To further confirm the linkage and reveal the genetic effects of these four candidate genes on milk fatty acid composition, genetic polymorphisms were identified and genotype-phenotype associations were performed in a Chinese Holstein cattle population.A previous genome-wide association study deduced that one (ARS-BFGL-NGS-39328), two (Hapmap26001-BTC-038813 and Hapmap31284-BTC-039204), two (Hapmap26001-BTC-038813 and BTB-00246150), and one (Hapmap50366-BTA-46960) genome-wide significant single nucleotide polymorphisms (SNPs) associated with milk fatty acids were close to or within the FASN, among which SNP rs41919985 was predicted to result in an amino acid substitution from threonine (ACC) to alanine (GCC), five SNPs  were synonymous mutations, and the remaining three  were found in FASN introns. Only one SNP each was identified for PPARGC1A, ABCG2 and IGF1.Nine SNPs were identified in FASN, PPARGC1A, ABCG2 and IGF1 were mainly associated with medium-chain saturated fatty acids and long-chain unsaturated fatty acids, especially FASN for C10:0, C12:0 and C14:0. Strong linkage disequilibrium was observed among ARS-BFGL-NGS-39328 and rs132865003 and rs134340637 in FASN (D´ > 0.9), and among Hapmap26001-BTC-038813 and Hapmap31284-BTC-039204 and rs109579682 in PPARGC1A (D´ > 0.9). Subsequently, haplotype-based analysis revealed significant associations of the haplotypes encompassing eight FASN SNPs  with C10:0, C12:0, C14:0, C18:1n9c, saturated fatty acids (SFA) and unsaturated fatty acids (UFA) (P = 0.0204 to P < 0.0001).Association studies revealed that FASN and PPARGC1A. SNPs within FASN, PPARGC1A, ABCG2 and IGF1 showed significant genetic effects on milk fatty acid composition in dairy cattle, indicating their potential functions in milk fatty acids synthesis and metabolism. The findings presented here provide evidence for the selection of dairy cows with healthier milk fatty acid composition by marker-assisted breeding or genomic selection schemes, as well as furthering our understanding of technological processing aspects of cows’ milk.Our study confirmed the linkage between the significant SNPs in our previous genome-wide association study and variants in The online version of this article (doi:10.1186/s12863-016-0418-x) contains supplementary material, which is available to authorized users. DGAT1, GHR, FASN and PPARGC1A genes as promising candidate genes for milk production traits [FASN, PPARGC1A, ABCG2 and IGF1 genes were associated with milk fatty acids in Chinese Holstein dairy cattle [FASN, PPARGC1A, ABCG2 and IGF1 genes were observed to be associated significantly with milk production traits in our previous candidate genes analysis in Chinese Holstein cattle [FASN, PPARGC1A, ABCG2 and IGF1 genes on traits of milk fatty acids in a Chinese Holstein cattle population. In addition, linkage disequilibrium (LD) analyses were conducted among the SNPs identified in our previous GWAS and in this study.Recently, an increasing number of genes have been reported as associated with milk production for dairy cattle breeding, and great improvements have been obtained. Many quantitative trait locus (QTL) analysis and association studies revealed the n traits . Neverthn traits –22 of asn traits , while pn traits , 25. Thuy cattle . In addin cattle –30. TherComplete details of the milk sample collection and the detection method for milk fatty acids have been reported previously . Brieflyted∗100, .The population in this study comprised 346 Chinese Holstein cows, which were the daughters of 13 sire families from 13 farms of the Beijing Sanyuan Dairy Farm Center. Sixteen main milk fatty acid traits were considered in this association study.The whole blood samples corresponding to the 346 Chinese Holstein cows with phenotypic values were collected. Genomic DNA was extracted from blood samples of the cows using a TIANamp Genomic DNA kit  according to the manufacturer’s instructions and frozen semen of the sires using a standard phenol-chloroform procedure. The quantity and quality of the extracted DNA were measured using a NanoDrop™ ND-2000c Spectrophotometer  and by gel electrophoresis.FASN, PPARGC1A, ABCG2 and IGF1 genes. For FASN, a total of 30 pairs of PCR primers  using Primer3 web program (v.0.4.0) [PPARGC1A (NCBI Reference Sequence: AC_000163.1): forward 5′- GCC GGT TTA TGT TAA GAC AG-3′ and reverse 5′- GGT ATT CTT CCC TCT TGA GC-3′. Primers were also designed from exon 7 and partial flanking intronic sequences of the ABCG2 gene (NCBI Reference Sequence: AC_000163.1): forward 5′- TAA AGG CAG GAG TAA TAA AG-3′ and reverse 5′- TAA CAC CAA ACT AAC CGA AG-3′, and the 5′-flanking region of the IGF1 gene (NCBI Reference Sequence: AC_000162.1): forward 5′- ATT ACA AAG CTG CCT GCC CC-3′ and reverse 5′- CAC ATC TGC TAA TAC ACC TTA CCC G-3′.A DNA pool was constructed from aforementioned 13 Holstein bulls  whose daughters were used for the association analysis to identify potential SNPs in the v.0.4.0) . FollowiPolymerase chain reaction (PCR) amplifications for the pooled DNA from the 13 sires were performed in a final reaction volume of 25 μL comprising of 50 ng of genomic DNA, 0.5 μL of each primer (10 mM), 2.5 μL of 10 × PCR buffer, 2.5 mM each of dNTPs, and 1 U of Taq DNA polymerase . The PCR protocol was 5 min at 94 °C for initial denaturation followed by 34 cycles at 94 °C for 30 s; 56 ~ 60 °C for 30 s; 72 °C for 30 s; and a final extension at 72 °C for 7 min for all primers. The PCR products were purified to remove residual primers, dNTPs and reagents from the amplification reaction. A gel purification kit  was used to extract the target DNA band. Then, 15 μL of each purified PCR product with 1 μL of each forward and reverse primer, was bi-directionally sequenced using an ABI3730XL sequencer .Matrix-assisted laser desorption/ionization time of flight mass spectrometry  was used for subsequent genotyping of the 346 Chinese Holstein cows.Pair-wise LD was measured between the genotyped SNPs of each gene and the corresponding adjacent SNPs that were significantly associated with target traits identified in our previous GWAS based on the criterion of D’ using the software Haploview . AccordiHardy-Weinberg equilibrium tests were performed on each identified SNP. A goodness-of-fit test (Chi-square) was used to compare the number of expected and observed genotypes, using 0.05 as significant threshold value.ijklmn was the phenotypic value of each trait of the cows; μ was the overall mean; Fi was the fixed effect of the farm; Pj was the fixed effect of parity; Lk was the fixed effect of the stage of lactation; Gl was the fixed effect corresponding to the genotype of polymorphisms or haplotype; αm was the random polygenic effect, distributed as N , with the additive genetic relationship matrix A and the additive genetic variance σa2; and eijklmn was the random residual, distributed as N , with identity matrix I and residual error variance σe2. Bonferroni correction was adopted to correct for multiple testing. The significance level of the multiple tests was equal to the raw P value divided by number of tests. In the present study, three genotypes were compared for each trait mean that three multiple comparisons needed to be performed, therefore, Bonferroni corrected significance levels of 0.05/3 = 0.0167 and 0.01/3 = 0.0033 were used. For the haplotype, the Bonferroni corrected significance levels were presented as 0.05/N, where N refers to the number of formed haplotypes. The additive (a), dominance (d) and allele substitution (α) effects were estimated according to the equation proposed by Falconer & Mackay [The mixed procedure of SAS 9.3 software  with the following animal model was performed to estimate the genetic effects of each candidate SNP or haplotype on the milk fatty acid traits.