RNA-Seq transcriptome profiling of immature grain wheat is a technique for understanding comparative modeling of baking quality.

Improving the baking quality is a primary challenge in the wheat flour production value chain, as baking quality represents a crucial factor in determining its overall value. In the present study, we conducted a comparative RNA-Seq analysis on the high baking quality mutant "O-64.1.10" genotype and its low baking quality wild type "Omid" cultivar to recognize potential genes associated with bread quality. The cDNA libraries were constructed from immature grains that were 15 days post-anthesis, with an average of 16.24 and 18.97 million paired-end short-read sequences in the mutant and wild-type, respectively. A total number of 733 transcripts with differential expression were identified, 585 genes up-regulated and 188 genes down-regulated in the "O-64.1.10" genotype compared to the "Omid". In addition, the families of HSF, bZIP, C2C2-Dof, B3-ARF, BES1, C3H, GRF, HB-HD-ZIP, PLATZ, MADS-MIKC, GARP-G2-like, NAC, OFP and TUB were appeared as the key transcription factors with specific expression in the "O-64.1.10" genotype. At the same time, pathways related to baking quality were identified through Kyoto Encyclopedia of Genes and Genomes. Collectively, we found that the endoplasmic network, metabolic pathways, secondary metabolite biosynthesis, hormone signaling pathway, B group vitamins, protein pathways, pathways associated with carbohydrate and fat metabolism, as well as the biosynthesis and metabolism of various amino acids, have a great deal of potential to play a significant role in the baking quality. Ultimately, the RNA-seq results were confirmed using quantitative Reverse Transcription PCR for some hub genes such as alpha-gliadin, low molecular weight glutenin subunit and terpene synthase (gibberellin) and as a resource for future study, 127 EST-SSR primers were generated using RNA-seq data.

Augmenting the Antitumor Efficacy of Natural Killer Cells via SynNotch Receptor Engineering for Targeted IL-12 Secretion.

Natural killer (NK) cells are crucial components of innate immunity, known for their potent tumor surveillance abilities. Chimeric antigen receptors (CARs) have shown promise in cancer targeting, but optimizing CAR designs for NK cell functionality remains challenging. CAR-NK cells have gained attention for their potential to reduce side effects and enable scalable production in cancer immunotherapy. This study aimed to enhance NK cell anti-tumor activity by incorporating PD1-synthetic Notch (synNotch) receptors. A chimeric receptor was designed using UniProt database sequences, and 3D structure models were generated for optimization. Lentiviral transduction was used to introduce PD1-Syn receptors into NK cells. The expression of PD1-Syn receptors on NK cell surfaces was assessed. Engineered NK cells were co-cultured with PDL1+ breast cancer cells to evaluate their cytotoxic activity and ability to produce interleukin-12 (IL-12) and interferon-gamma (IFNγ) upon interaction with the target cells. This study successfully expressed the PD1-Syn receptors on NK cells. CAR-NK cells secreted IL-12 and exhibited target-dependent IFNγ production when engaging PDL1+ cells. Their cytotoxic activity was significantly enhanced in a target-dependent manner. This study demonstrates the potential of synNotch receptor-engineered NK cells in enhancing anti-tumor responses, especially in breast cancer cases with high PDL1 expression.

Effects of Bacillus in Pectobacterium quorum quenching: A survey of two different acyl-homoserine lactonases.

Numerous functions in pathogenic Pectobacterium are regulated by quorum sensing (QS). Two different aiiA genes isolated from Bacillus sp. A24(aiiAA24) and Bacillus sp. DMS133(aiiADMS133) were used. Both genes encode acyl-homoserine lactonase (AiiA), which disrupts QS in Pectobacterium. To investigate the effect of different AiiAs on the inhibition of Pectobacterium carotovorum pathogenicity, two aiiA genes from different Bacillus strains were cloned and the resulting plasmids pME6863 (aiiAA24) and pME7080 (aiiADMS133) were transformed into P. carotovorum EMPCC cells. The effects of different lactonases on virulence features such as enzymatic activity, twitching and swimming motilities, and production of pellicle and biofilm formation were investigated. In EMPCC/pME6863, twitching and swimming motilities, and pellicle production were significantly reduced compared with EMPCC/pME7080. Quantitative real-time PCR (qRT-PCR) was used to measure virulence gene expression in transformed cells compared with expression levels in wild-type EMPCC. The expression of peh and hrpL genes was greatly reduced in EMPCC/pME6863 compared with EMPCC/pME7080. The sequence alignment and molecular dynamic modeling of two different AiiAA24 and AiiADMS133 proteins suggested that the replacement of proline 210 from AiiAA24 to serine in AiiADMS133 caused the reduction of enzyme activity in AiiADMS133.

