CAD Gene Research Articles

Staphylococcus saprophyticus Causing Infections in Humans Is Associated with High Resistance to Heavy Metals.

Staphylococcus saprophyticus is a common pathogen of the urinary tract, a heavy metal-rich environment, but information regarding its heavy metal resistance is unknown. We investigated 422 S. saprophyticus isolates from human infection and colonization/contamination, animals, and environmental sources for resistance to copper, zinc, arsenic, and cadmium using the agar dilution method. To identify the genes associated with metal resistance and assess possible links to pathogenicity, we accessed the whole-genome sequence of all isolates and used in silico and pangenome-wide association approaches. The MIC values for copper and zinc were uniformly high (1,600 mg/liter). Genes encoding copper efflux pumps (copA, copB, copZ, mco, and csoR) and zinc transporters (zinT, czrAB, znuBC, and zur) were abundant in the population (20 to 100%). Arsenic and cadmium showed various susceptibility levels. Genes encoding the ars operon (arsRDABC), an ABC transporter and a two-component permease, were linked to resistance to arsenic (MICs ≥ 1,600 mg/liter; 14% [58/422]; P < 0.05). At least three cad genes (cadA or cadC and cadD-cadX or czrC) and genes encoding multidrug efflux pumps and hyperosmoregulation in acidified conditions were associated with resistance to cadmium (MICs ≥ 200 mg/liter; 20% [85/422]; P < 0.05). These resistance genes were frequently carried by mobile genetic elements. Resistance to arsenic and cadmium were linked to human infection and a clonal lineage originating in animals (P < 0.05). Altogether, S. saprophyticus was highly resistant to heavy metals and accumulated multiple metal resistance determinants. The highest arsenic and cadmium resistance levels were associated with infection, suggesting resistance to these metals is relevant for S. saprophyticus pathogenicity.

Integrated Taxonomic Revision of AfrotropicalXyleborinus(Curculionidae: Scolytinae) Reveals High Diversity After Recent Colonization of Madagascar

AbstractThe ambrosia beetle genus Xyleborinus Reitter, 1913 is particularly species rich in Madagascar where the genus exhibits extraordinary morphological variation not seen elsewhere. This study provides the first detailed molecular phylogeny of the genus based on COI, 28S, and CAD gene fragments. Biogeographical and taxonomic hypotheses were tested for the Afrotropical fauna with a particular focus on the Malagasy radiation. Analyses revealed a single colonization of Madagascar no earlier than 8.5–11.0 Ma, indicating an extraordinary recent radiation on the island which has given rise to at least 32 species. Two recolonization events of the African mainland were strongly supported by the molecular data, with several other intraspecific dispersals to the mainland inferred from species distributions. A taxonomic re-evaluation of all Afrotropical Xyleborinus resulted in several taxonomic changes. We found that morphological differences associated with COI divergence higher than 7% indicated different species. Twelve new species are described: Xyleborinus castriformis Eliassen &amp; Jordal, sp. nov., Xyleborinus clivus Eliassen &amp; Jordal, sp. nov., Xyleborinus concavus Eliassen &amp; Jordal, sp. nov., Xyleborinus coronatus Eliassen &amp; Jordal, sp. nov., Xyleborinus diadematus Eliassen &amp; Jordal, sp. nov., Xyleborinus laevipennis Eliassen &amp; Jordal, sp. nov., Xyleborinus magnispinosus Eliassen &amp; Jordal, sp. nov., Xyleborinus margo Eliassen &amp; Jordal, sp. nov., Xyleborinus ntsoui Eliassen &amp; Jordal, sp. nov., Xyleborinus singularis Eliassen &amp; Jordal, sp. nov., Xyleborinus tuberculatus Eliassen &amp; Jordal, sp. nov., and Xyleborinus turritus Eliassen &amp; Jordal, sp. nov., all from Madagascar. New synonyms are proposed for Xyleborinus aemulus (Wollaston, 1869) [=Xyleborinus spinifer (Eggers, 1920)], Xyleborinus andrewesi (Blandford, 1896) [=Xyleborinus mimosae (Schedl, 1957)], Xyleborinus dentellus (Schedl, 1953) [=Xyleborinus forcipatus (Schedl, 1957)], Xyleborinus octospinosus (Eggers, 1920) [=Xyleborinus mitosomipennis (Schedl, 1953)], and Xyleborinus similans (Eggers, 1940) [=Xyleborinus sclerocaryae (Schedl, 1962)]. Two species were given new status: Xyleborinus profundus (Schedl, 1961) is elevated from subspecies of Xyleborinus aduncus (Schedl, 1961), and Xyleborinus mitosomus (Schedl, 1965) is reinstated from its previous synonymy with Xyleborinus spinosus (Schaufuss, 1891). Xyleborus gracilipennis Schedl 1957 is reverted to its original genus, and a similar status is confirmed for Xyleborus collarti Eggers 1932. The number of taxonomically valid Xyleborinus species in the Afrotropical region is now 47, which includes 3 adventive species. Revised diagnoses for all species and a key for species identification are provided.

