Search

Necroptosis, pro-inflammatory programmed necrosis, has been reported to exert momentous roles in pancreatic cancer (PC). Herein, the objective of this study is to construct a necroptosis-related prognostic model for detecting pancreatic cancer. In this study, the intersection between necroptosis-related genes and differentially expressed genes (DEGs) of pancreatic ductal adenocarcinoma (PDAC) was obtained based on GeneCards database, GEO database (GSE28735 and GSE15471), and verified using The Cancer Genome Atlas (TCGA). Next, a prognostic model with Cox and LASSO regression analysis, and divided the patients into high-risk and low-risk groups. Subsequently, the Kaplan–Meier (KM) survival curve and the receiver operating characteristic (ROC) curves were generated to assess the predictive ability of overall survival (OS) of PC patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the potential biofunction and possible mechanical pathways. The EMTome database and an immune analysis were applied to further explore underlying mechanism. Finally, clinical samples of PDAC patients were utilized to verify the expression of model genes via immunohistochemistry (IHC), and the normal human pancreatic ductal cell line, hTERT-HPNE as well as human pancreatic ductal carcinoma cell lines, PANC-1 and PL45, were used to identify the levels of model genes by Western blot (WB) and immunofluorescence (IF) in vitro. The results showed that 13 necroptosis-related DEGs (NRDEGs) were screened based on GEO database, and finally four of five prognostic genes, including KRT7, KRT19, IGF2BP3, CXCL5, were further identified by TCGA to successfully construct a prognostic model. Univariate and multivariate Cox analysis ultimately confirmed that this prognostic model has independent prognostic significance, KM curve suggested that the OS of low-risk group was longer than high-risk group, and the area under receiver (AUC) of ROC for 1, 3, 5 years was 0.733, 0.749 and 0.667, respectively. A GO analysis illustrated that model genes may participate in cell–cell junction, cadherin binding, cell adhesion molecule binding, and neutrophil migration and chemotaxis, while KEGG showed involvement in PI3K-Akt signaling pathway, ECMreceptor interaction, IL-17 signaling pathway, TNF signaling pathway, etc. Moreover, our results showed KRT7 and KRT19 were closely related to EMT markers, and EMTome database manifested that KRT7 and KRT19 are highly expressed in both primary and metastatic pancreatic cancer, declaring that model genes promoted invasion and metastasis potential through EMT. In addition, four model genes were positively correlated with Th2, which has been reported to take part in promoting immune escape, while model genes except CXCL5 were negatively correlated with TFH cells, indicating that model genes may participate in immunity. Additionally, IHC results showed that model genes were higher expressed in PC tissues than that in adjacent tumor tissues, and WB and IF also suggested that model genes were more highly expressed in PANC-1 and PL45 than in hTERT-HPNE. Tracing of a necroptosis-related prognostic model for pancreatic carcinoma reveals its invasion and metastasis potential through EMT and immunity. The construction of this model and the possible mechanism of necroptosis in PDAC was preliminarily explored to provide reliable new biomarkers for the early diagnosis, treatment, and prognosis for pancreatic cancer patients.