Enhancing productivity of Chinese hamster ovary (CHO) cells: synergistic strategies combining low-temperature culture and mTORC1 signaling engineering.

Introduction: The growing demand for recombinant proteins in medicine has prompted biopharmaceutical companies to seek ways to maximize the manufacturing process. Despite its known negative impact on cell growth, temperature shift (TS) has emerged as a cost-effective strategy to enhance protein quantity and quality in Chinese Hamster Ovary cells (CHO). As cells adapt their growth and protein synthesis rate to the environment through influencing mTOR complex 1 (mTORC1), here we evaluated the potential of mTORC1 signaling engineering to improve the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein in stable CHO cells at low temperature. Methods: First, the expression of genes that negatively control mTORC1 functions in response to environmental fluctuations, including TSC1, AMPK, MAPKAPK5, and MARK4 genes, was assessed via real-time qPCR in CHO-K1 after a temperature shift from 37°C to 30°C. Then, plasmids harboring the shRNAs targeting these genes were constructed into the PB513B-1 plasmid with expression driven by either the constitutive CMV promoter or the cold-inducible HSP90 promoter. Finally, the impact of transient gene downregulation was evaluated on GM-CSF and mTOR proteins productivity in GM-CSF-producing CHO-K1 cells using ELISA and Western-blot assays, respectively. The growth rate of the transfected cells at the two temperatures was evaluated using flow cytometry. Results: Hypothermic conditions promote the upregulation of mTORC1 inhibitor genes, especially TSC1 and MAPKAPK5, while downregulating S6K, a key effector of the mTORC1 signaling pathway, in CHO-K1 cells. Transcription and protein levels of mTOR increased upon transfection, "pB513-b CMV-P/4shRNAs/GFP" plasmid, "pB513-bHSP90-P/4sh-RNAs/GFP" and pB513B-1 plasmid as mock group in GM-CSF-producing CHO-K1 cells (approximately 60%), along with a high transcript level of S6K. Cell growth-related characteristics were improved, albeit with distinct effects at different temperatures. Notably, these changes were more efficient at 30°C when utilizing the HSP90 promoter, resulting in a three-fold increase in GM-CSF production after 3 days. Conclusion: This study highlights the importance of temperature regulation and mTORC1 modulation in CHO cellular processes, particularly in recombinant protein production. Understanding these mechanisms paves the way for developing innovative strategies to enhance cell growth, protein synthesis, and overall bioprocess performance, particularly in manufacturing human therapeutic proteins.

Evaluation of the impact of polypeptide-p on diabetic rats upon its cloning, expression, and secretion in Saccharomyces boulardii.

This study was designed to evaluate the effectiveness of recombinant polypeptide-p derived from Momordica charantia on diabetic rats. In this research, the optimized sequence of polypeptide-p gene fused to a secretion signal tag was cloned into the expression vector and transformed into probiotic Saccharomyces boulardii. The production of recombinant secretion protein was verified by western blotting, HPLC, and mass spectrometry. To assay recombinant yeast bioactivity in the gut, diabetic rats were orally fed wild-type and recombinant S. boulardii, in short SB and rSB, respectively, at two low and high doses as well as glibenclamide as a reference drug. In untreated diabetic and treated diabetic + SB rats (low and high doses), the blood glucose increased from 461, 481, and 455 (mg/dl), respectively, to higher than 600 mg/dl on the 21st day. Whereas glibenclamide and rSB treatments showed a significant reduction in the blood glucose level. The result of this study promised a safe plant-source supplement for diabetes through probiotic orchestration.

A simple and efficient method for cytoplasmic production of human enterokinase light chain in E. coli.