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant.

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose-to-glucose conversion of maize stover. Here, we analyzed a field-grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD-deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose-to-glucose conversion efficiency was determined after 10 different thermochemical pre-treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre-treatment. However, the relative increase in glucose yields after alkaline pre-treatment was not higher than the relative increase when no pre-treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p-coumarate levels in the cell wall. Taken together, our results reveal how phenolic metabolism is affected in CAD-deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar-platform biorefineries.

Characterization of Two New brown midrib1 Mutations From an EMS-Mutagenic Maize Population for Lignocellulosic Biomass Utilization.

Gene mutations linked to lignin biosynthesis are responsible for the brown midrib (bm) phenotypes. The bm mutants have a brown-reddish midrib associated with changes in lignin content and composition. Maize bm1 is caused by a mutation of the cinnamyl alcohol dehydrogenase gene ZmCAD2. Here, we generated two new bm1 mutant alleles (bm1-E1 and bm1-E2) through EMS mutagenesis, which contained a single nucleotide mutation (Zmcad2-1 and Zmcad2-2). The corresponding proteins, ZmCAD2-1 and ZmCAD2-2 were modified with Cys103Ser and Gly185Asp, which resulted in no enzymatic activity in vitro. Sequence alignment showed that CAD proteins have high similarity across plants and that Cys103 and Gly185 are conserved in higher plants. The lack of enzymatic activity when Cys103 was replaced for other amino acids indicates that Cys103 is required for its enzyme activity. Enzymatic activity of proteins encoded by CAD genes in bm1-E plants is 23–98% lower than in the wild type, which leads to lower lignin content and different lignin composition. The bm1-E mutants have higher saccharification efficiency in maize and could therefore provide new and promising breeding resources in the future.

The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda.

It is well known that insect larval midgut cadherin protein serves as a receptor of Bacillus thuringiensis (Bt) crystal Cry1Ac or Cry1Ab toxins, since structural mutations and downregulation of cad gene expression are linked with resistance to Cry1Ac toxin in several lepidopteran insects. However, the role of Spodoptera frugiperda cadherin protein (SfCad) in the mode of action of Bt toxins remains elusive. Here, we investigated whether SfCad is involved in susceptibility to Cry1Ab or Cry1Fa toxins. In vivo, knockout of the SfCad gene by CRISPR/Cas 9 did not increase tolerance to either of these toxins in S. frugiperda larvae. In vitro cytotoxicity assays demonstrated that cultured insect TnHi5 cells expressing GFP-tagged SfCad did not increase susceptibility to activated Cry1Ab or Cry1Fa toxins. In contrast, expression of another well recognized Cry1A receptor in this cell line, the ABCC2 transporter, increased the toxicity of both Cry1Ab and Cry1Fa toxins, suggesting that SfABCC2 functions as a receptor of these toxins. Finally, we showed that the toxin-binding region of SfCad did not bind to activated Cry1Ab, Cry1Ac, nor Cry1Fa. All these results support that SfCad is not involved in the mode of action of Cry1Ab or Cry1Fa toxins in S. frugiperda.

