Robust structured light 3D measurement method based on polarization-encoded projection patterns.

Fringe projection profilometry (FPP) is widely used in 3D vision measurement because of its high robustness and measurement accuracy. In the case of HDR objects, due to the problem of surface reflectivity, the obtained image will be overexposed. This will cause the sinusoidality of the fringes projected on the surface of the object in the acquired image to be interfered, resulting in a phase error in the calculated wrapped phase. Therefore, a polarization-encoded sinusoidal structured light is proposed to enhance the sinusoidality of the fringe. The phase information contained in the polarized sinusoidal structured light fringe is only related to the polarization state, not to the light intensity. A polarization coding assisted structured light measurement strategy (PASM) is proposed. This method uses polarization coding assisted polarization phase-shifting fringes for phase unwrapping. The angle of the linear polarizer is set to zero in this method, and it does not require rotating the polarizer. It only needs a single exposure to improve the fringe quality and obtain a more stable unwrapping phase. The experimental results show that the obtained polarization fringes have better sinusoidality, and the phase unwrapping can be more accurate. The reconstructed 3D point cloud also does not appear missing and has better accuracy. It is a reliable method for vision measurement of HDR objects.

Whole-exome sequencing identifies genes associated with Tourette's disorder in multiplex families.

Tourette's Disorder (TD) is a neurodevelopmental disorder (NDD) that affects about 0.7% of the population and is one of the most heritable NDDs. Nevertheless, because of its polygenic nature and genetic heterogeneity, the genetic etiology of TD is not well understood. In this study, we combined the segregation information in 13 TD multiplex families with high-throughput sequencing and genotyping to identify genes associated with TD. Using whole-exome sequencing and genotyping array data, we identified both small and large genetic variants within the individuals. We then combined multiple types of evidence to prioritize candidate genes for TD, including variant segregation pattern, variant function prediction, candidate gene expression, protein-protein interaction network, candidate genes from previous studies, etc. From the 13 families, 71 strong candidate genes were identified, including both known genes for NDDs and novel genes, such as HtrA Serine Peptidase 3 (HTRA3), Cadherin-Related Family Member 1 (CDHR1), and Zinc Finger DHHC-Type Palmitoyltransferase 17 (ZDHHC17). The candidate genes are enriched in several Gene Ontology categories, such as dynein complex and synaptic membrane. Candidate genes and pathways identified in this study provide biological insight into TD etiology and potential targets for future studies.

Whole genome sequence association with E-selectin levels reveals loss-of-function variant in African Americans.

E-selectin mediates the rolling of circulating leukocytes during inflammatory processes. Previous genome-wide association studies in European and Asian individuals have identified the ABO locus associated with E-selectin levels. Using Trans-Omics for Precision Medicine whole genome sequencing data in 2249 African Americans (AAs) from the Jackson Heart Study, we examined genome-wide associations with soluble E-selectin levels. In addition to replicating known signals at ABO, we identified a novel association of a common loss-of-function, missense variant in Fucosyltransferase 6 (FUT6; rs17855739,p.Glu274Lys, P = 9.02 × 10-24) with higher soluble E-selectin levels. This variant is considerably more common in populations of African ancestry compared to non-African ancestry populations. We replicated the association of FUT6 p.Glu274Lys with higher soluble E-selectin in an independent population of 748 AAs from the Women's Health Initiative and identified an additional pleiotropic association with vitamin B12 levels. Despite the broad role of both selectins and fucosyltransferases in various inflammatory, immune and cancer-related processes, we were unable to identify any additional disease associations of the FUT6 p.Glu274Lys variant in an electronic medical record-based phenome-wide association scan of over 9000 AAs.

Identification and validation of a regulatory mutation upstream of the BMP2 gene associated with carcass length in pigs.