Background: Breast cancer is the most prevalent malignancy in women globally, and apoptosis plays an important role in its pathological process. However, studies on the relationship between breast cancer prognosis and apoptosis-related genes are scarce. This study aimed to construct an apoptosis-related specific risk model for breast cancer and preliminarily explore the immunological differences between the high- and low-risk groups of this model to improve the prognosis and treatment of patients with breast cancer.Methods: The Cancer Gene Atlas (TCGA) and Gene Expression Omnibus (GEO) were used to analyze the correlation between apoptosis-related genes and differentially expressed genes. Apoptotic genes associated with prognosis were selected, and a risk model was constructed and validated using univariate and multivariate Cox proportional regression analyses. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO)analyses were used to predict potential mechanical pathways. Tumor lymphocyte infiltration was analyzed between the high- and low-risk groups. The CellMiner database, RNA: RNA-seq expression and Compound activity: DTP NCI-60 were used to assess the drug sensitivity of the model genes. Immunohistochemistry was used to validate the levels of risk model signatures in clinical samples.Results: Seventy-four differentially expressed apoptosis-related genes were screened between breast cancer tissues and adjacent normal tissues. Immune infiltration analysis suggested that CD8-positive T-cells and natural killer cells was considerably higher in the low-risk group than in the high-risk group and that the immune effect was higher in the low-risk group than in the high-risk group. Drug sensitivity analysis showed a positive correlation between the model genes and the sensitivity of multiple drugs, such as vemurafenib, dabrafenib, PD-98059, and palbociclib. Immunohistochemistry results were consistent with the above-mentioned results.Conclusion: Our findings suggest that the developed apoptosis-related specific risk model could be a novel predictive tool for use in patients with breast cancer and can serve as the basis for future breast cancer therapy.

Despite the extensive use of next-generation sequencing of RNA, simultaneous sequencing and quantitative mapping of multiple RNA modifications remain challenging. Herein, we develop MLC-Seq, a mass spectrometry-based direct sequencing method allowing for simultaneously unravelling the RNA sequences and quantitatively mapping different tRNA nucleotide modifications site-specifically. Importantly, MLC-Seq reveals the stoichiometric changes of tRNA modifications upon treatment with the dealkylating enzyme AlkB, and led to the discovery of new nucleotide modifications.

Mi Zhang,1 Wei Zhao,2 Shanshan Liu,1 Haichuan Liu,1 Liang Liu,2 Qiling Peng,2 Chengyou Du,1 Ning Jiang3 1Department of Hepatobiliary Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People’s Republic of China; 2School of Basic Medical Science, Chongqing Medical University, Chongqing, 400016, People’s Republic of China; 3Department of Pathology, Chongqing Medical University, Chongqing, 400016, People’s Republic of ChinaCorrespondence: Chengyou Du; Ning Jiang Email duchengyou@126.com; jiangning@cqmu.edu.cnBackground: The H/ACA small nucleolar ribonucleoprotein (snoRNP) gene family, including GAR1 ribonucleoprotein (GAR1), NHP2 ribonucleoprotein (NHP2), NOP10 ribonucleoprotein (NOP10), and dyskerin pseudouridine synthase 1 (DKC1), play important roles in ribosome biogenesis. However, the potential clinical value of the H/ACA snoRNP gene family in hepatocellular carcinoma (HCC) has not yet been reported.Methods: Bioinformation databases were used to analyze the expression and roles of the H/ACA snoRNP gene family in HCC. Survival analysis, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes enrichment pathway (KEGG) analyses were performed using R software. Tumor Immune Estimation Resource (TIMER) was used to analyze the correlation between the expression of the H/ACA snoRNP gene family and immune infiltration in HCC. Finally, immunohistochemistry and Western blotting were performed to verify the protein expression of the H/ACA snoRNP gene family in HCC tissues and adjacent tissues.Results: The expression of the H/ACA snoRNP gene family was significantly increased in HCC samples compared to normal tissues, and the area under the curve (AUC) of GAR1, NHP2, NOP10, and DKC1 was 0.898, 0.962, 0.884, and 0.911, respectively. Increased expression of the H/ACA snoRNP gene family was associated with poor prognosis in HCC patients (Hazard Ratio, HR = 1.44 [1.02– 2.04], 1.70 [1.20– 2.40], 1.53 [1.09– 2.17], and 1.43 [1.02– 2.03], respectively; log-rank P = 0.036, 0.003, 0.014, 0.039, respectively). GO and KEGG analyses showed that co-expressed genes were primarily enriched in ribosome biogenesis. In addition, upregulated expression of H/ACA snoRNP gene family was related to the infiltration of various immune cells and multiple T cell exhaustion markers in HCC patients. Immunohistochemical analysis and Western blotting showed that the protein expression of H/ACA snoRNP gene family was higher in HCC tissues than in adjacent tissues of clinical samples.Conclusion: H/ACA snoRNP gene family expression was higher in HCC tissues than in normal or adjacent tissues and was highly associated with poor prognosis of HCC patients and, therefore, has the potential to serve as diagnostic and prognostic biomarkers for HCC.Keywords: hepatocellular carcinoma, H/ACA snoRNP gene family, diagnosis, prognosis, biomarker