CAD Deficiency—Another Treatable Early Infantile Epileptic Encephalopathy

Early infantile epileptic encephalopathies (EIEEs) are caused by a variety of structural brain abnormalities, genetic defects and inborn errors of metabolism. Although most conditions do not have definitive treatment, a very few of these are treatable like pyridoxine dependency, pyridoxal phosphate–responsive seizures, folinic acid–responsive seizures, biotinidase deficiency, glucose transporter type 1 deficiency syndrome, creatine deficiency, and KCNQ2 and SCN2A encephalopathies.1 We now have another condition in EIEE that is easily treatable, i.e., EIEE-50 caused because of mutations in CAD gene that can be treated with uridine supplementation.

Correction: Characterization and analysis of CCR and CAD gene families at the whole-genome level for lignin synthesis of stone cells in pear (Pyrus bretschneideri) fruit.

There were errors in Biology Open (2017) 6, bio026997 (doi:10.1242/bio.026997).The wrong Fig. 8 was used in the published paper. The correct and original figure are shown below and both the online full-text and PDF versions of the article have been updated.Fig. 8 (correct image).Fig. 8 (original image).The authors apologise for these errors and any inconvenience they may have caused.This correction does not affect the results in the article or the conclusions of this study.

Identification of key genes and pathways affected in epicardial adipose tissue from patients with coronary artery disease by integrated bioinformatics analysis.

BackgroundCoronary artery disease (CAD) is a common disease with high cost and mortality. Here, we studied the differentially expressed genes (DEGs) between epicardial adipose tissue (EAT) and subcutaneous adipose tissue (SAT) from patients with CAD to explore the possible pathways and mechanisms through which EAT participates in the CAD pathological process.MethodsMicroarray data for EAT and SAT were obtained from the Gene Expression Omnibus database, including three separate expression datasets: GSE24425, GSE64554 and GSE120774. The DEGs between EAT samples and SAT control samples were screened out using the limma package in the R language. Next, we conducted bioinformatic analysis of gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways to discover the enriched gene sets and pathways associated with DEGs. Simultaneously, gene set enrichment analysis was carried out to discover enriched gene functions and pathways from all expression data rather than DEGs. The PPI network was constructed to reveal the possible protein interactions consistent with CAD. Mcode and Cytohubba in Cytoscape revealed the possible key CAD genes. In the next step, the corresponding predicted microRNAs (miRNAs) were analysed using miRNA Data Integration Portal. RT-PCR was used to validate the bioinformatic results.ResultsThe three datasets had a total of 89 DEGs (FC log2 > 1 and P value < 0.05). By comparing EAT and SAT, ten common key genes (HOXA5, HOXB5, HOXC6, HOXC8, HOXB7, COL1A1, CCND1, CCL2, HP and TWIST1) were identified. In enrichment analysis, pro-inflammatory and immunological genes and pathways were up-regulated. This could help elucidate the molecular expression mechanism underlying the involvement of EAT in CAD development. Several miRNAs were predicted to regulate these DEGs. In particular, hsa-miR-196a-5p and hsa-miR-196b-5p may be more reliably associated with CAD. Finally, RT-PCR validated the significant difference of OXA5, HOXC6, HOXC8, HOXB7, COL1A1, CCL2 between EAT and SAT (P value < 0.05).ConclusionsBetween EAT and SAT in CAD patients, a total of 89 DEGs, and 10 key genes, including HOXA5, HOXB5, HOXC6, HOXC8, HOXB7, COL1A1, CCND1, CCL2, HP and TWIST1, and miRNAs hsa-miR-196a-5p and hsa-miR-196b-5p were predicted to play essential roles in CAD pathogenesis. Pro-inflammatory and immunological pathways could act as key EAT regulators by participating in the CAD pathological process.