Human enterokinase light chain (hEKL) cDNA sequence was designed with the help of codon optimization towards Escherichia coli codon preference and ribosome binding site design and artificially synthesized with a thioredoxin fusion tag at the N-terminal and a five his-tag peptide at the C-terminal. The synthetic hEKL gene was cloned into the pET-15 expression vector and transferred into the three different expression strains of E. coli BL21(DE3), NiCo21, and SHuffle T7 Express. Different growth and induction conditions were studied using a statistical response surface methodology (RSM). Recombinant hEKL protein was expressed at high levels in soluble form with 0.71 mM IPTG after 4 h of induction at 25 °C. Autocatalytic process cleaved TRX tag with enterokinase recognition site by the impure hEKL and yielded the mature enzyme. The target protein was then purified to homogeneity (> 95%) by affinity chromatography. The activity of hEKL was comparable to the commercial enzyme. From 1 L culture, 80 mg pure active hEKL was obtained with the specific activity of 6.25 × 102 U/mg. Three main parameters that help us to produce the enzyme in the folded and active form are the type of strain, SHuffle T7 strain, TRX and histidine fusion tags, and growth conditions including the increase of OD of induction and IPTG concentration and the decrease of induction temperature.

RT-LAMP in SARS-CoV-2 detection: point to improve primer designing and decrease molecular diagnosis pitfalls.

BACKGROUND: Due to the high transmission rate of SARS-CoV-2, diagnostic tests have become tools for identifying patients. The key points were the virus genomes survey to design RT-LAMP primers; comparing the sensitivity and specificity of RT-LAMP and RT-qPCR; and determining the relationship among clinical symptoms, CT scan, RT-qPCR, and RT-LAMP results. METHODS: This cohort study included 444 symptomatic patients. The specificity and sensitivity of RT-LAMP were assayed. The five statistical models, simultaneously, by RapidMiner to find the best method for detecting the virus were done through the correlation between the clinical symptoms, RT-LAMP, RT-qPCR, and CT scan results. The chi-square test by SPSS 26.0 was used to calculate kappa agreement. RESULTS: The virus genome was detected in all the positive samples (198) by RT-qPCR and RT-LAMP. In addition, 246 samples were negative by RT-qPCR, while 88 were positive by RT-LAMP. Data mining analysis indicated that there were most associations between the RT-LAMP and CT scan data compared to RT-qPCR and CT scan data. CONCLUSIONS: RT-LAMP could detect SARS-CoV-2 with great simplicity, speed, and cheapness. Therefore, it is logical to screen, a large number of patients by RT-LAMP, and then RT-qPCR can be used on the limited samples.

Ion content, antioxidant enzyme activity and transcriptional response under salt stress and recovery condition in the halophyte grass Aeluropus littoralis.

OBJECTIVE: In contrast to glycophytes, halophyte plants have evolved unique morphological and physiological mechanisms to deal with abiotic stress. This study presents the physiological responses of Aeluropus littoralis, a halophyte grass, to salt stress and recovery conditions on the molecular level. RESULTS: Elemental analysis showed that Na+ concentration increased in the analyzed tissue during salt stress application, and declined at recovery condition. With the exception of root tissue, comparable trends of K+, Ca2+, and Mg2+ concentrations were observed (decreased during salt stress, increased during recovery). Salinity led to an increase in total chlorophyll (Chl), Chl a, and carotenoids content, while Chl b content decreased. The level of the proline amino acid associated with drought and salt stress was increased. Here APX, POD, and SOD activity were strongly detectable in roots and reduced later under recovery conditions. RT-qPCR revealed up-regulation of antioxidant genes at S1 and S3 in the root but down-regulation in recovery conditions. This study found a significant halophyte index for understanding the processes of salinity tolerance in A. littoralis. These findings may provide insight into the role of antioxidant enzymes during salt stress and the mechanism underlying the plant's response to stress.

Curcumin inhibits the replication of rotavirus in vitro.

Rotavirus is the most important etiological agent of infectious diarrhea in children under 5 years of age with more than 125,000 deaths occurring annually worldwide. The present study aims to determine the effect of curcumin, a natural polyphenol compound, on rotavirus in a cell culture model. The anti-viral activity of curcumin was evaluated by reverse-transcriptase quantitative PCR (RT-qPCR), TCID50, and western blot techniques to assess CC50 in curcumin-treated MA104 cells as well as EC50 and SI within the infected MA104 cell line. Our findings supported that curcumin exerted an inhibitory influence against rotavirus in a dose-dependent manner and decreased the viral titer and VP6 expression by ~99% at a concentration of 30 µM (p Keywords: curcumin; rotavirus; RT-qPCR; in vitro; anti-rotavirus agent.

Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.

Identification of salinity responsive genes in lavender through cDNA-AFLP.

Currently, a global demand exists forlavender as a significant medicinal plant and source of essential oils. Freshwater and arable lands are two major factors that inhibit extensive farming of medicinal plants in Iran. Saline water from seas and salty soil may be new resources for agricultural use, especially for medicinal plants. We sought to extend our knowledge of the Lavandula angustifolia genome and molecular basis of its salinity tolerance by using cDNA amplified fragment length polymorphism (cDNA-AFLP) to investigate the changes in plant transcriptomes in response to NaCl. All identified transcript derived fragments (TDF) were assigned as novel L. angustifolia genes related to signal transduction, regulation of gene expression, alternative splicing, autophagy, and secondary metabolite biosynthesis. qRT-PCR analysis of the TDFs in response to different concentrations of NaCl revealed various levels of mRNA of the identified genes in this plant. Our findings provided primary insights into the molecular response of L. angustifolia to salinity.

Identification of hub genes associated with RNAi-induced silencing of XIAP through targeted proteomics approach in MCF7 cells.

BACKGROUND: The X-linked inhibitor of apoptosis protein (XIAP) is the most potent caspase inhibitor of the IAP family in apoptosis pathway. This study aims to identify the molecular targets of XIAP in human breast cancer cells exposed to XIAP siRNA by proteomics screening. The expression of XIAP was reduced in MCF-7 breast cancer cells by siRNA. Cell viability and the mRNA expression level of this gene were evaluated by MTS and quantitative real-time PCR procedures, respectively. Subsequently, the XIAP protein level was visualized by Western blotting and analyzed by two-dimensional (2D) electrophoresis and LC-ESI-MS/MS. RESULTS: Following XIAP silencing, cell proliferation was reduced in XIAP siRNA transfected cells. The mRNA transcription and protein expression of XIAP were decreased in cells exposed to XIAP siRNA than si-NEG. We identified 30 proteins that were regulated by XIAP, of which 27 down-regulated and 3 up-regulated. The most down-regulated proteins belonged to the Heat Shock Proteins family. They participate in cancer related processes including apoptosis and MAPK signaling pathway. Reduced expression of HSP90B1 was associated with apoptosis induction by androgen receptor and prostate specific antigen. Suppression of XIAP resulted in the enhancement of GDIB, ENO1, and CH60 proteins expression. The network analysis of XIAP-regulated proteins identified HSPA8, HSP90AA1, ENO1, and HSPA9 as key nodes in terms of degree and betweenness centrality methods. CONCLUSIONS: These results suggested that XIAP may have a number of biological functions in a diverse set of non-apoptotic signaling pathways and may provide an insight into the biomedical significance of XIAP over-expression in MCF-7 cells.

Lead-induced oxidative stress and role of antioxidant defense in wheat (Triticum aestivum L.).

The aim of this study was to investigate soil lead pollution on biochemical properties and gene expression pattern of antioxidant enzymes in three wheat cultivars (Morvarid, Gonbad and Tirgan) at flag leaf sheath swollen stage. Lead (Pb(NO3)2) was used at four different concentrations (0, 15, 30 and 45 mg/kg of soil). The leaf and roots samples were taken at late-booting stage (Zadoks code, GS: 45). The results showed that lead heavy metal toxicity increased the expression of some genes and the activity of key enzymes of the antioxidant defense system in wheat. Moreover, the cell oxidation levels (MDA, LOX) enhanced under lead stress conditions. The relative gene expression and activity of antioxidant enzymes (CAT, SOD, GPX and APX) increased significantly in the both leaves and root tissues under lead stress conditions. The level of gene expression and enzymatic activity were higher in the root than the leaf tissue. There was no significant difference among cultivars in each of lead concentrations but Morvarid and Tirgan cultivars had more tolerance to toxic concentrations of lead when compared to Gonbad cultivar.

Proteomics evaluation of MDA-MB-231 breast cancer cells in response to RNAi-induced silencing of hPTTG.