& Mackay , i.e. \dFASN gene. Of these, three were located in the intronic region and six were in exons. The SNP in exon 39 (rs41919985) was predicted to result in an amino acid replacement (A2266T) from threonine (ACC) to alanine (GCC) in the FASN protein, and the other five SNPs in the coding region  were synonymous mutations. Regarding PPARGC1A, ABCG2 and IGF1, only one SNP was detected in each gene , of which rs109763947 is located in the 5′-untranslated region (UTR) and the other two SNPs are in intronic regions. The detailed SNP information is shown in Table FASN, PPARGC1A, ABCG2 and IGF1 identified in our previous GWAS [P > 0.01, Tables After sequencing the PCR products directly using the pooled genomic DNA, a total of nine SNPs were identified for the on 39 rs4919985 wacement A26T from tFASN and 16 milk fatty acid composition traits are presented in Table P < 0.0001, P = 0.0128, P = 0.0128), two SNPs (rs41919992 and rs133498277) showed strong associations with seven traits of C10:0, C12:0, C14:0, C18:1n9c, C16 index, SFA and UFA (P = 0.0190 to < 0.0001), three SNPs  were strongly associated with the above seven traits plus SFA/UFA (P = 0.045 to P <0.0001), and one SNP (rs41919999) showed significant association with C10:0 (P = 0.0012), C12:0 (P = 0.0041) and C14:0 (P = 0.0071). Meanwhile, for C14:1, C16:0, C16:1, C18:0, CLA, C14 index and C18 index, no significant SNPs in FASN were detected. Furthermore, the results showed that heterozygous genotypes of these SNPs were the dominant type for saturated fatty acids , and the homozygotic genotypes of these SNPs were dominant for unsaturated fatty acids .Associations between the nine SNPs of PPARGC1A, ABCG2 and IGF1 on 16 milk fatty acid compositions are shown in Table PPARGC1A was significantly associated with eight milk fatty acid traits, such as C10:0 (P = 0.0251), C12:0 (P = 0.0340), C14:0 (P = 0.0188), C16:1 (P = 0.0401), C18:1n9c (P = 0.0015), C16 index (P = 0.0010), SFA (P = 0.0065) and UFA (P = 0.0038). Correspondingly, the CC genotype was the dominant type for saturated fatty acids , and the TT genotype was dominant for unsaturated fatty acids .The effects of the three genotyped polymorphisms in ABCG2, SNP rs137757790 was significantly associated with C14:0 (P = 0.0026), C18:1n9c (P = 0.0048), SFA (P = 0.0343) and UFA (P = 0.0266). The AA genotype was dominant for saturated fatty acids (C14:0 and SFA), and the CC genotype was dominant for unsaturated fatty acids (C18:1n9c and UFA).For IGF1, SNP rs109763947 was significantly associated with C10:0 (P = 0.0342), C18:1n9c (P = 0.0024), C18:2n6c (P < 0.0001), C16 index (P = 0.0239), SFA (P = 0.0090) and UFA (P = 0.0023). The homozygous genotype of TT was the dominant type for saturated fatty acids (C10:0 and SFA), and the heterozygous genotype of CT was the dominant type for unsaturated fatty acids .For Additionally, the significant dominant, additive and allele substitution effects of the significant SNPs on the target milk fatty acid traits were observed Tables  and 7.TaFASN were highly linked (D’ > 0.9), and one haplotype block comprising eight SNPs was inferred . Subsequently, haplotype-based analysis showed significant associations of the haplotypes encompassing the eight FASN SNPs  with C10:0, C12:0, C14:0, C18:1n9c, SFA and UFA  detected in our previous GWAS  and the FASN, PPARGC1A, ABCG2 and IGF1 and milk fatty acid traits in Chinese Holstein cows.Information on the effects of DNA polymorphisms on milk fatty acid composition is scarce, because milk fatty acid composition data, unlike those of milk fat percentage and fat yield, are not collected routinely in milk recording schemes. Therefore, we attempted to explore the genetic variants of candidate genes identified by our previous GWAS on milk fatty acid composition . In thisFASN, showed significant association with C10:0 (P = 8.54E-06), C12:0 (P = 1.16E-07) and C14:0 (P = 6.01E-06) [FASN  that were significantly associated with C18:2n6c. Furthermore, if the haplotype block was defined based on the solid spine of the LD method, one haplotype block was constructed by the above three SNPs plus two SNPs, rs41921177 and rs41919999, that were associated with C10:0, C12:0 and C14:0. Similarly, strong linkages between the two significant SNPs (rs110131167 and rs108967640) for the C18 index, UFA and SFA/UFA identified in our previous GWAS and the SNP (rs109579682) in PPARGC1A for UFA and SFA identified in this study were observed. Probably as a result of the limited number of SNPs identified for ABCG2 and IGF1, and the farther distance between SNPs in the previous GWAS and their adjacent SNPs identified for ABCG2 and IGF1 in this study, no linkages with the significant SNPs identified in GWAS were observed.In our previous GWAS, the SNP rs41921177, at a distance of 58,172 bp away from .01E-06) . As expeFASN  were markedly associated with C10:0, C12:0 and C14:0, and five of these six SNPs  also showed significant associations with SFA, which suggested that the FASN gene mainly affects the medium-long chain saturated fatty acid traits. FASN is a complex, multifunctional enzyme that catalyzes de novo biosynthesis of long-chain saturated fatty acids [FASN gene is located in a quite large region associated with the medium-chain saturated milk fatty acids on BTA19, which is in agreement with our results that the SNPs in FASN mainly showed significant associations with C10:0, C12:0 and C14:0. Moreover, the five SNPs  also showed associations with the C18:1n9c, C16 index and UFA, and three SNPs  showed associations with C18:2n6, revealing that the FASN gene affects the long-chain unsaturated fatty acid traits. The haplotype-based association analysis showed their significant associations with C10:0, C12:0, C14:0, C18:1n9c, SFA and UFA, also confirming the genetic effects of the FASN gene on the medium-chain saturated and long-chain unsaturated milk fatty acids. Kim & Ntambi [FASN is a key gene involved in the pathway for MUFAs synthesis and incorporation into triacylglycerols and phospholipids, which is consistent with our results. However, the effect of FASN on PUFAs has not been reported elsewhere.Six out of nine SNPs in ty acids  and playty acids , 38. In ty acids , 40 and ty acids  have repFASN gene were associated with milk-fat percentage [FASN, including the non-synonymous SNP, rs41919985, observed in this study, which had been reported in different studies. The allele frequency of rs41919985 A (0.29) in our population is lower than that reported in Friesian and Jersey cattle  [FASN affected C14:0 and C18:2, which were consistent with our findings. Associations of the rs41919985 G allele with higher C14:0 and lower C18:1cis9 were also found in beef cattle [FASN gene had a significant effect on the fatty acid composition of backfat, intramuscular and intermuscular fat in an F2 population from Japanese Black and Limousin cattle. For all nine significant SNPs in FASN, the heterozygous genotypes were associated with a higher proportion of milk SFAs, while the homozygous genotypes were associated with much higher levels of long-chain MUFAs and PUFAs. Thus, decreasing the number of individuals with heterozygous genotypes for these target SNPs in FASN will be beneficial to produce high-quality milk with a high proportion of unsaturated fatty acids (UFAs).It was reported that the SNPs in different exons of the rcentage  and withrcentage  and beefrcentage . Morris rcentage  identifictively)  and 0.62ctively) . Morris f cattle . Abe et f cattle  revealedPPARGC1A correlates with milk fat content [SCD) mRNA levels in humans, mice and rats suggested that PPARs are able to regulate SCD [SCD [PPARGC1A was significantly associated with the C16 index. In our study, PPARGC1A mainly affected medium-chain saturated fatty acids and long-chain unsaturated fatty acids. Only a few studies have reported associations between PPARGC1A and milk fatty acid composition [PPARGC1A, c.1790 + 514G > A, was associated with the C16:1 and C16 index, and Bouwman et al. [PPARGC1A is associated significantly with the C16:1 and C16 index. The significant associations between PPARGC1A c.1790 + 514G > A and the C14:1, C14 index, and C18 index [PPARGC1A is involved in mammary gland metabolism, and the expression of  content . Moreove content . The finlate SCD . As the SCD [SCD . Our finposition , 43. Schn et al.  reportedABCG2 gene is located in the narrow region of chromosome 6 (BTA6), harboring a QTL with a large impact on milk production traits [IGF1 stimulates de novo fatty acid biosynthesis by Schwann cells during myelination. For ABCG2 and IGF1, most studies focused on investigating the association between the identified SNPs in these two genes and milk fat traits [ABCG2 gene showed significant effects on C12:1, C14:1 and C16:1. No association between IGF1 and milk fatty acids composition has been reported. Further studies will be necessary to confirm our results in different cattle population and to elucidate the mechanisms underlying the association found in this study.The bovine n traits , 49. Then traits , 51. Then traits  reportedt traits –58, whilt traits . Bouwmant traits  reportedFASN and PPARGC1A genes identified in our previous GWAS were strongly linked with the key mutations in these two candidate genes, but also presented a link of several variants of FASN, PPARGC1A, ABCG2 and IGF1 with milk fatty acid traits. In particular, FASN and PPARGC1A mainly affected medium-chain saturated fatty acids and long-chain unsaturated fatty acids. Our findings regarding genes and polymorphisms responsible for the variation of milk fatty acids composition provide useful information that can be combined with breeding programs to tailor the fatty acid content in cow’s milk.In this study, we not only confirmed the deduction that the significant SNPs close to the ABCG2, ATP-binding cassette, sub-family G, member 2; CAD, coronary artery disease; FASN, fatty acid synthase; GWAS, genome-wide association study; IACUC, institutional animal care and use committee; IGF1, insulin-like growth factor 1; LD, linkage disequilibrium; LDL, low density lipoprotein; MUFA, monounsaturated fatty acid; PPARGC1A, peroxisome proliferator-activated receptor gamma, coactivator 1 alpha; PUFA, polyunsaturated fatty acid; QTL, quantitative trait locus; SCD, stearoyl-CoA desaturase; SFA, saturated fatty acid; UFA, unsaturated fatty acids; UTR, untranslated region.


Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients.ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling.VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures.The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau  contains supplementary material, which is available to authorized users. VHL) gene via mutation, deletion or promoter methylation occurs in up to 91 % of sporadic ccRCC [Renal cell carcinoma (RCC) is dominated by the clear cell subtype (ccRCC), which makes up 70 % of all RCC and has a poor prognosis . Biallelic ccRCC  and drivic ccRCC .Cancer cell lines are used extensively to characterize novel anti-cancer therapeutics, but such studies have translated poorly from preclinical studies into clinically useful drugs. Genome-wide analysis shows that ccRCC cell lines have many more copy number alterations than primary ccRCC tissues , and traRecently researchers have developed methods for generation of primary tumor-derived cultures such as sphere-forming or adherent cells from brain tumors , 9 or 3-ccRCC samples were obtained from the University Health Network (UHN) and the Cooperative Health Tissue Network from patients providing written consent under UHN Research Ethics Board approval, protocol #09-0828-T  and incubated at 37 °C, in 5 % CO2, 2 % O2. Cell culture media was replaced every three to four days and cultures were passaged at confluence with 0.25 % Trypsin (Wisent) and split between 1:2 and 1:5 depending on growth rate. Cell cultures were monitored for Mycoplasma infection  and cell culture identity verification by short tandem repeat profiling (AmpFLSTR® Identifiler®).Cell suspensions were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with 10 % FBS or defined serum-free media (DSFM). DSFM contained DMEM/F12 + Glutamax, 1× B27 supplement (Gibco), 1× non-essential amino acids (Sigma-Aldrich), 1× Lipid Mixture 1 (Sigma-Aldrich), 4 μg/mL Heparin, 1 mM N-acetyl cysteine (Sigma-Aldrich), 10 mM HEPES, 10 ng/mL EGF (Invitrogen), and 10 ng/mL bFGF (Invitrogen). Both culture conditions included 100 Units/mL penicillin, and 100 μg/mL streptomycin. 786–0 and A-498 cells were cultured in IMDM with 10 % FBS, penicillin and streptomycin. All cultured cells were plated at a density of at least 5000 cells/cmTo determine doubling times, primary cell cultures were seeded in collagen-coated 96-well plates in IMDM/10 % FBS at 2500 and 5000 cells/well . 786–0 cells were seeded at a density of 300 cells/well. Plates were incubated in an IncuCyte ZOOM incubator and 4 images/well were taken at each time point. Growth curves based on cell confluence were compiled using IncuCyte ZOOM software from which estimates of doubling times were obtained.VHL was performed using primer sequences and melting temperatures in Additional file DNA was extracted using the Qiagen QIAamp DNA Mini kit. PCR for + and CA9− populations using a BD FACSAriaII cell sorter.Cells were suspended in Hank’s balanced salt solution with 2 % FBS, blocked with 20 μg/ml mouse IgG on ice for 10 min, then incubated on ice with anti-CD31-PECy7 , anti-CD45-PECy7  and anti-CA9-PE  for 30 min, washed, and resuspended in Hank’s + 2 % FBS with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). Viable (i.e. DAPI-negative) CD45/CD31-negative cells were sorted into CA9Adherent cell lines were grown in chamber slides to 50–90 % confluence, washed in PBS, fixed in 4 % paraformaldehyde for 15 min at 4 °C, and subsequently washed and permeabilized in PBS with 0.1 % Tween. Cells were then blocked with 0.5 % BSA, 5 % goat serum and 0.3 % hydrogen peroxide, incubated with primary antibody for 30 min at room temperature, washed, and incubated with a biotinylated goat anti-rabbit or goat anti-mouse secondary antibody, as appropriate, at 1:1000 for 30 min at room temperature. Cells were again washed, incubated with 1:1000 streptavidin-HRP (BD Biosciences) for 30 min at room temperature, washed again, and incubated with 3,3'-diaminobenzidine (DAB) for 5 to 10 min, as directed by the manufacturer , counterstained with hematoxylin, dehydrated, and coverslipped with histomount. Antibodies and dilutions were as follows: Pan-Cytokeratin, 1:100 (AbCAM); PAX-8, 1:500 (Protein Tech Group); Alkaline Phosphatase, 1:50 (Millipore); Aquaporin1, 1:100 (Abcam); E-Cadherin, 1:100 .VHLmut cells were re-suspended in 1:1 PBS/standard growth factor Matrigel (BD Biosciences) and injected under the renal capsule of male NOD/SCID/IL2Rγ−/− mice. 5 mice were injected with each cell line. Animal experimentation followed protocols approved by the University Health Network Animal Care Committee. After 10 weeks, mice were euthanized and tumors were harvested, fixed in formalin, paraffin-embedded and subjected to hematoxylin and eosin staining to assess histology.One million p-value cutoff of 0.05 were used.Genomic DNA was applied to Illumina Human Omni Express-12 or Omni2.5–8 SNP arrays, hybridized as instructed by the manufacturer, and scanned on the iScan Reader (Illumina). SNP Array data was analyzed with Nexus software using the SNP-FASST2 segmentation algorithm. Probe sets were centered to the median for all samples. Linear systematic correction was applied as follows: The bias values, including percent GC content and fragment length, were used to create a linear model whose parameters were estimated using the least squares method. The estimate was then subtracted from the probe Log2Ratio to obtain the corrected probe values. A minimum difference threshold of 25 % and a VHLmut and VHLwt cultures that were expanded in 10 % FBS conditions at passages 2 to 5. mRNA was isolated using a Qiagen RNeasy Kit and Poly-A enriched mRNA libraries were prepared following the Illumina TruSeq RNA Sample Preparation Kit v2 protocol. Libraries were sized on an Agilent Bioanalyzer, and their concentrations were validated by qPCR. Equimolar amounts of the eighteen different libraries were pooled, and subjected to 51 cycles of single-read sequencing on an Illumina HiSeq 2500 using Illumina V4 chemistry and reagents. The mean number of reads/sample was 40.4 M . Reads were aligned to the GRCh37 reference human genome, using Gencode V19 transcript models. Alignment was performed with Tophat (v2.0.13), using default parameters and with the Gencode V19 transcriptome index supplied. The median percentage of aligned reads was 95.8 % . Gene expression levels were estimated with Cufflinks (v.2.0.2) [GenomicFeatures’ (v1.18.7), using the UCSC hg19 “knownGene” transcripts table. Differential expression was determined using limma (v3.22.7).mRNA was extracted from snap-frozen tumor tissue and from v.2.0.2) , using dVHLmut and VHLwt signatures  MSigDB Collection C5, consisting of 1454 GO gene sets was queried against the patient tumor vs. VHLmut cells ranked gene list . For the latter two analyses, the outputs from GSEA were imported into Cytoscape v3.2.0 and visualized using the Enrichment Map plugin.Three GSEA analyses were performed using the RNAseq data: 1) Using the GSEA v2.2.1 PrerankedTool the res Fig.  were quel tissue ; 2) A gen = 39). Eight primary cultures with matched primary tumors and adjacent normal tissues were genotyped using single nucleotide polymorphism (SNP) arrays and, surprisingly, 6 out of 8 cultures established in FBS and all cultures in DSFM had a normal genotype  and only one of seven cases grown in FBS (RCC130) was VHL-mutant (VHLmut). Another FBS culture, RCC243 contained a mixture of VHLmut and VHLwt cells at passage two , which is constitutively upregulated in VHL-mutant cells and acts as a diagnostic biomarker in this disease [VHLmut cells expressed CA9, while RCC22 VHLwt cells did not cultured them in 10 % FBS  upon sorting of CA9+ cells from early passage cultures, and to 84.6 % (11 out of 13 attempts) upon sorting of CA9+ cells from primary cell suspensions  of their paired parental tumors, and display a few additional alterations that were not obvious in primary tumors: RCC22mut and RCC243mut have gain of chromosome 7, RCC243mut also has loss of chromosome 4, and RCC364mut has 2 deletions, one in chromosome 8 and one in chromosome 9. In comparison, 786–0 and A-498 had considerably more CNAs than primary ccRCCs.To assess genotype stability in vitro, we performed SNP arrays on three VHLmut/VHLwt cell culture pairs grown in 10 % FBS at passage 2 to 5, as well as RNA extracted from the matched primary tumor tissues. RNAseq data has been deposited in NCBI’s Gene Expression Omnibus [VHLmut and VHLwt cultures formed discrete subclusters, indicating distinct expression profiles within each sample type  were identified between VHLmut and VHLwt cell cultures  [VHLmut signature in ccRCC tumor tissues, and of the VHLwt signature in normal adjacent kidney tissues, respectively   showed sely Fig. .Fig. 5DiVHLmut vs. VHLwt cells, we also performed GSEA of a compiled database of gene sets relating to biological processes, molecular functions and pathways [VHLmut cells, including glycolysis and electron transport, hypoxia/oxygen response, epigenetics/histone modification and bile acid and bile salt transport  than gained (1531). As above, GSEA of gene ontology (GO) terms was performed using the ranked gene list of the primary tumor vs. VHLmut genes, and the results were again visualized using the Cytoscape Enrichment Map plugin  construct.Our results indicated that cultures established in DSFM were normal renal proximal tubule epithelial cells, the presumed cell of origin for ccRCC . The outVHLmut cultures had doubling times ranging from 44 to >200 h, in striking contrast to the 786–0 doubling time of 16 h. This more accurately reflects the heterogeneity and growth kinetics observed in patients and primary xenografts. Cultures were readily established from tumors of varying stages and grades  [VHLmut cultures, the majority of these were related to the loss of immune cells in the cultures, and the stimulation of cell proliferation in vitro.Comparison of transcriptional profiles between ion data  showed tatients) . Thus ovWe have exploited the unique biology of ccRCC to develop a protocol for the reproducible and accurate generation of ccRCC and patient-matched normal proximal tubule epithelial cell cultures Fig. . TranscrbFGF, basic fibroblast growth factor; CA9, carbonic anhydrase IX; ccRCC, clear cell renal cell carcinoma; CNA, copy number alteration; DNA, deoxyribonucleic acid; DSFM, defined serum-free media; EGF, epidermal growth factor; FBS, fetal bovine serum; GO, gene ontology; GSEA, gene set enrichment analysis; HIF, hypoxia inducible factor; IMDM, Iscove’s modified Dulbecco’s media; KEGG, Kyoto encyclopedia of genes and genomes; NOD/SCID/IL2Rγ−/−, Nonobese diabetic/severe combined immunodeficient/interleukin 2-receptor gamma knockout; RCC, renal cell carcinoma; RNA, ribonucleic acid; RNAseq, RNA sequencing; SNP, single nucleotide polymorphism; TCGA, the cancer genome atlas; TGFβ, transforming growth factor beta; TNF, tumor necrosis factor; VHL, von Hippel Lindau; VHLmut, von Hippel Lindau mutant; VHLwt, von Hippel Lindau wild-type


Pseudomonas abietaniphila KF701 utilizes biphenyl as a sole source of carbon and degrades polychlorinated biphenyls (PCBs). Here, we report the 6,886,250-bp draft genome sequence of KF701, which contains 6,315 coding sequences and 59.4 mol% G+C content. The strain possesses genes for biphenyl catabolism and other genes that mediate the degradation of benzoate, salicylate, and phenol. Biphenyes KF707 . Since tes KF707 . Some stph genes . The purP. abietaniphila KF701 was performed by the National Institute of Technology and Evaluation (NITE) using a combination of shotgun sequencing on a 454 Roche GS FLX+ system (Roche) and paired-end sequencing on a HiSeq sequencing system (Illumina). The Newbler software package  was used for genome assembly. The draft genome was composed of 140 contigs (>537 bp) totaling 6,886,250 bases, with a G+C content of 59.4 mol%. The N50 contig size and the largest contig size were 90,844 bp and 392,029 bp, respectively.Whole-genome shotgun sequencing of n = 328 CDSs), phages, prophages, transposable elements, and plasmids (n = 17 CDSs), motility and chemotaxis (n = 152 CDSs), metabolism of aromatic compounds (n = 146 CDSs), and metabolism of carbohydrates (n = 526 CDSs). A comparison of P. abietaniphila KF701 with other Pseudomonas strains within the RAST server database identified Pseudomonas syringae pv. syringae B728a  as its closest neighbor, with a score of 515, followed by P. syringae pv. phaseolicola 1448A , with a score of 481.Rapid genome annotation using the RAST annotation server  describebph genes very similar to those of Pseudomonas putida KF715  . Strain P. abietaniphila KF701 has been deposited at DDBJ/EMBL/GenBank under the accession numbers BBQJ01000001 to BBQJ01000140.The draft genome sequence of


Here we report details on the design and conformational analysis of two novel peptides showing antimicrobial properties, as reported in the research article, “New antimicrobial peptides against foodborne pathogens: from in silico design to experimental evidence” G. Palmieri, M. Balestrieri, Y.T.R. Proroga, L. Falcigno, A. Facchiano, A. Riccio, F. Capuano, R. Marrone, G. Campanile, A. Anastasio (2016)  Specifications TableValue of the data•These data details the molecular design and NMR characterization of two novel antimicrobial peptides.•NMR parameters, such as chemical shifts, in two different media can be useful for comparison with other peptides showing antimicrobial activities.•The structural features emerging from in silico analysis and peptide molecular models can used to guide the design of analogs with enhanced biological activities.•This data may provide insights for development of MTP-derived antimicrobials for food safety.2O 1:1  experiments, such as total correlation spectroscopy (TOCSY) Proton sequential assignments of the amino acid spin systems, obtained following the standard method proposed by Wuthrich 2O 1:1 are showed.In To compare the behavior of MTP1 and MTP2 peptides in the two different solvent systems, the αCH proton chemical shift deviations from random coil values <−0.1 ppm are indicative of helical structures, whilst deviations ranging from +1 to −1 point to random coil conformations Negative deviations of αCH proton chemical shift from random coil values 1.22O/TFE-d3 1:1, as reported in Structure calculations for MTP1 and MTP2 performed by the standard CYANA simulated annealing schedule 1.3Prediction of antimicrobial activity has been performed by using the Computational tools at the Antimicrobial Peptide Database web site (http://aps.unmc.edu/AP/).