Natural Variation in Lignin and Pectin Biosynthesis-Related Genes in Switchgrass (Panicum virgatum L.) and Association of SNP Variants with Dry Matter Traits

Switchgrass (Panicum virgatum), a C4 perennial grass native to North America and developed as a sustainable biofuel feedstock, occurs in two ecotypes, lowland and upland, which vary in their architecture as well as their range of adaptation. In this study, we assessed single nucleotide polymorphism (SNP) variation in 372 switchgrass genotypes for nine genes involved in lignin and pectin biosynthesis. STRUCTURE results at K = 3 differentiated the genotypes into three genetic subpopulations that corresponded largely to the previously characterized upland C1 and lowland C2 and C3 subpopulations. Out of the 146 SNPs identified, 19 SNPs were non-synonymous, including two non-conservative and common SNPs in cinnamyl alcohol dehydrogenase (CAD, Chr01N) and p-coumarate3-hydroxylase (C3H, Chr09N), two genes in the lignin biosynthesis pathway. Allele status at seven of the 19 non-synonymous SNPs, including the non-conservative SNP in C3H, was significantly associated with four dry matter traits within subpopulations. Dry matter traits appeared to be mostly dominant and three of them (acid detergent fiber, non-fiber carbohydrate, water-soluble carbohydrates) were the most frequently differentiated traits. In addition, a heterosis effect was detected at the non-conservative SNP in phenylalanine ammonia-lyase (PAL) for neutral detergent fiber. Association analysis revealed the CAD gene on Chr01N, its homoeolog on Chr01K and cinnamate 4-hydroxylase (C4H, Chr03K) as potential candidates associated with dry matter traits. Further analyses are needed to determine whether these candidate genes play a role in switchgrass lignin content and could be exploited to reduce recalcitrance in this bioenergy crop.

Identification of Cry1Ah-binding proteins through pull down and gene expression analysis in Cry1Ah-resistant and susceptible strains of Ostrinia furnacalis

Bacillus thuringiensis produces insecticidal Cry toxins used in the control of multiple insect pests. Evolution of insect resistance to Bt toxins endangers the use of Cry toxins for pest control. Analysis of the Cry1Ah-binding proteins from brush border membrane vesicles (BBMV) of Ostrinia furnacalis, Asian corn borer (ACB) from the Cry1Ah-resistant (ACB-AhR) and susceptible (ACB-BtS) strains was performed by an improved pull down assay that includes coupling Cry1Ah to NHS-activated Sepharose combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS). Our data show that Cry1Ah bound to alkaline phosphatase (ALP), cadherin-like (CAD), actin, aminopeptidase-N (APN), prophenoloxidase (proPO), serine proteinase inhibitor (SPI), immulectin, and V-ATPase and to other proteins that were not previously characterized as Cry-binding proteins in ACB-BtS strain. Analysis of Cry1Ah-pulled down proteins of the BBMV from ACB-AhR revealed that Cry1Ah toxin did not bind to ALP in ACB-AhR strain, suggesting that this protein may correlate with the resistant phenotype of this strain. Additionally, we analyzed the expression of representative genes coding for Cry1Ah-binding proteins such as ALP, APN, CAD, proPO, SPI, and immulectin by qRT-PCR. ACB-AhR showed increased expression levels of proPO (7.5 fold), ALP (6.2 fold) and APN (1.4 fold) in comparison to ACB-BtS strain. In contrast, the cad gene showed slight decreased expression in ACB-AhR strain (0.7 fold) compared with ACB-BtS strain. Our data suggest that differences in the susceptibility to Cry1Ah toxin in the ACB-AhR strain may be associated with reduced ALP binding sites and with an increased immune response. This study also brings evidence of a possible binding interaction of Cry1Ah toxin to immune related proteins like proPO.

Deer keds on wild ungulates in northern Italy, with a taxonomic key for the identification of Lipoptena spp. of Europe.

