The accessory gene regulator (agr) of Staphylococcus aureus is the central regulatory system that controls the gene expression for a large set of virulence factors. This global regulatory locus consists of two transcripts: RNAII and RNAIII. RNAII encodes four genes (agrA, B, C, and D) whose gene products assemble a quorum sensing system. RNAIII is the effector of the Agr response. Both the agrB and agrD genes are essential for the production of the autoinducing peptide, which functions as a signal for the quorum sensing system. In this study, we demonstrated the transmembrane nature of AgrB protein in S. aureus. A transmembrane topology model of AgrB was proposed based on AgrB-PhoA fusion analyses in Escherichia coli. Two hydrophilic regions with several highly conserved positively charged amino acid residues among various AgrBs were found to be located in the cytoplasmic membrane as suggested by PhoA-AgrB fusion studies. However, this finding is inconsistent with the putative transmembrane profile of AgrB by computer analysis. Furthermore, we detected an intermediate peptide of processed AgrD from S. aureus cells expressing AgrB and a 6 histidine-tagged AgrD. These results provide direct evidence that AgrB is involved in the proteolytic processing of AgrD. We speculate that AgrB is a novel protein with proteolytic enzyme activity and a transporter facilitating the export of the processed AgrD peptide.

The P2 operon of the staphylococcal accessory gene regulator (agr) encodes four genes (agrA, -B, -C, and -D) whose products compose a quorum sensing system: AgrA and AgrC resemble a two-component signal transduction system of which AgrC is a sensor kinase and AgrA is a response regulator; AgrD, a polypeptide that is integrated into the cytoplasmic membrane via an amphipathic ␣-helical motif in its N-terminal region, is the propeptide for an autoinducing peptide that is the ligand for AgrC; and AgrB is a novel membrane protein that involves in the processing of AgrD propeptide and possibly the secretion of the mature autoinducing peptide. In this study, we demonstrated that AgrB had endopeptidase activity, and identified 2 amino acid residues in AgrB (cysteine 84 and histidine 77) that might form a putative cysteine endopeptidase catalytic center in the proteolytic cleavage of AgrD at its C-terminal processing site. Computer analysis revealed that the cysteine and histidine residues were conserved among the potential AgrB homologous proteins, suggesting that the Agr quorum sensing system homologues might also exist in other Gram-positive bacteria.

The aim of this study was to determine the effects of high temperature stress on ammonium assimilation in leaves of two tall fescue cultivars (Festuca arundinacea), Jaguar 3 brand (J3) (heat-tolerant) and TF 66 (T6) (heatsensitive). High temperature stress for either 10 d or 20 d, and particularly the 20 d stress, produced dramatic changes in ammonium assimilation. After 20 d of stress treatment, the accumulations of total nitrogen, nitrate, soluble protein and total free amino acid (20 amino acids) decreased in both cultivars. Moreover, the activities of main regulatory enzymes, such as nitrate reductase, glutamine synthetase (GS), NADH-dependent glutamate synthase (GOGAT), as well as D 1 -pyrroline-5-carboxylate reductase (P5CR), also decreased in both cultivars when exposed to 20 d stress. Heat stress had little influence on ammonium accumulation in J3, but this was not the case with T6. The accumulations of nitrate, ammonium, soluble protein, and total free amino acid between the two cultivars were different. This suggests that accumulations of these nitrogen forms were associated with heat tolerance in both tall fescue cultivars. Changes of both NADH-glutamate dehydrogenase (NADH-GDH) activity and Glx (glutamine and glutamic acid) concentration in both cultivars indicated that there is an alternative system for assimilation of nitrogen through glutamate dehydrogenase (GDH) in T6 during longer high temperature stress periods. Our results provide an insight to further selection and breeding of heat-tolerant tall fescue turfgrass cultivars. KeywordsAmmonium assimilation AE Festuca arundinacea AE Free amino acid AE High temperature stress AE Nitrogen Abbreviations DW Dry weight FW Fresh weight GS Glutamine synthetase J3 Jaguar 3 brand NADH-GDH NADH-dependent glutamate dehydrogenase NADH-GOGAT NADH-dependent glutamate synthase