The use of cement emulsified asphalt mortar (CA mortar) in the track structure of high-speed speed railways has been gaining considerations by many researchers due to its coupled merits of the strength of cement as well as the flexibility of asphalt material. The asphalt to cement ratio (A/C) and the compatibility among constituent materials are crucial to the properties of CA mortar. To improve the performance properties and application of CA mortar, it is imperative to have a broad understanding of the composition mechanisms and compatibility between constituent materials. This paper summarizes interesting research outcomes related to the composition and properties of CA mortar. The consumption of water by cement promotes the breakdown of emulsified asphalt, likewise, the adsorption of asphalt droplets on the surface of cement grains retards the hydration process of cement. An appropriate A/C is required for the cement hydration rate to match the speed of demulsification of asphalt emulsion. Depending on the type and properties for which the CA mortar is designed to possess, the A/C ranges from 0.2 to 0.6 for type 1 (CAM I), and 0.6 to 1.2 for type 2 (CAM II). This paper also discusses measures taken to improve performance properties, compatibility, the interaction between constituent materials of CA mortar, and the use of additives as a partial replacement of cement in CA mortar production. The current review also suggests areas of interest for future research studies. This paper is useful to those who aim to understand or study the composition mechanisms and performance properties of CA mortar.

Facial expression recognition is a practical application in the field of artificial intelligence. This article focuses on the introduction of the principles of each facial expression recognition method based on deep learning in chronological order, as well as the advantages and disadvantages of each method. The methods of facial expression recognition based on deep learning introduced in this article are as follows: Li Dunyu [1] proposed a method of LBP and BP neural network to realize facial expression recognition. And then the convolutional neural network is widely used, Zhou Benjun [2] merged LBP and convolutional neural network to realize facial expression recognition; Liu [3] establish a model that combines a recurrent neural network with a 3D convolutional neural network, and added a timeline so that the machine can extract and recognize facial expressions in a video; Jung [4] proposed to combine the convolutional neural network and the deep neural network to form a new neural network DTAGN, by using this new network he completes facial expression recognition; Shi Xugan [5] proposed a model of combining deep belief network with multi-layer perceptron (MLP), and successfully applied it to the research of facial expression recognition. By utilizing joint training of feature points and multiple networks Xia Tian [6] finish the research; Zhang Feifei [7] and other scholars proposed multi-pose facial expression recognition based on generating an adversarial network.

In order to solve the problems of the sudden loss of fluidity and low expansion rate of CAM I (cement asphalt mortar type I) in a construction site with high environmental temperature, this paper studies the effect of temperature on the fluidity, expansion ratio and pH value of CAM I. The mechanism of action was analyzed by IR (infrared spectrometry), SEM (scanning electron microscopy) and other test methods. The results showed that a high temperature accelerates aluminate formation in cement paste. Aluminate adsorbs emulsifiers leading to demulsification of emulsified asphalt, and wrapped on the surface of cement particles, this causes CAM I to lose its fluidity rapidly. The aluminum powder gasification reaction is inhibited, resulting in an abnormal change in the expansion ratio. Based on findings, the application of an appropriate amount of superplasticizers can effectively improve the workability and expansion characteristics of CAM I at a high temperature.

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PAN(K217A)) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried – from expression host – six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.