Deer keds (Lipoptena spp.) are blood-sucking ectoparasites of domestic and wild animals, and also accidentally of humans. In Europe, five Lipoptena spp. have been recorded, although the lack of specific taxonomic keys has often led to mistaken identification or to missing data. The present study aimed to develop an identification key of the European species and also to identify Lipoptena spp. found on wild ungulates in northern Italy. In total, 390 hippoboscids were collected from Rupicapra rupicapra, Capreolus capreolus, Cervus elaphus and Ovis aries musimon in an Alpine area of Italy. After morphological identification, 140 specimens were subjected to phylogenetic analysis based on mitochondrial (CO1) and nuclear (CAD) gene sequences. Despite the expected presence of slight morphological variations, all specimens examined were identified both microscopically and molecularly as Lipoptena cervi (100% identity for both CO1 and CAD genes). The massive increase in wild ungulate populations can favour the possibility of detecting other species of Lipoptena. The identification keys proposed in the present study may help with monitoring the presence of Lipoptena species, particularly in European countries where this ectoparasite is neglected and for which various data (from diffusion to control methods) are still missing.

Quantitative Proteome Reveals Variation in the Condition Factor of Sea Urchin Strongylocentrotus nudus during the Fishing Season Using an iTRAQ-based Approach.

To investigate the variation in the condition factor of the sea urchin Strongylocentrotus nudus (S. nudus), gonads were collected in May (MAY), June (JUN), and July (JUL), at the beginning (AUG-b) and end of August (AUG-e). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) detection of the gonads revealed an obvious enhancement of the band at about 37 kDa from July, which was identified as transforming growth factor-beta-induced protein ig-h3 (TGFBI) by nanoLC-ESI-MS/MS. Gonadal proteins were identified by isobaric tagging for relative and absolute quantitation (iTRAQ), and regulation of the identified proteins in pairs of the collected groups was observed. A total of 174 differentially expressed proteins (DEPs) were identified. Seven of the DEPs showed significant correlations with both the gonad index (GI) and protein content. These correlations included 6-phosphogluconate dehydrogenase, decarboxylating isoform X2 (6PGD), CAD protein, myoferlin isoform X8, ribosomal protein L36 (RL36), isocitrate dehydrogenase [NADP], mitochondrial isoform X2 (IDH), multifunctional protein ADE2 isoform X3, sperm-activating peptides (SAPs) and aldehyde dehydrogenase, and mitochondrial (ALDH). However, TGFBI had no correlation with gonad index (GI) or protein content. 6PGD, IDH, multifunctional protein ADE2 isoform X3, and ALDH were shown to interact with each other and might play key roles in changing the condition factor of S. nudus gonads.

Expression and significance of Caspase-3 in hepatocellular carcinoma cells induced by high frequency square wave electric pulse with different intensities

Objective To investigate the expression level of Caspase-3and the damage effect in hepatoma cells under different intensity pulses. Methods The pulses were divided into 10 groups according to the electric field intensity. After pulsed discharge of Hep-G2 hepatoma cells cultured in vitro. The damage effect was judged by detecting the activity of Caspase-3, proliferation activity, apoptotic rate and the expression levels of cytochrome C (Cyt C), Cleaved-Caspase-3 and cysteine protease-activated DNA enzymes (CAD). Results High expression of Caspase-3 was found in hepatoma cells treated with electric pulse. The expression level of 750-1 750 V/cm increased with the increase of field intensity (P 0.05). Then the field strength continued to rise, the expression level gradually decreased. The electric pulse could inhibit the proliferation of hepatoma cells, and its inhibition ability was positively correlated with the field intensity. When the electric field intensity was 2 250-2 750 V/cm, the proliferative activity decreased to the lowest level (P>0.05). The effect of pulsed electric field on cells injury included induction of apoptosis and necrosis. The apoptosis effect reached the peak at the field strength of 1 750-2 000 V/cm (P>0.05). The necrosis effect increased significantly when the field strength was higher than that of 2 000 V/cm, and reached the highest at 2 750 V/cm. Western blotting suggests that pulsed electric field can induce the up-regulation of Cyt C, Cleaved-Caspase-3 and CAD protein expression, which leads to apoptosis (P<0.05). Conclusion High frequency square wave electric pulse can induce the increase of Caspase-3 expression in hepatoma cells, and activate Cyt C signaling pathway to induce apoptosis. The apoptotic effect was the most obvious when the filed strength was set on 1 750-2 000 V/cm. Key words: High frequency square wave; Electric pulse; Electric intensity; Damage effect; Apoptpsis