Quorum-sensing pheromones are signal molecules that are secreted from Gram-positive bacteria and utilized by these bacteria to communicate among individual cells to regulate their activities as a group through a cell density-sensing mechanism. Typically, these pheromones are processed from precursor polypeptides. The mechanisms of trafficking, processing, and modification of the precursor to generate a mature pheromone are unclear. In Staphylococcus aureus, AgrD is the propeptide for an autoinducing peptide (AIP) pheromone that triggers the Agr cell density-sensing system upon reaching a threshold and subsequently regulates expression of virulence factor genes. The transmembrane protein AgrB, encoded in the agr locus, is necessary for the processing of AgrD to produce mature AIP; however, it is not clear how AgrD interacts with AgrB and how this interaction results in the generation of mature AIP. In this study, we found that the AgrD propeptide was integrated into the cytoplasmic membrane by a conserved ␣-helical amphipathic motif in its N-terminal region. We demonstrated that membrane targeting of AgrD by this motif was required for the stabilization of AgrD and the production of mature AIP, although this region was not specifically involved in the interaction with AgrB. An artificial amphipathic peptide replacing the N-terminal amphipathic motif of AgrD directed the protein to the cytoplasmic membrane and enabled the production of AIP. Analysis of Bacillus ComX precursor protein sequences suggested that the amphipathic membrane-targeting motif might also exist in pheromone precursors of other Gram-positive bacteria.

Abiotic stress affects the growth and development of crops tremendously, worldwide. To avoid adverse environmental effects, plants have evolved various efficient mechanisms to respond and adapt to harsh environmental factors. Stress conditions are associated with coordinated changes in gene expressions at a transcriptional level. Dehydrins have been extensively studied as protectors in plant cells, owing to their vital roles in sustaining the integrity of membranes and lactate dehydrogenase (LDH). Dehydrins are highly hydrophilic and thermostable intrinsically disordered proteins (IDPs), with at least one Lys-rich K-segment. Many dehydrins are induced by multiple stress factors, such as drought, salt, extreme temperatures, etc. This article reviews the role of dehydrins under abiotic stress, regulatory networks of dehydrin genes, and the physiological functions of dehydrins. Advances in our understanding of dehydrin structures, gene regulation and their close relationships with abiotic stresses demonstrates their remarkable ability to enhance stress tolerance in plants.

The agr system is a global regulator of accessory functions in staphylococci, including genes encoding exoproteins involved in virulence. The agr locus contains a two-component signal transduction module that is activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the genus. The AIP has an unusual partially cyclic structure that is essential for function and that, in all but one case, involves an internal thiolactone bond between a conserved cysteine and the C-terminal carboxyl group. The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved cysteine. We demonstrate here that the S. intermedius AIP is processed by the S. intermedius AgrB protein to generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. intermedius strains examined also produce serine-containing AIPs.The agr locus, a global regulator of genes involved in pathogenesis and other accessory functions (27), is widely conserved among staphylococci (2) and has homologs in other species (11). It consists of two divergent operons, driven by promoters P2 and P3, respectively (6,22,24,26). The P2 operon contains four genes: agrA, -B, -C, and -D. AgrA and -C comprise a two-component signaling module, of which AgrC is the receptor and AgrA the response regulator (22), which, when activated, upregulates the transcription of both the P2 and the P3 operons. The P3 transcript, RNAIII, encodes delta-hemolysin and is the effector of the agr response (5, 24). The AgrC ligand is an autoinducing peptide (AIP) (9, 14, 15) that is proteolytically processed by AgrB from a propeptide encoded by agrD and probably also secreted by AgrB (9, 37). Although structurally conserved, agrB, -D, and -C have diverged widely among staphylococci, giving rise to multiple specificity groups, in which heterologous AIP-receptor interactions are inhibitory. The mature AIPs are seven to nine amino acids long and have variable amino acid sequences. All of the seven native staphylococcal AIPs thus far analyzed, including the four known Staphylococcus aureus variants plus strains of Staphylococcus warneri, Staphylococcus epidermidis, and Staphylococcus lugdunensis (7,8,23,25), contain a five-amino-acid thiolactone ring, essential for function, in which a conserved cysteine is attached to the C-terminal carboxyl by a thioester linkage. AgrD has been sequenced for ca. 20 other non-S. aureus staphylococci, and all but one are predicted to contain a cysteine at the same position (2), suggesting that the mature AIPs would each contain the same thiolactone ring (8,9,(16)(17)(18)25). The exception is a strain of Staphylococcus intermedius that is predicted to contain a serine in place of the usual cysteine (2,20). AIPs with a serine replacing cysteine and therefore having a lactone rather than a thiolactone ring have been synthesized in vitro. These do not activate the cognate receptor, and they inhibit agr activation in heterologous combinations (17...