BACKGROUND: Carcass length is very important for body size and meat production for swine, thus understanding the genetic mechanisms that underly this trait is of great significance in genetic improvement programs for pigs. Although many quantitative trait loci (QTL) have been detected in pigs, very few have been fine-mapped to the level of the causal mutations. The aim of this study was to identify potential causal single nucleotide polymorphisms (SNPs) for carcass length by integrating a genome-wide association study (GWAS) and functional assays. RESULTS: Here, we present a GWAS in a commercial Duroc × (Landrace × Yorkshire) (DLY) population that reveals a prominent association signal (P = 4.49E-07) on pig chromosome 17 for carcass length, which was further validated in two other DLY populations. Within the detected 1 Mb region, the BMP2 gene stood out as the most likely causal candidate because of its functions in bone growth and development. Whole-genome gene expression studies showed that the BMP2 gene was differentially expressed in the cartilage tissues of pigs with extreme carcass length. Then, we genotyped an additional 267 SNPs in 500 selected DLY pigs, followed by further whole-genome SNP imputation, combined with deep genome resequencing data on multiple pig breeds. Reassociation analyses using genotyped and imputed SNP data revealed that the rs320706814 SNP, located approximately 123 kb upstream of the BMP2 gene, was the strongest candidate causal mutation, with a large association with carcass length, with a ~ 4.2 cm difference in length across all three DLY populations (N = 1501; P = 3.66E-29). This SNP segregated in all parental lines of the DLY (Duroc, Large White and Landrace) and was also associated with a significant effect on body length in 299 pure Yorkshire pigs (P = 9.2E-4), which indicates that it has a major value for commercial breeding. Functional assays showed that this SNP is likely located within an enhancer and may affect the binding affinity of transcription factors, thereby regulating BMP2 gene expression. CONCLUSIONS: Taken together, these results suggest that the rs320706814 SNP on pig chromosome 17 is a putative causal mutation for carcass length in the widely used DLY pigs and has great value in breeding for body size in pigs.

Transcriptome sequencing analysis of porcine MDM response to FSL-1 stimulation.

Mycoplasma infection can cause many diseases in pigs, resulting in great economic losses in pork production. Innate immune responses are thought to play critical roles in the pathogenesis of mycoplasma disease. However, the molecular events involved in immune responses remain to be determined. Hence, the object of this study was to use RNA-Seq to investigate the gene expression profiles of the innate immune response mediated by FSL-1 in pig monocyte-derived macrophages (MDMs). The results revealed that 1442 genes were differentially expressed in the FSL-1 group compared with the control groups, of which 777 genes were upregulated and 665 genes were downregulated. KEGG pathway analysis showed that the upregulated genes were mainly involved in innate immune-related pathways including the TNF signaling pathway, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, Jak-STAT signaling pathway, chemokine signaling pathway, NOD-like receptor signaling pathway and NF-kappa B signaling pathway. The downregulated genes were only involved in the cGMP-PKG signaling pathway and glycerophospholipid metabolism. Our results showed that FSL-1 stimulation activated the TLR2 signaling pathway and resulted in diverse inflammatory responses. FSL-1 induced the transcription of numerous protein-coding genes involved in a complex network of innate immune-related pathways. We speculate that TNF, IL1B, IL6, NFKB1, NFKBIA, CXCL2, CXCL8, CXCL10, CCL2, CCL4 and CCL5 were the most likely hub genes that play important roles in the above pathways. This study identified the differentially expressed genes and their related signaling pathways, contributing to the comprehensive understanding of the mechanisms underlying host-pathogen interactions during mycoplasma infection and providing a reference model for further studies.

Muscle glycogen level and occurrence of acid meat in commercial hybrid pigs are regulated by two low-frequency causal variants with large effects and multiple common variants with small effects.

BACKGROUND: Meat production from the commercial crossbred Duroc × (Landrace × Yorkshire) (DLY) pig is predominant in the pork industry, but its meat quality is often impaired by low ultimate pH (pHu). Muscle glycogen level at slaughter is closely associated with pHu and meat technological quality, but its genetic basis remains elusive. The aim of this study was to identify genes and/or causative mutations associated with muscle glycogen level and other meat quality traits by performing a genome-wide association study (GWAS) and additional analyses in a population of 610 DLY pigs. RESULTS: Our initial GWAS identified a genome-wide significant (P = 2.54e-11) quantitative trait locus (QTL) on SSC15 (SSC for Sus scrofa chromosome) for the level of residual glycogen and glucose (RG) in the longissimus muscle at 45 min post-mortem. Then, we demonstrated that a low-frequency (minor allele frequency = 0.014) R200Q missense mutation in the PRKAG3 (RN) gene caused this major QTL effect on RG. Moreover, we showed that the 200Q (RN-) allele was introgressed from the Hampshire breed into more than one of the parental breeds of the DLY pigs. After conditioning on R200Q, re-association analysis revealed three additional QTL for RG on SSC3 and 4, and on an unmapped scaffold (AEMK02000452.1). The SSC3 QTL was most likely caused by a splice mutation (g.8283C>A) in the PHKG1 gene that we had previously identified. Based on functional annotation, the genes TMCO1 on SSC4 and CKB on the scaffold represent promising candidate genes for the other two QTL. There were significant interaction effects of the GWAS tag SNPs at those two loci with PRKAG3 R200Q on RG. In addition, a number of common variants with potentially smaller effects on RG (P < 10-4) were uncovered by a second conditional GWAS after adjusting for the two causal SNPs, R200Q and g.8283C>A. CONCLUSIONS: We found that the RN- allele segregates in the parental lines of our DLY population and strongly influences its meat quality. Our findings also indicate that the genetic basis of RG in DLY can be mainly attributed to two major genes (PRKAG3 and PHKG1), along with many minor genes.