Acinetobacter baumannii is of serious concern in the hospital setting. Here, we report draft genome sequences of 11 A. baumannii isolates that were isolated from a single patient over a 65-day period, during which time the isolates exhibited increased antimicrobial resistance.The development of multidrug-resistant  Acinetobacter baumannii is recognized as a serious threat in the hospital environment, particularly in the intensive care unit (ICU) setting , mediated through acquired resistance determinants, or a mutant . Here, reads were trimmed at their 5′ and 3′ ends until an average base quality of 30 was reached in a window of 20 bases, and the assembly was performed with Velvet version 1.1.04 using optimized k-mer size and coverage cutoff values based on the average length of contigs with >1,000 bp. All genomes were sequenced to a minimum depth of 20×, with a median coverage of 41× across all 11 isolates. The median genome size was 3.980 Mb and ranged from 3.878 Mb (isolate C) to 3.996 Mb (isolate K). The resulting number of contigs per genome ranged from 122 (isolate J) to 836 (isolate C). Contigs were annotated with Prokka 1.11 (Acinetobacter-specific database for annotation. The number of predicted tRNAs per genome ranged from 39 (isolate C) to 58 (isolate F). The range of predicted coding sequences (CDSs) per genome was from 3,543 CDSs (isolate C) to 3,797 CDSs (isolate I), with a median of 3,780 CDSs from all 11 genomes.Bacterial genomic DNA was prepared from cultures using the Qiagen DNeasy kit, according to the manufacturer’s instructions, and sequenced using MiSeq . One nanogram of genomic DNA was prepared using the Nextera XT DNA sample preparation kit (Illumina) prior to sequencing on the MiSeq platform using the paired-end 2 × 250-bp (version 2) protocol. The resulting FASTQ files were quality trimmed and assembled http://pubmlst.org/abaumannii/) shows that all isolates belong to sequence type 1 (ST1), which corresponds to the international clone lineage 1  revealed that these isolates possess multiple aminoglycoside-modifying enzymes: all possessed aac(3)-Ia and aph(3′)-Ic, while nine amikacin-resistant isolates had aph(3′)-via, which was missing in two amikacin-susceptible isolates.Multilocus sequence typing using the Pasteur scheme (ineage 1 . The genineage 1 . FurtherA. baumannii isolates will aid our understanding of genetic changes that occur during a prolonged infection treated with multiple antimicrobial therapies.The genome information of these 11 sequential FITR01000001, FCNC01000001, FCND01000001, FCNG01000001, FCNJ01000001, FCNF01000001, FCNK01000001, FCNH01000001, FCNI01000001, FCNE01000001, and FJMY01000001, respectively.The draft genome sequences of isolates B, C, D, E, F, G, I, J, K, L, and M have been deposited in DDBJ/ENA/GenBank under the accession numbers


Saccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation.gup1∆ mutant were used as previously described to generate biofilm-like mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis. S. cerevisiae W303-1A wt strain and S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133.The proteome of moonlighting or actual metabolism occurs extracellularly. The gup1∆ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.yECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive  Saccharomyces cerevisiae is the most studied lower Eukaryote. As all microorganisms, it is predominantly regarded as a unicellular organism. Yet, S. cerevisiae, as any microbe, can form large multicellular communities: biofilms, colonies and stalks [S. cerevisiae [C. albicans [S. cerevisiae colonies ECM displays large amounts of glycoproteins [C. albicans biofilms has been reported to contain proteins, sugars and DNA [The yeast d stalks . All thed stalks –5. This d stalks , 6–8, wialbicans . These oproteins , namely proteins , while E and DNA , 11. In  and DNA , 13.C. albicans [O-Acyltransferases family, although neither displays the correspondent enzymatic activity [S. cerevisiae and C. albicans, the deletion of GUP1 has been associated with a vast number of phenotypes from major biological processes, namely plasma membrane and cell wall molecular composition, biogenesis and structure [S. cerevisiae gup1∆ and wt strains.The yeast proteins Gup1 and Gup2 are highly conserved in all Eukaryotes , 15, namalbicans , presentactivity . In spitactivity . For thiactivity , and Gupactivity –25. Hedgactivity , commandactivity . ECM theactivity , 29 and activity . In yeastructure , 32, endtructure , 34, diftructure , 36. Themoonlighting. The comparison between the S. cerevisiae wt strain and the mutant defective in the GUP1 gene displaying abnormal ECM texture, was fruitful in which it contributed to recognize yECM composition is dynamically regulated and allowed to unveil putative players in structural integrity maintenance.The proteins secreted into the yECM from a homogenous overlay/mat  were ideS. cerevisiae ECM, a biofilm-like overlay/mat of cells was cultured as previously described [The overall strategy of the present work is presented in Fig. escribed . This alescribed . To pinpescribed . The useS. cerevisiae strains were chosen for comparison, a wild type strain and a correspondent mutant strain defective for GUP1 gene in view of its role in higher Eukaryotes in Hedgehog pathway signalling and consequent indirect ECM composition regulation. Compared to wild type strain, the mats developed by gup1∆ presented a differently textured soft jelly-like yECM (not shown). The comparison of yECM proteome from gup1∆ mutant and wt should therefore indicate candidate proteins involved in yECM physicochemical and molecular properties and functions, while the comparison with the proteome secreted into LGM by either strain should enable the identification of proteins specific from yECM.Two gup1∆ mutant strains was extracted and the protein fraction identically purified and analysed , only 62 are presently uncharacterized ORFs or have no attributed function. The remaining 630 have known or predicted roles spread by a wide range of functional groups, from which Metabolism, broadly taken, and Protein Fate/Remodelling account for 51 % of the yECM proteins, while ≈ 10 % are proteins involved in Cell Rescue and Defence mechanisms . Furthermore, the glyoxylate and/or TCA cycles, aconitase Aco1, citrate synthase Cit1, malate synthase Mls1 and dehydrogenase Mdh1, isocitrate liase Icl2, and isocitrate dehydrogenases Idh1/2/ and Idp2, were also found (db PXD001133). Actually, from the 12 top scored proteins , 8 belong to these groups: Eno1 and Eno2, Tdh3, Fba1, Pgk1, Tdh1, Pyk1 and Pdc1. Enolase 1 (Eno1) and glyceraldehyde-3-phosphate dehydrogenase (Tdh1) are well known for targeting the cell surface of 3. Furtheerevisae . Eno1 alerevisae .S. cerevisiae, there has been reference to the existence of two metabolically diverse sub populations promoting nutrients flow inside the colony [C. albicans biofilms display sub-populations of morphologically and metabolically distinct cells [Besides glycolysis/gluconeogenesis, also the metabolism of amino acids, nucleotides and lipids were represented. yECM therefore displays large sets of enzymes from both glycolytic and gluconeogenic pathways. The presence of proteins which genes are known to be repressed by glucose, like Pck1, Mls1 and Icl2, suggests that yeast cells might have developed into two metabolically distinct populations, one under glucose repression, using glucose available directly from the growth medium, and another derepressed, putatively feeding on the ethanol generated by the first population. In colonies of e colony , 43. Idect cells . In liquct cells , 45, 46.S. cerevisiae ECM (db PXD001133), 150 are involved in the synthesis, folding and degradation of other proteins . In particular, Bmh1 and Bmh2 also known as 14-3-3 proteins were found. These are important proteins transversal to a large number of signalling pathways in mammalian cells, including TOR pathway and retrograde response, HOG, PKC and Ras/cAMP pathways, and chitin synthesis control [S. cerevisiae and C. albicans [intein homing endonuclease Vde, also known as PI-SceI [The group of proteins that were ascertained to the broad designation of  control , 58. Botalbicans , 45, 59. PI-SceI . In the  PI-SceI . Western PI-SceI . In all gup1∆ yielded 587 proteins (db PXD001133), 15 % less than wt. Still, the two strains shared 35 % of the total yECM proteome (213 proteins). In opposition, gup1∆ LGM yielded 311 (three times more proteins than wt), 215 of which common to the mutant yECM . This shows that ECM, regardless to the strain, is richer in proteins than the LGM counterpart, and that the deletion of GUP1 selectively interferes with protein availability in the extracellular space both in solid and liquid growth conditions.As mentioned above, using Nano LC-MS/MS, yECM extracts from gup1Δ LGM samples presented a great number of cell surface proteins, including cell wall and plasma membrane proteins (db PXD001133). These include plasma membrane integral proteins as Pma1 and GPI-anchored proteins, namely several members of the Gas family that are crucial for the cell wall remodelling, Gas1, Gas3 and Gas5. Gas1 was already identified as one of the proteins the mutant secretes into LGM more abundantly than wt strain [gup1Δ mutant cells [gup1Δ LGM samples also presented a high number of proteins intervening in the carbon metabolism (db PXD001133), namely Eno1 (enolase), Fbp1 , and Tpi1 (triosephosphate isomerase). These proteins were already reported in the cell surface of other yeast strains during liquid growth [gup1Δ mutant sample (db PXD001133).The t strain . The abnnt cells , 61, 62.d growth , 59, butgup1∆ mutant ECM.In opposition to LGM, mutant cells secreted into the yECM 15 % less proteins than wt Fig. , a largeth protein was the putative glucanase Scw4. Extracting the proteins common to yECM and LGM in either strain, yECM-only proteins for either strain were identified  and Mls1 .Globally, the proteins found in yECM of both strains belong to very diverse pathways, mechanisms and structures Fig. . The 4 ogup1∆ mutant suggest that the mutant could not form two metabolically distinct subpopulations, one fermenting the glucose present in the medium and the other respiring the ethanol and/or glycerol produced by the first. This is corroborated by the further absence from the mutant yECM of the pyruvate decarboxylase isoforms Pdc5 and Pdc6, while these and Pdc1 isoforms were all detected in the wt yECM. Pdc5 and Pdc6 are enzymes involved in glucose fermentation into ethanol, which expression is repressed by Pdc1 [The differences in protein identified in the wt and the  by Pdc1 , 66.gup1∆ mutant also differ in the expression of other proteins. In order to deeper characterize these differences, the yECM protein samples of both wt and gup1∆ strains were subjected to two-dimensional differential gel electrophoresis (DIGE)  Fig. . This algup1Δ yECM samples. A total of 56 protein spots presented significantly different p < 0.05, abundance variation 1.5-fold or greater between the two strains. The mutant strain presented 28 spots with increased abundance, and another 28 spots that were significantly less abundant than in the wt. Among these 56 protein spots, the 15 ones presenting an abundance variation of 2-fold or greater, orange-marked spots in Fig. The proteomic profile of DIGE Fig.  analysedgup1Δ replicates. The variations between each replicate are displayed as an abundance matrix, represented in a standardized logarithmic scale of abundance ranging from −0.5 (green) to +0.5 (red).A pattern analysis was performed to assess the differential abundance of proteins in the several replicates and validate the reproducibility of the extraction methodology Fig. . The Prigup1Δ mutant in relation to wt Table gup1∆ could be the cause of releasing from the cell surface proteins weakly attached to the wall. Finally, two uncharacterized proteins, Hmf1 and Ypl225w, were also identified. These two proteins were not sufficiently resolved in the 2DE and were identified from the same spot . Hsp60 is a mitochondrial chaperone involved in the de novo folding of mitochondrial proteins and respiration complexes assembly [i.e., the proteins from yECM that were not found also in LGM . The presence of sulphated sugars in the yECM has been previously suggested [gup1∆-associated change in ECM structure.Hri1 has no known biological function, although it was found to interact with members of the yeast Sec 63 complex used for translocation of pre-secretory proteins into the ER . Hri1 pruggested . A direcS. cerevisiae yECM, grown onto biofilm-like mats. This yeast, in opposition to the human pathogen C. albicans, has been poorly assessed in what regards the biology of multi-cellular aggregates (for a review see [This work presents a first comprehensive assessment of the proteome from the extracellular matrix of view see ). Most oview see  that allview see , which fS. cerevisiae mats, as previously described for this yeast colonies [C. albicans biofilms [moonlighters [C. albicans and its implication in their development [C. albicans infections, Eno1 binds host cells plasminogen and plasmin therefore facilitating tissue invasion [C. albicans biofilm development and yeast-to-hyphae dimorphic transition [S. cerevisiae or C. albicans cell wall outer surface [yECM displays large sets of enzymes from both glycolytic and gluconeogenic pathways, suggesting that colonies , 43 or Cbiofilms , actuallbiofilms , 83), thbiofilms ) or withbiofilms ). Thereflighters . This telighters  and Eno1lighters , which plighters , mediatielopment . On the elopment , as wellelopment . Identicinvasion , playinginvasion . Concomiansition . Besides surface , 59, 90, surface , 56, andzincins Prd1, Ape2 and Zps1, as well as the HSP70 chaperones Ssa1/2/3/4, Ssb1, Ssc1, Sse1/2 and Kar2. Furthermore, again in analogy to higher Eukaryotes, well-established signalling effectors like Bmh1 and Bmh2, or candidate signalling proteins like Tfp1, Vma1 and/or Vde were found in yECM, suggesting that broad signalling events and molecular cell-to-cell communication could occur through the matrix from yeast multi-cellular aggregates.In analogy to metalloproteinases from higher Eukaryotes extracellular matrix, yECM presents candidate proteinases that could functionally fulfil a role in matrix remodelling, the Zn-dependent exopeptidases Lap4, Dug1 and Ecm14, the metalloproteases GUP1. Many proteins from wt ECM were absent from the mutant ECM. These comprise several functional classes: carbon metabolism, cell rescue and defence, protein fate and cellular organization. In particular, in the mutant ECM no fermentation or gluconeogenic enzymes were found, as well as proteins that regulate this pathway, namely the Fbp1  and Pyc2 (pyruvate carboxylase). This suggests that the mutant mats do not harbour metabolically distinct populations. Moreover, the mutant ECM has under-represented the enzymes that allow the production of acetate and citrate, which properties as cross linkers might underlie ECM structure and justify mutant’ sliminess.The results were expanded by using a mutant strain defective in the yeast orthologue of a negative regulator of the Hedgehog signal from high Eukaryotes, moonlighting for their unexpected roles, although this designation has been mainly applied to proteins of higher Eukaryotes [The yECM emerges as a dynamic and protein rich environment, with high molecular diversity. The majority of the proteins identified are ascribed to intracellular compartments in databases. In fact, only 15 % from the 693 proteins were already annotated to the cell surface, cell wall and/or plasma membrane, and 3.8 % have no ascribed location. Nevertheless, some of the supposedly intracellular proteins were already reported to appear in the cell surface, namely several of the glycolytic/fermentation enzymes , 45. Actkaryotes .S. cerevisiae ECM, which includes several categories of proteins. Sets of intracellular enzymes covering whole metabolic pathways glycolysis, fermentation and gluconeogenesis through the glyoxylate cycle, were found, including the simultaneous presence of glucose induced and repressed enzymes, suggesting that the yECM might allow active metabolism, while receiving proteins from two distinct metabolically active cell populations. Moreover, multiple chaperones and metalloproteinases, and several broad signalling cross-talkers and putative signalling proteins were identified, which suggests the yECM encompasses remodelling and signalling events, analogously to the biological roles from higher Eukaryotes ECM, possibly controlling the fate of the imbedded cells, their survival, differentiation and spatial distribution.The acknowledgement of the existence of structural organization, and differential expression of genes with concomitant metabolic and morphological specialization across yeast colonies or biofilms, is in accordance with yeasts having complex multicellular behaviour. The existence of an extracellular matrix that, in analogy to the higher Eukaryotes, may operate as scaffold but also actively contribute to this behaviour agrees with the suggested complexity. The present work presents the first identification of the proteome of S. cerevisiae wt strain and the mutant defective in the GUP1 gene, which displays an abnormally loose ECM, showed the profound effect that a single gene mutation can have on the yECM. This suggests that yECM composition is highly dynamic and should vary according to microbial species/strain and type of multicellular aggregate. This work opens a door into the understanding of yeast extracellular matrix as a model of primordial processes associated with multicellular life, taking yeast colonies, biofilms, or other types of such large cellular aggregates, as tissue-like communities.The comparison between the S. cerevisiae strains W303-1A MATa leu2-3 leu2-112 ura3-1 trp1-1 his3-11 his3-15 ade2-1 can1-100 [gup1::His5 [600 1 was spread evenly onto Ø90 mm YPD plates, supplemented with 2 % agarose . Four i  was carried out in a Mini PROTEAN® 3 Cell apparatus (Bio-Rad), in 1.5 mm thick polyacrylamide gel, with a 4 % stacking gel and a 10 % resolving gel. The sample (5 μg) was brought up to 10 μl with MilliQ water and mixed with 5X Laemmli loading buffer . The mixFor identification of the proteins, 50 μg of total protein extract was brought up to 40 μl with MilliQ water, mixed with Laemmli loading buffer , and thegup1Δ, and a Cy2 labelled pooled standard, i.e., a mixture of equal amounts of all the samples used for each DIGE experiment. To each mixture, an equal volume of 2X hydration buffer ; 2 % dithiothreitol (w/v); 4 % pharmalytes (v/v); pH 3–11) was added for the cup loading process.2D Difference Gel Electrophoresis (DIGE) was performed using four independent cultures of wt and mutant strains, generating eight individual samples. Proteins were labelled with Cy dye derivatives according to manufacturer instructions . Briefly, each protein sample (50 μg) was labelled with 400 pmol of Cy dye in 1 μl of anhydrous N,N-dimethylformamide . The mixture was incubated on ice in the dark for 30 min. The reaction was stopped with the addition of lysine (10 mM), followed by a second incubation for 10 min on ice also in the dark. The labelled samples were combined as in Table For 2DE first dimension protein separation, 24 cm IPG strips in the pH range of 3–11 were used. The strips were previously hydrated overnight with 7 M urea, 2 M thiourea, 4 % CHAPS (w/v), 100 mM DeStreak, and 2 % pharmalytes (v/v), at pH 3–11. Isoelectric focusing was performed at 20 °C using the following programme: 120 V for 1 h, 500 V for 2 h, 500–1000 V for 2 h, 1000–5000 V for 6 h, and 5000 V for 10 h. Subsequently, the strips were equilibrated for 12 min in reducing solution ; 2 % SDS (w/v); 2 % DTT (w/v); pH 6.8) and then for 5 min in alkylating solution ; 2 % SDS (w/v); 2.5 % iodoacetamide (w/v); pH 6.8). The second-dimension SDS-PAGE was run on homogeneous 10 % T and 2.6 % C polyacrylamide gels casted in low-fluorescent glass plates. Electrophoresis was carried out in the dark at 20 °C, with a potency of 2 W/gel for 18 h, using an Ettan-Dalt six unit .gup1Δ mutant were carried out using average ratio and unpaired Student’s t-test. In order to reduce the false positive, False Discovery Rate [Proteins were visualized using a Typhoon 9400™ scanner GE Healthcare, with CyDye filters. Cy3, Cy5 and Cy2 image acquisition was done at specific excitation/emission wavelengths, respectively 532 nm/580 nm, 633 nm/670 nm and 488 nm/520 nm, and using 100 μm as pixel size. Image analysis was carried out with DeCyder™ differential analysis software . The Differential In-gel Analysis (DIA) module was used to assign spot boundaries and to calculate parameters such as normalized spot volumes. Inter-gel variability was corrected using a biological variance analysis (BVA) module. The internal standard image gel with the greatest number of spots was used as a master gel. Comparisons between wt and SDS-PAGE and 2DE gels were both stained with Colloidal Coomassie Blue according to published procedures , 96, andSamples of total protein extract  were loaded onto a C18-A1 ASY-Column 2 cm pre-column (Thermo Scientific) and then eluted onto a Biosphere C18 column . The proteins were separated using a gradient on a nanoEasy HPLC (Proxeon) coupled to a nanoelectrospray ion source (Proxeon) at a flow-rate of 250 nl/min. The mobile phase A consisted of 0.1 % formic acid in 2 % acetonitrile, and mobile phase B of 0.1 % formic acid in 100 % acetonitrile. A solvent gradient was applied for 140 min, ranging from 0 % to 35 % phase B. Mass spectra were acquired on the LTQ-Orbitrap Velos (ThermoScientific) in the positive ion mode. Full-scan MS spectra (m/z 400–1800) were acquired with a target value of 1,000,000, at a resolution of 30,000 (at m/z 400), and the 15 most intense ions were selected for Collision Induced Dissociation (CID) fragmentation in the LTQ with a target value of 10,000 and normalized collision energy of 38 %. Precursor ion-charge state screening, and monoisotopic precursor ion selection, were enabled. Singly charged ions and unassigned charge states were rejected. Dynamic exclusion was enabled with a repeat count of 1 and exclusion duration of 30 ms.S. cerevisiae database (7798 sequences). The search parameters used were the following: peptide tolerance - 10 ppm; fragment ion tolerance - 0.8 Da; missed cleavage sites - 2; fixed modification - carbamidomethyl cysteine; and variable modifications - methionine oxidation. Mascot ion score 20 and a 99 % peptide confidence were set as filters.Proteome Discoverer 1.2 with MASCOT 2.3 was used to search in the Uniprot/Swissprot taxonomy The supernatants from spots excised from 2DE gels were collected, and 1 μl was spotted onto a MALDI target plate and allowed to air-dry at room temperature. Subsequently, 0.5 μl of α̣-cyano-4-hydroxytranscinnamic acid matrix (3 mg/ml) in 50 % (v/v) ACN (Sigma Aldrich), was added to the dried peptide digest spots, and allowed to air-dry again at room temperature. Analyses were performed in a 4800 Plus MALDI TOF/TOF™ and Proteomics Analyzer , using 4000 Series Explorer™v 3.5 software (ABSciex). The instrument was operated in reflector mode, with an accelerating voltage of 20,000 V. All mass spectra were internally calibrated using peptides from the auto-digestion of the trypsin. The MS spectra of all the spotted fractions were acquired in positive reflector mode for peak selection signal to noise ratio >12. The suitable precursors for MS/MS sequencing analysis were selected, and fragmentation was carried out using the CID atmospheric gas, on 1 kV ion reflector mode, and precursor mass windows ±4 Da. The plate model and default calibration were optimized for the MS-MS spectra processing.www.matrixscience.com), and the NCBInr database . The MASCOT search parameters were: i) species - S. cerevisiae, ii) allowed number of missed cleavages – 1, iii) fixed modification - carbamidomethyl cysteine, iv) variable modifications - methionine oxidation, v) peptide tolerance - ±50 ppm for PMF and 80 ppm for MSMS searches, vi) MS/MS tolerance - ±0.3 Da, and vii) peptide charge - +1. In all identified proteins, the probability score was p < 0.05, i.e., greater than the one fixed by Mascot as significant.The search of peptides was performed in batch mode, using GPS Explorer v3.5 software (ABSciex) MASCOT version 2.3 (The Western blot was performed as described before . The rabThere are no ethics concerns involved with the experimental design from this manuscript.


Distinct decrease in particulate matter (PM) and carbon monoxide (CO) levels was experienced in exhaust emissions from engine operating on biodiesel blends POB-5, POB-20, POB-40, POB-50, POB-80, and POB-100 comparative to conventional petroleum diesel. Percentage change in CO and PM emissions for different biodiesel blends ranged from −2.1 to −68.7% and −6.2 to −58.4%, respectively, relative to conventional diesel, whereas an irregular trend was observed for NOx emissions. Only POB-5 and POB-20 showed notable reductions, whereas all other blends (POB-40 to POB-100) showed slight increase in NOx emission levels from 2.6 to 5.5% comparative to petroleum diesel.Current study presents RSM based optimized production of biodiesel from palm oil using chemical and enzymatic transesterification. The emission behavior of biodiesel and its blends, namely, POB-5, POB-20, POB-40, POB-50, POB-80, and POB-100 was examined using diesel engine (equipped with tube well). Optimized palm oil fatty acid methyl esters (POFAMEs) yields were depicted to be 47.6 ± 1.5, 92.7 ± 2.5, and 95.4 ± 2.0% for chemical transesterification catalyzed by NaOH, KOH, and NaOCH At present, the world is looking for appropriate solution of energy crises as its demand is escalating day by day due to rapidly growing population and global industrialization. Currently, fossil based nonrenewable resources like petroleum, coal, natural gas, and so forth are mostly fulfilling the energy requirements of the world but their limited availability is inversely proportional to the rising energy demands. Besides, the consumption of these fossil fuels is intimidating the whole world with environmental impacts like global warming and depletion of the ozone shield .μm, result in deposition in alveolar region of lungs [Typically, the diesel engine exhaust  has been categorized as carcinogenic and several studies revealed an augmented health risk like lung cancer upon long term exposure to diesel engine exhaust . The maiof lungs –6. Reseaof lungs . Previouof lungs –10.Optimization of biodiesel production through chemical or enzymatic transesterification process is another area of consideration. In this context, different optimization methods have been developed by the researchers with the main target of offering high yield and good quality of biodiesel product. Response surface methodology (RSM) is gaining much recognition these days as an imperative tool for the optimization of various biochemical and technological processes. Usually, RSM is considered as a combination of mathematical and statistical protocols utilized for process development, improvement, and optimization. RSM is also proved to be very helpful technique for the analysis of specific problems where a response of interest is influenced by various process variables with the objective to optimize the said response –13.Although palm oil is in use as a feedstock for biodiesel production, no earlier studies are reported on the RSM based simultaneous optimization of chemical and enzymatic transesterification for production of biodiesel using palm oil. Therefore, the current study was designed with the main objective to optimize both of the chemical and enzymatic transesterification processes for palm oil biodiesel production using RSM as well as evaluation of exhaust emission levels from diesel engine operated on the synthesized biodiesel and its blends to check its compatibility as a green fuel.3, methanol, toluene, isopropanol, phenolphthalein, starch, acetic acid, HCl, sodium thiosulfate, n-hexane, acetone, potassium iodide, iodine trichloride, potassium dichromate, and chloroform were obtained from Merck Chemicals, (Germany), while NOVOZYME-435 (lipase acrylic resin from Candida Antarctica) and lipase from Aspergillus niger were purchased from Sigma Chemical Co. . A.n. lipase (lipase from Aspergillus niger immobilized on lewatite) was obtained from Process Biotechnology Laboratory, Department of Chemistry, GC University, Lahore, Pakistan.All the experimental work was executed using analytical/research grade chemicals and reagents. Palm oil was procured from DESCON chemicals (Pvt.) Ltd. Davis Road Lahore, Pakistan. KOH, NaOH, NaOCHPalm oil was physicochemically analyzed for the quality parameters including refractive index, density, acid value, iodine value, peroxide value, saponification value, and unsaponifiable matter (%) using standard methods of AOCS.3 using specified reaction conditions according to central composite response surface design (CCRD) [Biodiesel was produced using both chemical and enzymatic transesterification of palm oil. Chemical transesterification reactions of oil were catalyzed by NaOH, KOH, and NaOCHn (CCRD) , 14–16, n (CCRD)  and Shimn (CCRD) .A), reaction time (B), reaction temperature (C), and methanol : oil molar ratio (D) on percentage yield of biodiesel, was evaluated through CCRD. The investigated ranges for variables A, B, C, and D are described in For alkali catalyzed chemical transesterification, the reactions were carried out in a laboratory scale reactor, that is, three neck glass fabricated flask fitted with a stirrer, sampling inlet, chiller, condenser, thermometer, and heating plate . Chemica4 full-factorial CCRD design was employed during optimization using four variables with five different levels leading to 30 experiments. Each experiment was repeated three times. The data generated by 30 experiments was further statistically analyzed and used for optimization of biodiesel yield. The experimental results were analyzed by Design Expert 7 and SPSS for ANOVA, response surface plots, and diagnostics checks. The appropriate response surface models for optimization purposes based on experimental biodiesel yields were chosen for both chemical and enzymatic transesterification on the basis of ANOVA with high statistical significance, lack of fit tests, and high values of R2. Furthermore, significance of the selected models and the individual coefficients were tested by f and t-tests [A 2 t-tests , 15, 16.