AIMS: Breast cancer is the most common cancer in women worldwide. Several genes are up-regulated in breast cancer such as human pituitary tumor transforming gene (hPTTG). This study aims to evaluate cell proliferation and the downstream expression pattern of hPTTG1 gene at the mRNA and protein levels after specific down-regulation of hPTTG1 by siRNA. MAIN METHODS: The human breast cancer MDA-MB-231 cell line was transfected with siRNA against hPTTG1. The mRNA and protein expression levels were examined by Real-time PCR and Western blot, respectively. The cell proliferation was assayed by MTS. To investigate the pattern of protein expression, total cellular protein was analyzed by 2D gel electrophoresis and mass spectroscopy. Subsequently, the possible biological consequences were determined by the bioinformatics databases. KEY FINDINGS: Subsequent of hPTTG1 silencing in the MDA_MB-231 cells, the proliferation of cells decreased obviously. In response to hPTTG1 silencing, the levels mRNA and protein were effectively down-regulated 80% and 50%, respectively, at 48 h post-transfection. The proteomics evidenced that PTTG1 increased the expression of 5 proteins. The reduced expression of PTTG1 was functionally involved in hypoxia (NPM1, ENO1), cell proliferation and apoptosis (ENO1, NPM1, NME1, STMN1), and metastasis (NPM1, NME1). SIGNIFICANCE: We identified the hPTTG1-regulated proteins and its molecular mechanism in pathogenesis of breast cancer. Further study emphasis is to understand the association of hPTTG1 with other genes in cancer progression. This novel modality might also consider for identification of targeted drugs, prognosis and follow up in breast cancer gene therapy.

New insights into the roles of the FOXO3 and P27Kip1 genes in signaling pathways.

Background: The forkhead box O3 (FOXO3) and p27Kip1 are two important genes in breast cancer progression. In the present study we analyzed the effect of simultaneous FOXO3 silencing and p27Kip1 activation on breast cancer cell survival and the potential targets of these changes in cancer molecular pathways. Materials and methods: The present study involved the cloning of FOXO3a shRNA and p27Kip1 genes under the control of the bidirectional survivin promoter to down- and up-regulate FOXO3 and p27Kip1 genes, respectively. After transfection of the recombinant expression vector into the breast cancer cell line, the inhibition of cell growth was assessed by MTS and flow cytometry assays. Following the extraction of total mRNA and protein, the expression of target genes was evaluated by qPCR and Western blotting in both treated and untreated cell lines. Then, the downstream protein responses were examined by 2 D electrophoresis. The differentially expressed proteins were also identified by mass spectrometry. Results: Rates of cell proliferation were significantly inhibited in the transfected cell line 72 h post-transfection. Proteomic profiling of the cell line resulted in the identification of seven novel protein markers in breast cancer responsive to these changes in expression of FOXO3 and p27Kip1. The changes in expression of these markers suggested that certain signaling pathways contribute to the development of breast cancer. Conclusion: Simultaneous silencing of FOXO3 and activation of p27Kip1 in MDA-MB-231 cells caused alterations in the expression level of several genes involved in apoptosis, cell proliferation, cell cycle control, tissue invasion, drug resistance, and metastasis. It seems that the identified genes might serve as useful biomarkers for breast cancer.

CDC25A pathway toward tumorigenesis: Molecular targets of CDC25A in cell-cycle regulation.

The cell division cycle 25 (CDC25) phosphatases regulate key transitions between cell-cycle phases during normal cell division, and in the case of DNA damage, they are key targets of the checkpoint machinery that ensure genetic stability. Little is known about the mechanisms underlying dysregulation and downstream targets of CDC25. To understand these mechanisms, we silenced the CDC25A gene in breast cancer cell line MDA-MB-231 and studied downstream targets of CDC25A gene. MDA-MB-231 breast cancer cells were transfected and silenced by CDC25A small interfering RNA. Total messenger RNA (mRNA) was extracted and analyzed by quantitative real-time polymerase chain reaction. CDC25A phosphatase level was visualized by Western blot analysis and was analyzed by 2D electrophoresis and LC-ESI-MS/MS. After CDC25A silencing, cell proliferation reduced, and the expression of 12 proteins changed. These proteins are involved in cell-cycle regulation, programmed cell death, cell differentiation, regulation of gene expression, mRNA editing, protein folding, and cell signaling pathways. Five of these proteins, including ribosomal protein lateral stalk subunit P0, growth factor receptor bound protein 2, pyruvate kinase muscle 2, eukaryotic translation elongation factor 2, and calpain small subunit 1 increase the activity of cyclin D1. Our results suggest that CDC25A controls the cell proliferation and tumorigenesis by a change in expression of proteins involved in cyclin D1 regulation and G1/S transition.