A missense mutation (p.Tyr452Cys) in the CAD gene compromises reproductive success in French Normande cattle

We scanned the genome of 77,815 Normande cattle with different Illumina SNP chips (Illumina Inc., San Diego, CA) to map recessive embryonic lethal mutations using homozygous haplotype deficiency. We detected 2 novel haplotypes on chromosomes 11 and 24 but did not confirm 6 previously reported haplotypes. The one on chromosome 11 showed a marked reduction in conception rates and moderate decrease in nonreturn rate in at-risk versus control mating, supporting late embryonic mortality. After fine mapping and analyzing whole-genome sequences, we prioritized a missense mutation in CAD (g.72399397T>C; p.Tyr452Cys)-a gene encoding a protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) essential for de novo pyrimidine biosynthesis-as a candidate causal variant. This transition mutation replaces a tyrosine residue, which is perfectly conserved among living organisms, with a cysteine residue in the carbamoyl-phosphate synthetase 2 domain of the protein. A single animal was confirmed to be homozygous for the mutation based on Sanger sequencing. However, large-scale genotyping of the candidate variant with the Illumina EuroG10k BeadChip revealed an absence of live homozygotes in a panel of 33,323 Normande animals and an absence of carriers in 348,593 animals from 19 other cattle breeds. These results support recessive embryonic lethality with nearly complete penetrance, as was previously reported in CAD mutants in several eukaryote species. The only homozygous cow had extremely poor udder conformation, suggesting a potential role of CAD in udder development, but no effect was detected when comparing daughter yield deviations of 250 heterozygous bulls with that of 2,912 homozygotes for the ancestral allele. Together, our results showed the importance of large-scale screening for homozygous haplotype deficiency with hundreds of thousands of animals, validating results with an independent data set, and considering unexpected live homozygotes, to avoid both false-positive and false-negative discoveries. These discoveries will be used primarily in mating decisions to avoid at-risk mating. In addition, we recommend including CAD in the breeding objectives of Normande cattle.

Lignin biosynthesis as a key mechanism to repress Polystigma amygdalinum, the causal agent of the red leaf blotch disease in almond

The red leaf blotch (RLB) of almond, caused by the fungus Polystigma amygdalinum, is considered as one of the most important leaf diseases of this fruit tree. Differential cultivar susceptibility to the RLB has been described based on field observations, while its molecular and biochemical bases remain unknown to date. We aimed to explore the plant defence mechanisms related to the cultivar susceptibility by identifying some relevant physical and chemical strategies for the pathogen control. Thus, we studied the regulation of seven defence-related genes as well as the lignin deposition in two almond cultivars with highly differential response to RLB: the highly tolerant ‘Mardía’ and the susceptible ‘Tarraco’ cultivars. ‘Mardía’ displayed an up-regulation of the CAD and DFN1 genes at early stages of RLB symptom expression, with further lignin deposition in the fungal-colonized area that was visualized by microscopy. Thus, ‘Mardía’ uses both physical and chemical responses to effectively repress the pathogen. In contrast, ‘Tarraco’ triggered the up-regulation of HQT and LDOX genes, related to chlorogenic acid and anthocyanin biosynthesis pathways, respectively, while lignin deposition was not clearly noticed. This strategy recorded in ‘Tarraco’ at later stages of RLB symptoms failed to control the fungal infection and colonization. Our results suggested a major role of the phenylpropanoids pathway in the defence response against RLB, by showing that an early production of lignin might be a major mechanism to control the spread of P. amygdalinum within the host leaf tissues.