−1 [FTIR spectroscopic monitoring of transesterification of palm oil was executed using Interspec 200-X FTIR spectrophotometer  equipped with mountable FTIR liquid cells. The spectra were recorded over scanning range of 500 to 5000 cm−1 , 20.μm). A 1.0 μL biodiesel sample was injected into the column through split injection mode with a split ratio 1 : 100. Extra pure helium was used as carrier gas (mobile phase) with flow rate of 1.2 mL/min. Column oven temperature was programmed from 150 to 250°C @ 4°C/min while initial and final hold up times were set to be 1 and 5 min, respectively. Temperature of the injector and MS transfer line were maintained at 250 and 260°C, respectively. An electron ionization system was employed for GC/MS detection while scanning mass range varied from 30 to 550 m/z. Identification of individual fatty acids of palm oil biodiesel was executed by comprehensive comparison of their relative retention times with those of authentic standards of fatty acid methyl esters . For confirmation of FAMEs profile of palm oil biodiesel, MS spectra of the unknown sample were also compared with MS spectra of the same from the NIST mass spectral library provided with GC/MS system. Quantification of individual fatty acids was carried out by Agilent Technologies data handling software (Chem Station 6890) and composition was reported as relative percentage of the total peak area [Fatty acids profile of the prepared palm oil biodiesel was analyzed qualitatively and quantitatively using GC/MS (Agilent Technologies 6890N) network gas chromatographic system having an inert XL mass and autoinjector. Fatty acid methyl esters of the biodiesel produced were separated using polar capillary column  of power 20 HP and weight 210 kg equipped with a tube well situated in village nearby University of Gujrat, Gujrat, Pakistan. Palm oil biodiesel as synthesized in the current study along with its blends was employed for estimation of their emission levels. Biodiesel samples were blended with conventional fossil based petroleum diesel and different blends, that is, B5, B20, B40, B50, B80, and B100 with biodiesel percentage 5, 10, 20, 40, 50, 80, and 100%, respectively, were prepared.2) flue gas analyzer, that is, LANCOM-III  fabricated with infrared and electrochemical sensor of nondispersive nature was used, whereas the estimation of particulate matter was executed using “The Casella” (particulate sampling system instrument) in compliance with ISO-9096 and BS-3405. Cellulosic filter media with pore size <10 μ were used in the instrument provided that it resulted in quantitative retention of PM10 for definite time intervals. The filter media were well assembled in a leak proof dust collecting port. The whole assembly was connected with a steal probe with the help of special tuning to withstand high temperatures associated with flue gases. After definite time interval PM retained on the surface of the filter media was measured [Exhaust emissions monitoring from diesel engine, operated on different biodiesel blends, was carried out from the monitoring holes already provided on the diesel engine stacks. All the measurements were executed thrice when the engine was operating at their optimum load at different times to ascertain the emission behavior. For the monitoring of CO and NOx , cetane number (ASTM D 613), pour point (ASTM D 97), cloud point (ASTM D 2500), kinematic viscosity (ASTM D 445), and ash content (ASTM D 874), and so forth, were determined using standards methods.Fuel properties of the produced palm oil biodiesel,2/100 g of oil, 10.85 ± 1.17 meq/kg of oil, 198.7 ± 2.5 mg KOH/g of oil and 3.40 ± 0.02% for refractive index, density, acid value, iodine value, peroxide value, saponification value, and unsaponifiable matter (%), respectively.Physicochemical characteristics of palm oil used as feedstock for the production of biodiesel were evaluated which were found to be 1.454 ± 0.003, 0.88 ± 0.18, and 0.45 ± 0.04 mg KOH/g of oil, 54.60 ± 2.51 g I3 ranged from 22.5 to 47.4, 84.0 to 92.7, and 84.9 to 95.4%, respectively, whereas it ranged from 62.5 to 94.2 and 27.5 to 62.8% for enzymatic transesterification catalyzed by NOVOZYME-435 and A.n. lipase, respectively.Based upon experimental outputs developed from enzymatic and chemical transesterification of palm oil as per defined CCRD, comparative descriptions of biodiesel yields (%) were carried out and presented in 3, respectively, whereas 94.2 ± 3.1 and 62.8 ± 2.4%, respectively, for NOVOZYME-435 and A.n. lipase catalyzed transesterification of palm oil  and methanol to oil molar ratio (7.5 : 1.0) at reaction temperature of 52.5°C for 75 minutes .3 catalyzed transesterification of palm oil, offering optimum POFAMEs yield, were found to be 0.75%, 6 : 1 methanol to oil molar ratio, 45°C reaction temperature, and 90 minutes reaction time. The highest POFAMEs yield was obtained for NOVOZYME-435 catalyzed transesterification of palm oil using 1.0% NOVOZYME-435 concentration, 6 : 1 methanol : oil molar ratio, 32.5°C reaction temperature, and 60 hours reaction time, whereas for A.n. Lipase catalyzed transesterification the optimal POFAMEs yield was recorded by using 1.25% A.n. lipase concentration, 9 : 1 methanol : oil molar ratio, 30°C reaction temperature, and 96 hours reaction time  to fatty acid methyl esters, as these IR bands were not present in the parent oil  IR spectra as shown in FTIR spectroscopic analysis was performed for monitoring transesterification reactions of palm oil. FTIR spectra of palm oil and palm oil derived biodiesel were recorded. The presence of IR bands in the region 1425–1447 cm−1 for O–CH2 groups in glycerol  was found to be in IR spectrum of palm oil, while in biodiesel spectrum this band was absent. Furthermore, the regions 1700–1800 cm−1 for C=O stretch and 2800–3000 cm−1 for symmetric C–H stretching were present in both the palm oil and the biodiesel IR spectra. These findings are in agreement with those of our previous studies [The region 1370–1400 cm studies , 20 as w studies , 23.P values for all three models  were less than the level of significance 0.05, meaning that the quadratic models were significantly fit for the experimental results of transesterification of palm oil using three different alkaline catalysts. For model a, the main effects, that is, catalyst concentration (A), reaction time (B), and methanol to oil molar ratio (D), were found to be significant with P values lesser than 0.05, while for model b methanol : oil molar ratio (D) and catalyst concentration (A) were among the main significant effects, whereas for model c all the main effects were found to be significant.ANOVA for response surface quadratic model  clearly AD) for model a, and for model b catalyst concentration × reaction time (AB), catalyst concentration × reaction temperature (AC), catalyst concentration × methanol to oil molar ratio (AD), reaction time × methanol to oil molar ratio (BD), and reaction temperature × methanol to oil molar ratio (CD), whereas, for model c, catalyst concentration × reaction temperature (AC) and reaction time × reaction temperature (BC) were depicted to be significant. Among quadratic terms reaction time (B2) and methanol to oil molar ratio (D2) were ascertained to be significant for model a, while for model b catalyst concentration (A2), reaction time (B2), and methanol to oil molar ratio (D2) were significant quadratic terms with P values lesser than 0.05, while for model c only methanol to oil ratio (D2) was significant quadratic term.Among all first order interaction terms, catalyst concentration × methanol to oil molar ratio  and enzyme concentration (A), were found to be noteworthy, while for model e the main effects, that is, enzyme concentration (A), reaction time (B), reaction temperature (C), and methanol to oil molar ratio (D) were found to be significant. Among all first order interaction terms, enzyme concentration × reaction time (AB), enzyme concentration × reaction temperature (AC), and enzyme concentration × methanol to oil molar ratio (AD) were significant for model d, whereas for model e enzyme concentration × reaction time (AB), enzyme concentration × reaction temperature (AC), and reaction time × reaction temperature (BC) were found to be significant first order interaction terms. Similarly, for model d, P values for quadric terms, that is, enzyme concentration (A2) and reaction time (B2), were lesser than 0.05 which ascertained their significant contribution; on the other hand, for model e among quadratic terms, reaction time (B2), reaction temperature (C2), and methanol to oil molar ratio (D2) were ascertained as nonsignificant quadratic terms.On the other hand, ANOVA for quadratic models d and e and for enzymatic transesterification of palm oil catalyzed by NOVOZYME-435 and A.n. lipase, respectively, are described in 3 catalyzed transesterification of palm oil for optimized production of POFAME's; the response surface plots depicted that maximum POFAME's yield was achieved when transesterification of palm oil was performed using NaOCH3, that is, 0.75% for a time period of 90 minutes at 45°C, whereas beyond these levels lesser POFAME's yield was observed. Response surface plots  was revealed to consist of