RNA-Seq analysis revealed genes associated with drought stress response in kabuli chickpea (Cicer arietinum L.).

Drought is the most important constraint that effects chickpea production globally. RNA-Seq has great potential to dissect the molecular mechanisms of tolerance to environmental stresses. Transcriptome profiles in roots and shoots of two contrasting Iranian kabuli chickpea genotypes (Bivanij and Hashem) were investigated under water-limited conditions at early flowering stage using RNA-Seq approach. A total of 4,572 differentially expressed genes (DEGs) were identified. Of these, 261 and 169 drought stress responsive genes were identified in the shoots and the roots, respectively, and 17 genes were common in the shoots and the roots. Gene Ontology (GO) analysis revealed several sub-categories related to the stress, including response to stress, defense response and response to stimulus in the tolerant genotype Bivanij as compared to the sensitive genotype Hashem under drought stress. In addition, several Transcription factors (TFs) were identified in major metabolic pathways such as, ABA, proline and flavonoid biosynthesis. Furthermore, a number of the DEGs were observed in "QTL-hotspot" regions which were reported earlier in chickpea. Drought tolerance dissection in the genotypes revealed that the genes and the pathways involved in shoots of Bivanij were the most important factor to make a difference between the genotypes for drought tolerance. The identified TFs in the experiment, particularly those which were up-regulated in shoots of Bivanij during drought stress, were potential candidates for enhancing tolerance to drought.

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA.

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species. Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species.

A novel purple acid phytase from an earthworm cast bacterium.

BACKGROUND: Phytases are a diverse group of enzymes initiating the dephosphorylation of phytate. Phytate is considered as an anti-nutritional compound because of its capability to chelate nutrients such as Fe2+ , Zn2+ , Mg2+ , and Ca2+ . In this study, several bacterial isolates obtained from earthworm casts were evaluated for their phytate degrading capability. Enzymatic properties and the sequence of the corresponding phytase-encoding gene of the selected isolate were determined. RESULTS: The phytase exhibited its highest activity at pH 4.0 and was stable from pH 3 up to pH 9. The temperature optimum was determined to be 65 °C. The strongest inhibitors of enzymatic activity were identified as vanadate, Cu2+ , and Zn2+ . High-performance ion chromatography analysis of enzymatic phytate dephosphorylation revealed that the first dephosphorylation product was d/l-myo-inositol(1,2,3,4,5)pentakisphosphate. CONCLUSION: Owing to its enzymatic properties, such as tolerance to tartrate and the presence of the consensus motifs PDTVY, GNHE, DLG, VLFH, and GHDH, this phytase could be classified as a purple acid phytase. To the best of our knowledge, this is the first report describing a bacterial purple acid phytase. © 2017 Society of Chemical Industry.

Proteomics analysis of Medicago truncatula response to infection by the phytopathogenic bacterium Ralstonia solanacearum points to jasmonate and salicylate defence pathways.

The infection of the model legume Medicago truncatula with Ralstonia solanacearum GMI1000 gives rise to bacterial wilt disease via colonisation of roots. The root and leaf responses to early infection (1 and 3 days post infection) were characterised to investigate the molecular mechanisms of plant resistance or susceptibility. A proteomics approach based on pools of susceptible and resistant recombinant inbred lines was used to specifically target the mechanisms for tolerance. Differential abundances were evidenced for proteins involved in defence (e.g., PR5, PR10, or Kunitz protease inhibitors) and signalling pathways (such as cyclophilin). R. solanacearum inoculation modifies expression levels of those genes, either in both genotypes (AOS1, LOX4, and proteinase inhibitors) or specifically in the resistant line (PR proteins). Exogenous application of salicylic acid (SA) enhanced tolerance to the bacteria, whereas methyl jasmonate (MeJA) enhanced short-term tolerance then promoted disease in the susceptible ecotype, suggesting that they may mediate defence responses. Conversely, proteomics-identified genes were also shown to be SA or MeJA responsive. This is the first description of differential response to R. solanacearum in M. truncatula. Our results suggest that root basal defence is activated at 1 dpi, together with the JA pathway. Specific resistance is then evidenced at three dpi, with the up-regulation of SA-dependent PR proteins.