Krüppel-Like Factor 4 Inhibits Pancreatic Cancer Epithelial-to-Mesenchymal Transition and Metastasis by Down-Regulating Caveolin-1 Expression.

BACKGROUND/AIMS: Krüppel-like factor 4 (KLF4), a member of the KLF family of zinc finger transcription factors, has been identified as a tumor suppressor gene in a variety of tumors. However, the molecular mechanisms by which KLF4 inhibits epithelial-to-mesenchymal transition (EMT) and metastasis in pancreatic cancer remain unclear. METHODS: KLF4 expression in pancreatic cancer was analyzed using public datasets (Oncomine and The Cancer Genome Atlas). The expression of KLF4, caveolin-1 (Cav-1), E-cadherin, and vimentin, and their correlations with clinicopathological characteristics were evaluated by immunohistochemistry in pancreatic cancer tissues. The biological functions and underlying mechanisms of KLF4 expression on EMT and metastasis were also investigated in vitro and in vivo. RESULTS: Public datasets showed that KLF4 expression was significantly decreased in pancreatic cancer and correlated with the depth of invasion and disease stage. The expression of KLF4, Cav-1, E-cadherin, and vimentin protein in pancreatic cancer tissues was closely associated with pathological grade, disease stage, and metastasis. KLF4 expression was also positively correlated with E-cadherin expression and negatively correlated with vimentin expression, whereas Cav-1 expression was negatively associated with E-cadherin expression and positively correlated with vimentin expression. Knockdown of KLF4 expression promoted EMT and facilitated pancreatic cancer cell growth and metastasis in vitro and in vivo. In addition, immunohistochemistry (IHC) results indicated that KLF4 expression was negatively correlated with Cav-1 expression. Furthermore, down-regulating KLF4 expression increased Cav-1 and vimentin expression and decreased E-cadherin expression. Mechanistically, KLF4 could transcriptionally inhibit Cav-1 expression by binding directly to the promoter domain of Cav-1. CONCLUSIONS: KLF4 inhibits pancreatic cancer EMT and metastasis by down-regulating Cav-1 expression, suggesting that the KLF4/Cav-1 signaling pathway may be a novel diagnostic and therapeutic target.

Investigation of Eph-ephrin A1 in the regulation of embryo implantation in sows.

Eph A1 and ephrin A1 (Eph-ephrin A1) is a key receptor-ligand pair of Eph-ephrin system, which plays important roles in the migration and adhesion of cells, tissue morphogenesis and vasculogenesis in mammals. In order to investigate the regulation of Eph-ephrin A1 during porcine embryo implantation, the expressions of mRNA and protein of Eph-ephrin A1 were detected in different reproductive tissues from twelve sows during embryo implantation period on pregnancy day 13, 18 and 24, respectively. Functions of Eph-ephrin A1 on the migration and adhesion of porcine endometrial epithelial cells were analysed by RNA interference (RNAi), transwell migration assays and MTT assays. Results showed that mRNA levels of Eph-ephrin A1 were highly expressed in endometrial attachment site when compared to other reproductive tissues (p < 0.05) and were peaked on pregnancy day 18 during embryo implantation (p < 0.05). Protein levels of Eph-ephrin A1 were highly expressed in endometrial attachment site and were peaked on pregnancy day 18 (p < 0.05). Eph-ephrin A1 proteins were located in endometrial luminal epithelium, stroma of attachment site and inter-attachment site during embryo implantation, and the protein levels were higher during implantation compared to pre-implantation or post-implantation. Furthermore, silencing ephrin A1 gene significantly reduced the migration and adhesion capacity of porcine endometrial epithelial cells. These findings suggest that the Eph-ephrin A1 protein likely targets endometrial attachment site to enhance the migration and adhesion of porcine endometrial epithelial cells around pregnancy day 18 during pregnancy in sows.

