acids are indicated by red triangles. α-Helices and β-strands are represented by black coils and arrows, respectively. Figure S2. SDS-PAGE analysis of the purified recombinant XYL10C, XYL10C-ΔN, XylE, XynE2, and their mutants. Lanes: M, the standard protein molecular weight markers; 1, 3, 10, and 45, the crude enzyme of wild-type XYL10C, XYL10C-ΔN, XylE and XynE2; 2, 4, and 11, the deglycosylated XYL10C, XYL10C-ΔN, and XylE; 5-9 and 13-26, the saturated mutants of XYL10C-ΔN; 12 and 27-44, the saturated mutants of XylE; and 46, the mutant XynE2-Q85E. Figure S3. Comparison of thermostability of XYL10C and XYL10C-ΔN. A, the kinetic stability assayed at 80 °C and 90 °C; B, the thermodynamic stability . Figure S4. pH stability of XYL10C and XYL10C-ΔN. Figure S5. HPLC analysis of the hydrolysis products by XYL10C (A) and XYL10C-ΔN (B). X1, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose; X6, xylohexaose; beechwood, beechwood xylan. Figure S6. The X-ray diffraction pattern of the XYL10C-ΔN crystal. The resolution edges are shown by different concentric circles. Figure S7. The dimeric structure (Chains A and B) of the XYL10C-ΔN. A, The hydrogen bonds between monomers. The residues involved in forming hydrogen bonds are indicated, and the red dotted line represents hydrogen bond. B, The van der Waals force network between monomers. The residues in blue are from chain A, and those red are from chain B. The residues involved are represented by yellow dashed lines. Figure S8. The XYL10C-ΔN docked with xyloheptaose. The enzyme and substrate interactions are indicated by yellow dotted lines. Figure S9. The active-site architecture of mutant XylE-Q116E.