In order to improve the stability of air bubbles in fresh concrete, it is of great significance to have a better understanding of the mechanisms and main influencing factors of bubble stability. In the present review, the formation and collapse process of air bubbles in fresh concrete are essentially detailed; and the advances of major influencing factors of bubble stability are summarized. The results show that the surface tension of air–liquid interface exerts a huge impact on bubble stability by reducing surface free energy and Plateau drainage, as well as increasing the Gibbs surface elasticity. However, surface tension may not be the only determinant of bubble stability. Both the strength of bubble film and the diffusion rate of air through the membrane may also dominate bubble stability. The application of nano-silica is a current trend and plays a key role in ameliorating bubble stability. The foam stability could be increased by 6 times when the mass fraction of nano-particle reached 1.5%.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.

To realize the win-win objective of environmental conservation and waste recycling, oil-contaminated soil was utilized as an additive in cement-based materials. The effect of diesel and engine oil and the corresponding oil-contaminated soil on cement-based materials were studied, including the heat release of cement hydration, rheological and flow properties, flexural and compressive strength, hydration products and oil leaching values. The results showed that oil-contaminated soil increased the heat release of hydration of unit mass cement and reduced rheological and flow properties of cement paste and mortars. However, when the dosage of oil-contaminated soil is about 4%, the optimum values of the flexural and compressive strength of mortar, in standard curing 7 and 28 days, were obtained. The leaching values of oil in the disposition satisfied the requirement of China standards. The results confirmed that utilizing an appropriate dosage of oil-contaminated soil in cement-based materials improved the flexural and compressive strength, which is stable to dispose of the waste. This shows that using disposed oil-contaminated soil in cement-based materials will serve as a cost-effective and environmental solution.

Semen enhances HIV infection in vitro , but how long it retains this activity has not been carefully examined. Immediately postejaculation, semen exists as a semisolid coagulum, which then converts to a more liquid form in a process termed liquefaction. We demonstrate that early during liquefaction, semen exhibits maximal HIV-enhancing activity that gradually declines upon further incubation. The decline in HIV-enhancing activity parallels the degradation of peptide fragments derived from the semenogelins (SEMs), the major components of the coagulum that are cleaved in a site-specific and progressive manner upon initiation of liquefaction. Because amyloid fibrils generated from SEM fragments were recently demonstrated to enhance HIV infection, we set out to determine whether any of the liquefaction-generated SEM fragments associate with the presence of HIV-enhancing activity. We identify SEM1 from amino acids 86 to 107 [SEM1(86-107)] to be a short, cationic, amyloidogenic SEM peptide that is generated early in the process of liquefaction but that, conversely, is lost during prolonged liquefaction due to the activity of serine proteases. Synthetic SEM1(86-107) amyloids directly bind HIV-1 virions and are sufficient to enhance HIV infection of permissive cells. Furthermore, endogenous seminal levels of SEM1(86-107) correlate with donor-dependent variations in viral enhancement activity, and antibodies generated against SEM1(86-107) recognize endogenous amyloids in human semen. The amyloidogenic potential of SEM1(86-107) and its virus-enhancing properties are conserved among great apes, suggesting an evolutionarily conserved function. These studies identify SEM1(86-107) to be a key, HIV-enhancing amyloid species in human semen and underscore the dynamic nature of semen's HIV-enhancing activity. IMPORTANCE Semen, the most common vehicle for HIV transmission, enhances HIV infection in vitro , but how long it retains this activity has not been investigated. Semen naturally undergoes physiological changes over time, whereby it converts from a gel-like consistency to a more liquid form. This process, termed liquefaction, is characterized at the molecular level by site-specific and progressive cleavage of SEMs, the major components of the coagulum, by seminal proteases. We demonstrate that the HIV-enhancing activity of semen gradually decreases over the course of extended liquefaction and identify a naturally occurring semenogelin-derived fragment, SEM1(86-107), whose levels correlate with virus-enhancing activity over the course of liquefaction. SEM1(86-107) amyloids are naturally present in semen, and synthetic SEM1(86-107) fibrils bind virions and are sufficient to enhance HIV infection. Therefore, by characterizing dynamic changes in the HIV-enhancing activity of semen during extended liquefaction, we identified SEM1(86-107) to be a key virus-enhancing component of human semen.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Numerous affinity purification-mass spectrometry (APMS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli. Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