Bmp4 inhibits goose granulosa cell apoptosis via PI3K/AKT/Caspase-9 signaling pathway

Bone morphogenetic protein 4 (BMP4) has an important role in regulating cellular proliferation, differentiation and apoptosis. It, however, is still unclear as to the mechanisms by which BMP4 regulates the apoptosis of granulosa cells (GCs) in geese. In the present study, there was cloning of the full-length coding sequence of goose BMP4 gene, which consisted of 1212 nucleotides encoding 403 amino acids. Its deduced amino acid sequence comprised one signal peptide, one TGFβ pro-peptide and one mature peptide domain. Results from conducting the quantitative real-time PCR (qPCR) indicated the relative abundances of BMP4 mRNA in geese GCs increased gradually from the relative abundances in pre-hierarchical follicles that were 4 to 6 mm in diameter to that in the fifth largest (F5) follicle and then relative abundances of BMP4 mRNA decreased with further development as the largest (F1) follicle. Results from use of the TUNEL assay indicated that overexpression of the goose BMP4 gene suppressed GC apoptosis and this was confirmed when relative abundances of the CAD, Caspase-9 and Caspase-3 proteins were determined using western blotting. In addition, overexpression of the BMP4 gene induced phosphorylation of AKT, which was inhibited with use of the PI3K inhibitor, LY294002. Co-transfection of BMP4 and LY294002 resulted in increased relative abundances of Caspase-9 and CAD proteins but had no effect on that of Caspase-3. Taken together, these results suggested that expression of the BMP4 gene resulted in a reduction in Caspase-9 protein leading to inhibition of GC apoptosis via the PI3K/AKT signaling pathway in geese.

A Multi-Gene Analysis and Potential Spatial Distribution of Species of the Strodei Subgroup of the Genus Nyssorhynchus (Diptera: Culicidae).

Nyssorhynchus strodei (Root) is an understudied species of potential epidemiological importance, having been found naturally infected in Brazil with Plasmodium falciparum Welch, Plasmodium vivax Grassi & Feletti, and Plasmodium malariae Feletti & Grassi. It belongs to the strodei subgroup that includes Nyssorhynchus albertoi (Unti), Nyssorhynchus arthuri (Unti), Nyssorhynchus rondoni (Neiva & Pinto), Nyssorhynchus striatus (Sant'Ana & Sallum), and three unnamed species, Nyssorhynchus arthuri B, Nyssorhynchus arthuri C, and Nyssorhynchus arthuri D. As the accurate identification of vector species is of fundamental importance for public health entomology, the aim of the study was to provide additional information for the presence of seven species that had been previously misidentified as Ny. strodei. Bayesian phylogenetic analyses using DNA sequences of the genes COI mtDNA, white, CAD and CAT nuclear genes confirmed Ny. albertoi, Ny. arthuri, Ny. strodei, and Ny. striatus as distinct groups within the strodei subgroup and corroborated the presence of three undescribed species under the name Ny. arthuri. Results of the GMYC model analysis corroborated Ny. arthuri B, Ny. arthuri C, and Ny. arthuri D; however, they did not distinguish between Ny. strodei and Ny. albertoi. Predicted distribution of seven species based on maximum entropy in MaxEnt showed that each species has its specific ecological niche suitability.

Discrimination of Anopheles species of the Arribalzagia Series in Colombia using a multilocus approach

The Arribalzagia Series of the Anopheles Subgenus comprises morphologically similar species or members of species complexes which makes correct species identification difficult. Therefore, the aim of this work was to discriminate the morphospecies of the Arribalzagia Series present in Colombia using a multilocus approach based on ITS2, COI and CAD sequences. Specimens of the Arribalzagia Series collected at 32 localities in nine departments were allocated to seven species. Individual and concatenated Bayesian analyses showed high support for each of the species and reinforced the previous report of the Apicimacula species Complex with distribution in the Pacific Coast and northwestern Colombia. In addition, a new molecular operational taxonomic unit-MOTU was identified, herein denominated near Anopheles peryassui, providing support for the existence of a Peryassui species Complex. Further, the CAD gene, just recently used for Anopheles taxonomy and phylogeny, demonstrated its power in resolving phylogenetic relationships among species of the Arribalzagia Series. The divergence times for these species correspond to the early Pliocene and the Miocene. Considering the epidemiological importance of some species of the Series and their co-occurrence in malaria endemic regions of Colombia, their discrimination constitutes an important step for vector incrimination and control in the country.