A splice mutation in the PHKG1 gene causes high glycogen content and low meat quality in pig skeletal muscle.

Glycolytic potential (GP) in skeletal muscle is economically important in the pig industry because of its effect on pork processing yield. We have previously mapped a major quantitative trait loci (QTL) for GP on chromosome 3 in a White Duroc × Erhualian F2 intercross. We herein performed a systems genetic analysis to identify the causal variant underlying the phenotype QTL (pQTL). We first conducted genome-wide association analyses in the F2 intercross and an F19 Sutai pig population. The QTL was then refined to an 180-kb interval based on the 2-LOD drop method. We then performed expression QTL (eQTL) mapping using muscle transcriptome data from 497 F2 animals. Within the QTL interval, only one gene (PHKG1) has a cis-eQTL that was colocolizated with pQTL peaked at the same SNP. The PHKG1 gene encodes a catalytic subunit of the phosphorylase kinase (PhK), which functions in the cascade activation of glycogen breakdown. Deep sequencing of PHKG1 revealed a point mutation (C>A) in a splice acceptor site of intron 9, resulting in a 32-bp deletion in the open reading frame and generating a premature stop codon. The aberrant transcript induces nonsense-mediated decay, leading to lower protein level and weaker enzymatic activity in affected animals. The mutation causes an increase of 43% in GP and a decrease of>20% in water-holding capacity of pork. These effects were consistent across the F2 and Sutai populations, as well as Duroc × (Landrace × Yorkshire) hybrid pigs. The unfavorable allele exists predominantly in Duroc-derived pigs. The findings provide new insights into understanding risk factors affecting glucose metabolism, and would greatly contribute to the genetic improvement of meat quality in Duroc related pigs.

Genome-wide association study identifies 22 new loci for body dimension and body weight traits in a White Duroc×Erhualian F2 intercross population.

OBJECTIVE: Growth-related traits are important economic traits in the swine industry. However, the genetic mechanism of growth-related traits is little known. The aim of this study was to screen the candidate genes and molecular markers associated with body dimension and body weight traits in pigs. METHODS: A genome-wide association study (GWAS) on body dimension and body weight traits was performed in a White Duroc×Erhualian F2 intercross by the illumina PorcineSNP60K Beadchip. A mixed linear model was used to assess the association between single nucleotide polymorphisms (SNPs) and the phenotypes. RESULTS: In total, 611 and 79 SNPs were identified significantly associated with body dimension traits and body weight respectively. All SNPs but 62 were located into 23 genomic regions (quantitative trait loci, QTLs) on 14 autosomal and X chromosomes in Sus scrofa Build 10.2 assembly. Out of the 23 QTLs with the suggestive significance level (5×10-4), three QTLs exceeded the genome-wide significance threshold (1.15×10-6). Except the one on Sus scrofa chromosome (SSC) 7 which was reported previously all the QTLs are novel. In addition, we identified 5 promising candidate genes, including cell division cycle 7 for abdominal circumference, pleiomorphic adenoma gene 1 and neuropeptides B/W receptor 1 for both body weight and cannon bone circumference on SSC4, phosphoenolpyruvate carboxykinase 1, and bone morphogenetic protein 7 for hip circumference on SSC17. CONCLUSION: The results have not only demonstrated a number of potential genes/loci associated with the growth-related traits in pigs, but also laid a foundation for studying the genes' role and further identifying causative variants underlying these loci.

A GWA study reveals genetic loci for body conformation traits in Chinese Laiwu pigs and its implications for human BMI.