Enamel fluorosis has been related to an increase in the amount of amelogenin in fluorosed enamel compared with normal enamel in the maturation stage. In this study we tested the hypothesis that fluoride incorporated into carbonated apatite alters amelogenin hydrolysis. Recombinant human amelogenin (rh174) was allowed to bind to 0.15 mg of carbonated hydroxyapatite (CAP) or to fluoride-containing carbonated hydroxyapatite (F-CAP) synthesized to contain 100, 1,000, or 4,000 ppm F(-). After 3 h of digestion with recombinant human matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4), bound protein was characterized by reverse-phase high-performance liquid chromatography (HPLC). Proteolytic fragments of amelogenin formed after 24h of digestion with MMP20 of KLK 4 were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The hydrolysis, by both MMP20 and KLK4, of amelogenin bound to F100-CAP was significantly reduced in a dose-dependent manner compared with the hydrolysis of amelogenin bound to CAP. After 24 h of hydrolysis, a similar number of MMP20 cleavage sites was found for amelogenin bound to CAP and amelogenin bound to F100-CAP; however, 24 fewer KLK4 cleavage sites were identified for amelogenin bound to F100-CAP than for amelogenin bound to CAP. These results suggest that the reduced hydrolysis of amelogenins in fluorosed enamel may be partially caused by the increased fluoride content in fluoride-containing apatite, contributing to the hypomineralized enamel matrix phenotype observed in fluorosed enamel.

κ-Carrageenan sulfated oligosaccharides containing one, two, and four sulfate groups were complexed with divalent metal cations and electrosprayed in positive ion mode. The resulting metal complexes were subjected to sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) and electron capture dissociation (ECD). The presence of divalent metal cations in the electrospray solution facilitates formation of doubly positively charged precursor ions in the gas phase. Unsurprisingly, abundant sulfate group (SO3) loss is observed in SORI CAD of sulfated oligosaccharides complexed with divalent metal cations. By contrast, sulfation is retained in ECD of metal-sulfated oligosaccharide complexes, providing information on the site of sulfation. Most product ions resulting from ECD are C/Z- or B/Y-type ions, corresponding to cleavage of glycosidic bonds linking two sugar units. However, several A/X-type product ions corresponding to sugar cross-ring cleavage were also observed in ECD. Such cross-ring fragments are important in carbohydrate analysis because they provide linkage information.

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.

Curacin A is a mixed polyketide/nonribosomal peptide possessing anti-mitotic and anti-proliferative activity. In the biosynthesis of curacin A, the N-terminal domain of the CurF multifunctional protein catalyzes decarboxylation of 3-methylglutaconyl-acyl carrier protein (ACP) to 3-methylcrotonyl-ACP, the postulated precursor of the cyclopropane ring of curacin A. This decarboxylase is encoded within an "HCS cassette" that is used by several other polyketide biosynthetic systems to generate chemical diversity by introduction of a beta-branch functional group to the natural product. The crystal structure of the CurF N-terminal ECH(2) domain establishes that the protein is a crotonase superfamily member. Ala(78) and Gly(118) form an oxyanion hole in the active site that includes only three polar side chains as potential catalytic residues. Site-directed mutagenesis and a biochemical assay established critical functions for His(240) and Lys(86), whereas Tyr(82) was nonessential. A decarboxylation mechanism is proposed in which His(240) serves to stabilize the substrate carboxylate and Lys(86) donates a proton to C-4 of the acyl-ACP enolate intermediate to form the Delta(2) unsaturated isopentenoyl-ACP product. The CurF ECH(2) domain showed a 20-fold selectivity for ACP-over CoA-linked substrates. Specificity for ACP-linked substrates has not been reported for any other crotonase superfamily decarboxylase. Tyr(73) may select against CoA-linked substrates by blocking a contact of Arg(38) with the CoA adenosine 5'-phosphate.