myristic acid methyl esters (C14:0), palmitic acid methyl esters (C16:0), palmitoleic acid methyl esters (C16:1), stearic acid methyl esters (C18:0), oleic acid methyl esters (C18:1), linoleic acid methyl esters (C18:2), and linolenic acid methyl esters (C18:3) with % compositions 1.40, 41.5, 0.20, 3.90, 38.6, 10.6 and 1.09%, respectively . Palmiti2 are considered as the main factors of concern. Reductions in pulmonary functions, asthma, lung function decrements, and premature deaths, and so forth are reported to be associated with these pollutants. Furthermore, high affinity of CO for hemoglobin to form carboxyhaemoglobin is assumed as main attributor of CO toxicity causing acute poisoning leading to death [Pollution-free air is one of the key requirements for healthy society , 26. Mucto death , 27–31. −2/s) 40°C), ash content (%), cloud point (°C), pour point (°C), higher heating value (MJ/Kg), and cetane number for POFAME, were depicted to be 4.31 ± 0.23 mm−2/s, 0.032 ± 0.025%, 11.8 ± 1.5°C, 7.49 ± 1.20°C, 42.66 ± 0.54 MJ/Kg, and 52.44 ± 2.29, respectively. Benjamin et al. [−2/s, 16.0°C, and 50.0 for kinematic viscosity, cloud point, and cetane number of palm oil biodiesel, respectively. The results presented by Benjumea et al. [The fuel characteristics, that is, kinematic viscosity  employed, whereas NOVOZYME-435 was the most effective lipase catalyst. Furthermore, synthesized biodiesel was found to be green fuel in terms of considerable reduction in exhaust emissions comparative to conventional petrodiesel and it showed fuel properties technically in compliance with ASTM D6751 and EN 14214 standards.Results ascertained the optimized production of palm oil based biodiesel using response surface methodology. The optimal levels of palm oil biodiesel via base catalyzed transesterification was 95.4 ± 2.0% using NaOCH


B. thuringiensis strain BGSC 4BT1. Thsα and Thsβ (or Thsβ') exhibit optimal antimicrobial activity at a 1:1 ratio and act sequentially to affect target cells, and they are all highly thermostable (100°C for 30 min) and pH tolerant (pH 2.0 to 9.0). Thusin shows remarkable efficacy against all tested Gram-positive bacteria and greater activities than two known lantibiotics thuricin 4A-4 and ticin A4, and one antibiotic vancomycin against various bacterial pathogens , Staphylococcus sciuri, Enterococcus faecalis, and Streptococcus pneumoniae). Moreover, thusin is also able to inhibit the outgrowth of B. cereus spores. The potent antimicrobial activity of thusin against some Gram-positive pathogens indicates that it has potential for the development of new drugs.Due to the rapidly increasing prevalence of multidrug-resistant bacterial strains, the need for new antimicrobial drugs to treat infections has become urgent. Bacteriocins, which are antimicrobial peptides of bacterial origin, are considered potential alternatives to conventional antibiotics and have attracted widespread attention in recent years. Among these bacteriocins, lantibiotics, especially two-component lantibiotics, exhibit potent antimicrobial activity against some clinically relevant Gram-positive pathogens and have potential applications in the pharmaceutical industry. In this study, we characterized a novel two-component lantibiotic termed thusin that consists of Thsα, Thsβ, and Thsβ'  and that was isolated from a  The introduction of antibiotics for the treatment of infections is the most significant scientific achievement of the twentieth century with regard to reducing human morbidity and mortality  are small peptides that undergo extensive posttranslational modification and are considered the best-characterized bacteriocins , vancomycin-resistant Enterococcus faecalis (VRE), penicillin-resistant Pneumococcus (PRP), Propionibacterium acnes, Streptococcus mutans, Clostridium difficile, and Mycobacteria , acted synergistically against most of the tested Gram-positive bacteria and were able to inhibit the outgrowth of Bacillus cereus spores. In addition, comparison of the antimicrobial activities of thusin, thuricin 4A-4, ticin A4, and vancomycin against six species of Gram-positive bacterial pathogens suggested that thusin may be a promising therapeutic agent.In our previous research, we demonstrated that the B. thuringiensis strain BGSC 4BT1 was kindly provided by the Bacillus Genetic Stock Center (BGSC) and propagated on Luria-Bertani (LB) agar plates at 28°C. The other strains, which served as indicator strains, are listed in Table The The bacteriocin biosynthetic gene clusters were predicted using BAGEL 3.0  broth at 28°C. At the early stationary phase (OD600≈3.0), the cells were removed by centrifugation at 12,000 rpm for 10 min. The concentration of active substances was measured using Amberlite XAD-7HP  as described previously . Purification of thuricin 4A-4 and ticin A4 was carried out as described previously  that contained varying amounts of Thsα and Thsβ (or Thsβ') at 28°C with agitation at 220 rpm for 5 h. Bacterial growth was evaluated by measuring the culture OD600.The activated 5 cfu/mL. Tests were performed using 96-well microtiter plates. Each well was filled with 100 μL of serial two-fold dilutions of the antimicrobials and 100 μL of the inoculum and incubated at 37°C for 24 h. All experiments were performed in triplicate. The MIC was defined as the lowest concentration of samples that could inhibit visible growth of the tested strains.The antimicrobial activity of the fermental supernatant of strain BGSC 4BT1 and the eluate in HPLC analysis were assessed using the agar well diffusion method as described previously  accurate-mass quadrupole time of flight (Q-TOF) LC-MS system. The MS operating conditions were as follows: capillary voltage, 3500 V; flow rate of drying gas, 9 L/min; nebulizer pressure, 35 lb/inB. thuringiensis BMB171 was used as the indicator strain in these two experiments.To determine the sensitivities of Thsα, Thsβ, and Thsβ' to pH, aliquots of the bacteriocin preparations (5 × MIC) were adjusted to pH 2.0, 3.0, 7.0, 8.0, 9.0, and 10.0 with 1 M NaOH or 1 M HCl solution followed by incubation at 28°C for 2 h, and the residual antimicrobial activity was measured after neutralizing the sample to pH 6.0. For the thermal stability assay, aliquots of the bacteriocin preparations (5 × MIC) were exposed to 80, 100, and 121°C for 30 min and used for an antimicrobial activity assay. B. thuringiensis BMB171 cultures (OD600≈0.5) were diluted 10-fold, and 200 μL of the diluted culture was added to 1.5 mL Eppendorf tubes that contained Thsα or Thsβ . The tubes were incubated at 37°C for 30 min prior to centrifugation at 12,000 rpm for 1 min. The supernatants were removed from each tube, and the cell pellets were washed twice with LB broth, then resuspended in 200 μL of LB broth. Cells that had been treated with only Thsα were added to microtiter wells that contained Thsβ, and cells that had been exposed to only Thsβ were added to microtiter wells that contained Thsα . The microtiter plates were incubated at 28°C and monitored at hourly intervals for 5 h. In addition, cells exposed to Thsα and Thsβ in combination at a ratio of 1:1 served as a control.The B. cereus ATCC14579 spores were activated by incubation at 70°C for 15 min and were then transferred into LB broth at an optical density at 600 nm of 0.8. After adding different amounts of antimicrobials, the germination process was followed by monitoring the optical density at 600 nm every 10 min.Germination assays were conducted as described previously , two genes encoding a posttranslational modification enzyme (thsM1 and thsM2), two genes encoding an ABC transporter that could be involved in immunity (thsEF), and one gene (thsT) encoding a transporter . The optimal synergy of Thsα and Thsβ (or Thsβ') at a 1:1 ratio is consistent with reports of other two-component lantibiotics, such as staphylococcin C55, plantaricin W, lacticin 3147, and haloduracin  that maximized the bioactivity was determined. The ability of mixtures containing various amounts of Thsα and Thsβ (or Thsβ') to inhibit the growth of B. amyloliquefaciens, B. cereus, B. thuringiensis, B. subtilis, B. pumilus, E. faecalis, L. monocytogenes, Staph. aureus, Staph. Sciuri, and Strep. pneumoniae, but not against any of the tested Gram-negative bacteria , act synergistically to potently inhibit several Gram-positive pathogens, including B. cereus, L. monocytogenes, Staph. aureus (MRSA), Staph. sciuri, E. faecalis, and Strep. pneumoniae. To our knowledge, thusin is the first reported two-component lantibiotic in the B. cereus group and has potential applications in the pharmaceutical industry.The increasing incidence of drug-resistant pathogens has prompted the pharmaceutical and scientific communities to investigate alternatives to traditional antibiotics, such as bacteriocins. Bacteriocins exhibit several desirable properties that make them suitable as alternatives to traditional antibiotics: (i) they are potent (in vitro and in vivo), (ii) they are minimally toxic, (iii) they can be produced in situ by probiotics, and (iv) they are amenable to bioengineering  that maximized bioactivity was 1:1. Due to the differences in the maximum absorption wavelengths of Thsα and Thsβ, the absorption peak areas of Thsα, Thsβ and Thsβ' at 220 nm differed . The production of thurisin 4A-4 and ticin were both ~1 mM/L, but thusin was only 0.1 to 0.2 mM/L. Comparison of antimicrobial activity between thusin, thuricin 4A-4, and ticin A4 showed that thusin had higher activity against all tested indicator bacteria   had no hemolytic activity (data not shown), and more detailed testing will be performed to assess the feasibility of its application in further study.Finally, we have to emphasize that the systematic safety testing of thusin and its antimicrobial activity BX, JZ, and MS designed research; BX, HL, JL, and MSa performed research; BX, JZ, LR, DP, and MS analyzed data; BX and MS wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.