Pigs share numerous physiological and phenotypic similarities with human and thus have been considered as a good model in nonrodent mammals for the study of genetic basis of human obesity. Researches on candidate genes for obesity traits have successfully identified some common genes between humans and pigs. However, few studies have assessed how many similarities exist between the genetic architecture of obesity in pigs and humans by large-scale comparative genomics. Here, we performed a genome-wide association study (GWAS) using the porcine 60 K SNP Beadchip for BMI and other four conformation traits at three different ages in a Chinese Laiwu pig population, which shows a large variability in fat deposition. In total, 35 SNPs were found to be significant at Bonferroni-corrected 5 % chromosome-wise level (P = 2.13 × 10-5) and 88 SNPs had suggestive (P < 10-4) association with the conformation traits. Some SNPs showed age-dependent association. Intriguingly, out of 32 regions associated with BMI in pigs, 18 were homologous with the loci for BMI in humans. Furthermore, five closest genes to GWAS peaks including HIF1AN, SMYD3, COX10, SLMAP, and GBE1 have been already associated with BMI in humans, which makes them very promising candidates for these QTLs. The result of GO analysis provided strong support to the fact that mitochondria and synapse play important roles in obesity susceptibility, which is consistent with previous findings on human obesity, and it also implicated new gene sets related to chromatin modification and Ig-like C2-type 5 domain. Therefore, these results not only provide new insights into the genetic architecture of BMI in pigs but also highlight that humans and pigs share the significant overlap of obesity-related genes.

Genome-wide association analysis reveals genetic loci and candidate genes for meat quality traits in Chinese Laiwu pigs.

Meat quality traits have economically significant impacts on the pig industry, and can be improved using molecular approaches in pig breeding. Since 1994 when the first genome-wide scan for quantitative trait loci (QTLs) in pig was reported, over the past two decades, numerous QTLs have been identified for meat quality traits by family based linkage analyses. However, little is known about the genetic variants for meat quality traits in Chinese purebred or outbred populations. To unveil it, we performed a genome-wide association study for 10 meat quality traits in Chinese purebred Laiwu pigs. In total, 75 significant SNPs (P < 1.01 × 10(-6)) and 33 suggestive SNPs (P < 2.03 × 10(-5)) were identified. On SSC12, a region between 56.22 and 61.49 Mb harbored a cluster of SNPs that were associated with meat color parameters (L*, lightness; a*, redness; b*, yellowness) and moisture content of longissimus muscle (LM) and semimembranosus muscle at the genome-wide significance level. A region on SSC4 also has pleiotropic effects on moisture content and drip loss of LM. In addition, this study revealed at least five novel QTLs and several candidate genes including 4-linked MYH genes (MYH1, MYH2, MYH3, and MYH13), MAL2, LPAR1, and PRKAG3 at four significant loci. Except for the SSC12 QTL, other QTLs are likely tissue-specific. These results provide new insights into the genetic basis of meat quality traits in Chinese Laiwu pigs and some significant SNPs reported here could be incorporated into the selection programs involving this breed.

Genome-wide association analyses for meat quality traits in Chinese Erhualian pigs and a Western Duroc × (Landrace × Yorkshire) commercial population.

BACKGROUND: Understanding the genetic mechanisms that underlie meat quality traits is essential to improve pork quality. To date, most quantitative trait loci (QTL) analyses have been performed on F2 crosses between outbred pig strains and have led to the identification of numerous QTL. However, because linkage disequilibrium is high in such crosses, QTL mapping precision is unsatisfactory and only a few QTL have been found to segregate within outbred strains, which limits their use to improve animal performance. To detect QTL in outbred pig populations of Chinese and Western origins, we performed genome-wide association studies (GWAS) for meat quality traits in Chinese purebred Erhualian pigs and a Western Duroc × (Landrace × Yorkshire) (DLY) commercial population. METHODS: Three hundred and thirty six Chinese Erhualian and 610 DLY pigs were genotyped using the Illumina PorcineSNP60K Beadchip and evaluated for 20 meat quality traits. After quality control, 35 985 and 56 216 single nucleotide polymorphisms (SNPs) were available for the Chinese Erhualian and DLY datasets, respectively, and were used to perform two separate GWAS. We also performed a meta-analysis that combined P-values and effects of 29 516 SNPs that were common to Erhualian, DLY, F2 and Sutai pig populations. RESULTS: We detected 28 and nine suggestive SNPs that surpassed the significance level for meat quality in Erhualian and DLY pigs, respectively. Among these SNPs, ss131261254 on pig chromosome 4 (SSC4) was the most significant (P = 7.97E-09) and was associated with drip loss in Erhualian pigs. Our results suggested that at least two QTL on SSC12 and on SSC15 may have pleiotropic effects on several related traits. All the QTL that were detected by GWAS were population-specific, including 12 novel regions. However, the meta-analysis revealed seven novel QTL for meat characteristics, which suggests the existence of common underlying variants that may differ in frequency across populations. These QTL regions contain several relevant candidate genes. CONCLUSIONS: These findings provide valuable insights into the molecular basis of convergent evolution of meat quality traits in Chinese and Western breeds that show divergent phenotypes. They may contribute to genetic improvement of purebreds for crossbred performance.