The asbABCDEF gene cluster from Bacillus anthracis is responsible for biosynthesis of petrobactin, a catecholate siderophore that functions in both iron acquisition and virulence in a murine model of anthrax. We initiated studies to determine the biosynthetic details of petrobactin assembly based on mutational analysis of the asb operon, identification of accumulated intermediates, and addition of exogenous siderophores to asb mutant strains. As a starting point, in-frame deletions of each of the genes in the asb locus ( asbABCDEF ) were constructed. The individual mutations resulted in complete abrogation of petrobactin biosynthesis when strains were grown on iron-depleted medium. However, in vitro analysis showed that each asb mutant grew to a very limited extent as vegetative cells in iron-depleted medium. In contrast, none of the B. anthracis asb mutant strains were able to outgrow from spores under the same culture conditions. Provision of exogenous petrobactin was able to rescue the growth defect in each asb mutant strain. Taken together, these data provide compelling evidence that AsbA performs the penultimate step in the biosynthesis of petrobactin, involving condensation of 3,4-dihydroxybenzoyl spermidine with citrate to form 3,4-dihydroxybenzoyl spermidinyl citrate. As a final step, the data reveal that AsbB catalyzes condensation of a second molecule of 3,4-dihydroxybenzoyl spermidine with 3,4-dihydroxybenzoyl spermidinyl citrate to form the mature siderophore. This work sets the stage for detailed biochemical studies with this unique acyl carrier protein-dependent, nonribosomal peptide synthetase-independent biosynthetic system.

Gas phase complexes Mg*+ (2,6-difluoropyridine) (1) and Mg*+ (pentafluoropyridine) (2) have been subjected to photodissociation in the spectral range of approximately 230-440 nm. Except for the evaporative photofragment Mg*+ , the primary photoproduct for is C(5)H(3)N*(+), which is associated with the rupture of two C-F bonds by the photoexcited Mg*+ , forming very stable MgF(2). In contrast, the direct loss of MgF(+) is more favorable for due to fluorine substitution. Given enough energy, C(5)H(3)N*(+) can undergo decomposition to form C(4)H(2)*(+) and HCN. These results are very different from those for Mg*+ (2-fluoropyridine), highlighting the significance of the additional F at C6 of and . Density functional theory (DFT) calculations have been employed to examine the geometries and energetics of the complexes as well as relevant reaction mechanisms. All of the complexes feature the direct attachment of Mg*+ to the N atom. The key intermediate is found to be FMg(+) (C(5)H(x)F(4-x)N) (x = 3 or 0), which can lead to the formation of MgF(+) directly or MgF(2) through activation of another C-F bond adjacent to N, producing the pyridyne radical cations. However, hydrogen-transfer prior to the rupture of the second C-F bond followed by ring-opening of C(5)H(3)N*(+) may result in the formation of chain forms of C(5)H(3)N*(+). The influence of the fluorine substitution on the competition of the two routes have been demonstrated.

The metal cation-molecule complex of Mg+ cation with C6H5OCF3 molecule was produced in a laser-ablation supersonic expansion nozzle source. The intra-complex reaction in Mg+-C6H5OCF3 by photons in the spectral region of 230–440 nm has been studied. The formation of Mg+ was found to be the predominant dissociation path way, and at the same time, the other products MgF + , C 6 H 5 + , and C 6 H 5 OCF 3 + were also observed. We monitored all of the photodissociation products as a function of the excitation wavelength in the 230–440 nm spectral region. The action spectra of the all products except the dissociative charge-transfer product C 6 H 5 OCF 3 + are very similar. They consist of two pronounced bands, one of which is on the red side and the other is on the blue side of the Mg+ 32P ← 32S atomic transition. The two peaks of these products are all located at about 243 nm and 332 nm, respectively. The calculated results at the B3LYP/6-31+G∗∗ level show that there are three isomers: one is with Mg+ attached to the O atom, while it also partially interacts with one of F atoms (4), the next is with Mg+ simultaneously to the two F atoms (4′), and the last is with Mg+ sitting above the aromatic ring (4″). The isomer 4 is more stable than 4′ and 4″ by ∼3–5 kcal/mol. Although the three isomers are comparable in energy, on the basis of the formation channels of the products and their action spectra, the photoreaction products in our experiment are deduced to originate from 4. The branching fraction ratios for the all product except C 6 H 5 OCF 3 + were nearly constant. A photoreaction mechanism is proposed that the conical intersection opens an efficient pathway for the nonadiabatic transition from the excited state to the ground state, producing an intermediate, which yields the photoproducts with a constant branching ratio. The charge-transfer product ion C 6 H 5 OCF 3 + is ascribed to a predissociative charge transfer process.