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated !500Â coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at !5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n Z 27), deletions (n Z 5), insertions (n Z 3), and multinucleotide variants (n Z 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/ 80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines. (J Mol Diagn 2016, 18: 299e315; http:// dx

IntroductionThe core binding factor (CBF) transcription factor family consists of heterodimers forming between 1 of 3 CBF␣ subunits, RUNX1/ AML1, RUNX2, or RUNX3 and CBF␤. 1 RUNX1 contacts CBF␤ and binds DNA via its Runt domain. 2,3 CBF activities are commonly reduced in acute myeloid leukemia cases due to expression of AML1-ETO or CBF␤-SMMHC or to deletion or point mutation of the RUNX1 gene, and TEL-AML1 dominantly inhibits CBF in a subset of pediatric acute lymphoblastic leukemias. 1 RUNX1 regulates genes specific to the lymphoid, myeloid, and megakaryocyte lineages, 4-7 and mice lacking RUNX1 do not develop definitive hematopoiesis, indicating a role in adult hematopoietic stem cell (HSC) formation. 8,9 In contrast, deletion of the RUNX1 gene in adult mice leads to increased HSCs, a reduction in common lymphoid progenitor numbers and in platelet formation, and increased myelopoiesis. 10 In addition to regulating lineage-specific genes and cytokine receptors, 1 RUNX1 directly stimulates G1 to S cell-cycle progression. CBF␤-SMMHC or AML1-ETO dominantly inhibit RUNX1 and slow G1 progression in hematopoietic cell lines or in murine or human marrow progenitors, 11-14 cdk4, cyclin D2, or c-Myc overcome inhibition of proliferation by these CBF oncoproteins, 13,15,16 exogenous RUNX1 stimulates G1 progression, 14,15,17 and stimulation of G1 via deletion of p16INK4a or expression of E7 cooperates with CBF␤-SMMHC or TEL-AML1 to induce acute leukemia. 18,19 Induction of cdk4 or cyclin D3 transcription may underlie stimulation of G1 progression by RUNX1. 15,20 Not only does RUNX1 regulate cell-cycle progression, but in addition RUNX1 levels increase as hematopoietic cells progress from G1 to S and from S to G2/M. 20 Phosphorylation of S303 by cdks leads to RUNX1 degradation during G2/M. 21 We now provide additional evidence that cdks phosphorylate S303 and demonstrate that S48 and S424 are also substrates of cdk1/ cyclin B and cdk6/cyclin D3. Moreover, phosphorylation of S48, S303, and S424 strengthens the ability of RUNX1 to activate transcription and to stimulate hematopoietic cell proliferation. Methods PlasmidsHuman RUNX1 cDNA segments were subcloned downstream of glutathionine S-transferase (GST) in pGEX-4T-1 (Amersham, Piscataway, NJ). , containing RUNX1 amino acids 28-86, was generated using an XbaI/SmaI RUNX1 cDNA fragment. , , , , and were generated by ligation of XbaI/BamHI, BamHI/ EcoRI, SmaI, SalI/EcoRI, or NcoI/EcoRI fragments into the pGEX-4T-1 vector. Mutations S424A, T41A, S48A, and T41A/S48A were introduced into these plasmids by polymerase chain reaction (PCR)-based sitedirected mutagenesis. RUNX1 in the context of CMV-Myc-RUNX1 was also subjected to site-directed mutagenesis to generate S48A, S303A, S424A, S48D, S303D, and S424D. All mutants were confirmed by DNA sequencing. DNA subcloning allowed the generation of mutant combinations in CMV-Myc-RUNX1, and the S48A/S303A/S424A (tripleA) or S48D/S303D/S424D (tripleD) mutants were transferred to the pBabePuro-RUNX1-ER retroviral vector or to pMTCB6 down...