CircRNA-5335 Regulates the Differentiation and Proliferation of Sheep Preadipocyte via the miR-125a-3p/STAT3 Pathway.

The content of intramuscular fat (IMF) from preadipocytes is proportional to meat quality in livestock. However, the roles of circRNAs in IMF deposition in sheep are not well known. In this study, we show that circRNA-5335/miR-125a-3p/STAT3 play a crucial adjective role in the proliferation and differentiation of sheep preadipocytes. In this study, we characterized the roles of differentially expressed circRNA-5335/miR-125a-3p/STAT3, which were screened from sheep of different months of age and based on sequencing data. Firstly, the expression profiles of circRNA-5335/miR-125a-3p/STAT3 were identified during the differentiation of preadipocytes in vitro by RT-qPCR and WB. Then, the targeting relationship of the circRNA-5335/miR-125a-3p/STAT3 was verified by dual-luciferase reporter assays. The results of RT-qPCR, CCK8, EdU and Oil Red O staining assay showed that miR-125a-3p suppressed the differentiation and raised the proliferation of preadipocytes by targeting STAT3. As a competing endogenous RNA, the downregulation of circRNA-5335 decreased the expression of STAT3 by increasing miR-125a-3p, which inhibited the differentiation of preadipocytes and promoted proliferation. Our present study demonstrates the functional significance of circRNA-5335/miR-125a-3p/STAT3 in the differentiation of sheep preadipocytes, and provides novel insights into exploring the mechanism of IMF.

microRNA-148a suppresses human gastric cancer cell metastasis by reversing epithelial-to-mesenchymal transition.

MicroRNAs (miRNAs) are important regulators of gastric cancer development and progression. miR-148a is one of the most frequently and highly downregulated miRNAs in gastric cancer and is associated with advanced clinical stage and poor prognosis. In this study, we investigated the role of miR-148a in gastric cancer metastasis. Levels of miR-148a were determined by qRT-PCR in 60 gastric cancer samples. Cell migration and invasion assays were performed in a stably expressing miRNA-148a gastric cancer cell line established using a lentivirus expression system. Epithelial-mesenchymal transition (EMT) was evaluated using qRT-PCR and Western Blots to detect epithelial marker E-cadherin and mesenchymal marker, vimentin. Luciferase reporter assays were used to identify downstream targets and biological function of miR-148a. Gastric cancer tissue had significantly lower expression of miR-148a compared to non-tumor tissue. Low miR-148a levels were associated with lymph node metastasis, N stage, and blood vessel invasion. miR-148a overexpression inhibited metastasis of gastric cancer cells. miR-148a overexpression also downregulated vimentin expression and upregulated E-cadherin expression, suggesting that miR-148a inhibited EMT. Finally, the SMAD2 gene was identified as the direct and functional target of miR-148a. MiR-148a suppresses gastric cancer metastasis and EMT, likely via SMAD2. Restoration of miR-148a expression could have important implications in gastric cancer therapy.

Novel fluorescent risedronates: synthesis, photodynamic inactivation and imaging of Bacillus subtilis.

Novel fluorescently-labeled conjugates of risedronate were synthesized using an epoxide linker, enabling conjugation of risedronate via its pyridyl nitrogen with the aromatic succinimidyl esters. The compounds were characterized by using (1)H NMR, (13)C NMR, (31)P NMR, UV-vis and fluorescence emission spectroscopies. Biological activity assays showed that the conjugates 14 and 15 exhibited photodynamic inactivation of Bacillus subtilis (ATCC 6633) with 91% and 47% bacterial lethality at 10 µM upon visible light irradiation, respectively. Both 14 and 15 could be also used for fluorescence imaging of Bacillus subtilis.

A novel fluorogenic substrate for dinuclear Zn(II)-containing metallo-ß-lactamases.

In an effort to prepare a fluorogenic substrate to be used in activity assays with metallo-ß-lactamases, (6R,7R)-8-oxo-7-(2-oxo-2H-chromene-3-carboxamido)-3-((4-(2-oxo-2H-chromene-3-carboxamido)-phenylthio)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (CA) was synthesized and characterized. CA exhibited a fluorescence quantum yield (φ) of 0.0059, two fluorescence lifetimes of 3.63×10(-10) and 5.38×10(-9)s, and fluorescence intensity that is concentration-dependent. Steady-state kinetic assays revealed that CA is a substrate for metallo-ß-lactamases (MßLs) L1 and CcrA, exhibiting Km and kcat values of 18µM and 5s(-1) and 11µM and 17s(-1), respectively.