Analysis of CmCADs and three lignifying enzymes in oriental melon (‘CaiHong7’) seedlings in response to three abiotic stresses

Abstract Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195), a key enzyme in lignin monomer synthesis pathway, is responsible for the conversion of cinnamyl aldehydes to cinnamyl alcohols. Our previous work identified five CAD genes in the melon genome and named CmCAD1 to CmCAD5, respectively. The further promoter analysis of CmCADs revealed elements involved in stress response and signaling pathway. However, what abiotic stresses the genes respond to remain unclear. Thus three abiotic stresses were conducted on oriental melon seedlings (‘CaiHong7’) as drought, salt and wound. In our study, lignin deposition was significantly promoted in stems and roots, and stem sections showed deeper staining with phloroglucinol-HCl under the three abiotic stresses than the control, indicating a higher lignin accumulation promoted by abiotic stress. Activities of CAD and peroxidase (POD; EC 1.11.1.7), as well as phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), were generally induced to significant levels by the abiotic stresses, implying that lignin synthesis pathway may be activated. CmCAD2 was significantly up-regulated in stems and roots under salt treatment but was most strongly induced in whole seedlings under drought stress. Also, CmCAD2 was significantly induced by wound treatment as well as the other CmCADs. However, the other four CmCAD genes exhibited tissue-specific inductions under drought and salt stresses. Expression analysis suggested that CmCAD2 might be a major gene in lignin deposition responding to the abiotic stresses.

Bacterial Genes of 2,4-Dichlorophenoxyacetic Acid Degradation Encoding α-Ketoglutarate-Dependent Dioxygenase Activity

The tfdA gene encodes α-ketoglutarate-dependent dioxygenase, which catalyzes the first step of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation pathway. The entire range of 2,4-D-degrading bacteria is divided into three groups based on their phylogeny, physiological and biochemical features, and isolation source. Each of these groups has its own version of the tfdA gene. The first group is the most studied and consists of fast-growing copiotrophic β- and γ-Proteobacteria isolated from anthropogenic habitats. The bacteria of this group possess the canonical sequence of this gene. Within this group, tfdA forms at least three classes (I, II, and III) of highly homologous gene families. The tfdA gene of Cupriavidus necator JMP134 was recognized as a Class I type. Class II consists of tfdA sequences belonging only to the genus Burkholderia of β-Proteobacteria, whereas Class III includes tfdA sequences belonging to the genera Delftia, Cupriavidus, Variovorax, Achromobacter, Comamonas, Rhodoferax, Halomonas, and Pseudomonas of β- and γ-Proteobacteria. The similarity of full-length nucleotide sequences between Class I and other classes was about 77–78%, and that between Class II and Class III was 93%. The second and third groups of 2,4-D-degrading bacteria are closely related to the genera Sphingomonas and Bradyrhizobium, which belong to α-Proteobacteria. tfdA-Like genes were identified only in four Sphingomonas spp. This fact and their phylogenetically distinct position make it possible to suggest that these genes evolved independently from each other through vertical gene transfer. The tfdAα gene was identified in the third group of bacteria of the genus Bradyrhizobium, which are both able and unable to degrade 2,4-D. Nevertheless, 2,4-D-degrading bacteria of the genera Bradyrhizobium and Sphingomonas have cad genes, which initiate the first step of the chlorophenoxyacetic acid degradation pathway. Based on the data on tfdAα localization in bacteria isolated from pristine ecosystems, a theory has been proposed that the tfdAα gene is ancestral for tfdA. Horizontal transfer and further adaptation to anthropogenic habitats probably led to the emergence of tfdA. However, tfdA and tfdAα may have diverged from a common ancestor, because they show a high similarity (51–57%) and separate distribution in the β-Proteobacteria, γ-Proteobacteria, and α-Proteobacteria, respectively.