Single-variant and multi-variant trend tests for genetic association with next-generation sequencing that are robust to sequencing error.

As with any new technology, next-generation sequencing (NGS) has potential advantages and potential challenges. One advantage is the identification of multiple causal variants for disease that might otherwise be missed by SNP-chip technology. One potential challenge is misclassification error (as with any emerging technology) and the issue of power loss due to multiple testing. Here, we develop an extension of the linear trend test for association that incorporates differential misclassification error and may be applied to any number of SNPs. We call the statistic the linear trend test allowing for error, applied to NGS, or LTTae,NGS. This statistic allows for differential misclassification. The observed data are phenotypes for unrelated cases and controls, coverage, and the number of putative causal variants for every individual at all SNPs. We simulate data considering multiple factors (disease mode of inheritance, genotype relative risk, causal variant frequency, sequence error rate in cases, sequence error rate in controls, number of loci, and others) and evaluate type I error rate and power for each vector of factor settings. We compare our results with two recently published NGS statistics. Also, we create a fictitious disease model based on downloaded 1000 Genomes data for 5 SNPs and 388 individuals, and apply our statistic to those data. We find that the LTTae,NGS maintains the correct type I error rate in all simulations (differential and non-differential error), while the other statistics show large inflation in type I error for lower coverage. Power for all three methods is approximately the same for all three statistics in the presence of non-differential error. Application of our statistic to the 1000 Genomes data suggests that, for the data downloaded, there is a 1.5% sequence misclassification rate over all SNPs. Finally, application of the multi-variant form of LTTae,NGS shows high power for a number of simulation settings, although it can have lower power than the corresponding single-variant simulation results, most probably due to our specification of multi-variant SNP correlation values. In conclusion, our LTTae,NGS addresses two key challenges with NGS disease studies; first, it allows for differential misclassification when computing the statistic; and second, it addresses the multiple-testing issue in that there is a multi-variant form of the statistic that has only one degree of freedom, and provides a single p value, no matter how many loci.

The role of miR-369-3p in proliferation and differentiation of preadipocytes in Aohan fine-wool sheep.

MicroRNAs (miRNAs) are a large class of non-coding RNAs that play important roles in the proliferation and differentiation of adipocytes. Our previous sequencing analysis revealed higher expression of miR-369-3p in the longissimus muscle of 2-month-old Aohan fine-wool sheep (AFWS) compared to 12-month-old sheep (P<0.05), suggesting that miR-369-3p may regulate fat deposition in AFWS. To test this, miR-369-3p mimics, inhibitors, and negative controls (NCs) were constructed and transfected into AFWS preadipocytes. After transfection with miR-369-3p mimics, we found a decrease (P<0.05) in the expression of genes and proteins related to cell proliferation and differentiation, detected by RT-qPCR (quantitative reverse transcription PCR) and western blot analyses. Moreover, EdU (5-ethynyl-2'-deoxyuridine) detection and Oil Red O staining showed a decrease (P<0.05) in cell proliferation and lipid accumulation, respectively. The opposite trends (P<0.05) were obtained after transfection with miR-369-3p inhibitors. In conclusion, the results showed that miR-369-3p can inhibit the proliferation and differentiation of AFWS preadipocytes, providing a theoretical basis to further explore the molecular mechanism of fat deposition in sheep and other domestic animals.

Synthesis, characterization and activity of new phosphonate dipeptides as potential inhibitors of VanX.

VanX, a Zn(II)-dependent D-ala-D-ala dipeptidase, is essential for vancomycin resistance in Enterococcus faecium. The enzymatic activity of VanX was previously found to be inhibited competitively by 2-{[(1-aminoethyl) (hydroxy) phosphoryl]oxy} propanoic acid (1B). Here we report the synthesis and characterization of seven phosphonate dipeptide analogs of D-ala-D-ala with various substituent, the activity evaluation indicated that six of these phosphonate analogs inhibit VanX with IC(50) of 0.48-8.21mM. These data revealed a structure-activity relationship which is that the large substituent group on ß-carbon resulted in low binding affinity of the phonphonate analog to VanX. This information will be helpful to guide the design and synthesis of the tightly-binding inhibitors for VanX.