The intra-complex reaction in Mg+-nitromethane by photons in the spectral region of 240–400 nm has been studied. The product Mg+, Mg+O, Mg+OH, and Mg+NO2 were observed. The products Mg+ and Mg+OH appeared throughout the whole wavelength range, but the product Mg+O emerged only in the shorter wavelength region, whereas the product Mg+NO2 in the longer one. The calculated results show that there are the two isomers. The absorption spectrum of the two isomers were calculated, and the isomer, which is with Mg+ attached to single O atom, was in good agreement with the experimental data. On the basis of the branching fraction data, the orientation of the electronic orbital, and reaction energies, the photoreaction mechanisms were explained for the Mg+ insertion the chemical bonds and the state-specificity.

We compare electron capture dissociation (ECD) of doubly protonated and divalent metal-adducted tyrosine O-sulfated peptides without basic amino acid residues. ECD of doubly protonated Tyr2-sulfated cholecystokinin (CCKS) and doubly protonated Tyr12-sulfated gastrin II (GST) resulted in complete loss of SO3 from all product ions. Thus, contrary to typical ECD behavior, localization of the sulfate groups was not possible. By contrast, ECD of Ca-, Mn-, Zn-, and Fe-adducted CCKS and ECD of deprotonated GST with two calcium adducts, i.e., [GST + 2Ca - H]3+, resulted in sulfated c'- and z.-type product ions with high sequence coverage, thereby allowing both sequencing and sulfate localization. In addition, divalent metal adduction provided improved positive mode ionization efficiency for these peptides. The drastically different fragmentation behavior observed in ECD of protonated and metal-adducted CCKS and GST, respectively, is proposed to be a consequence of the absence of basic amino acid residues, promoting a mobile proton-like fragmentation mechanism, including abundant sulfate loss, for protonated species. Retention of sulfate groups was also observed in electron detachment dissociation (EDD) of CCKS and GST. However, the EDD fragmentation efficiency was much lower than that of ECD and very limited fragmentation was observed in EDD of GST, precluding localization of the sulfate group in that peptide.

Complexes of fluorinated benzenes (o-C6H4−nF2+n) and Mg˙+ are subjected to ultraviolet photodissociation (260–340 nm), producing efficiently benzyne radical cations (C6H4−nFn˙+) besides Mg˙+ and MgF+. We show that the consecutive dissociation of C6H4−nFn˙+ follows the [C4+ + C2] pattern exclusively for n ≤ 2 after the parent complexes absorb one or two photons. However, the dissociation pattern is switched to [C5+ + C1] and [C1+ + C5] for n ≥ 3. In particular, upon two-photon absorption at 340 nm by the complexes of Mg˙+(C6HF5) (1) and Mg˙+(C6F6) (2), photoproducts of CF+, C5H+, and C5HF˙+ from C6HF3˙+ and CF+, C5F+, C5F2˙+, and C5F3+ from C6F4˙+ are detected, respectively. Theoretical calculations are used to explain the switching of the dissociation patterns induced by the fluorine substitutions. It was found that the formation of C5+ + C1 is energetically more favorable than that of C4+ + C2 from C6HF3˙+ and C6F4˙+ and of C1+ + C5. Except for C5H2F+ + CF, all the channels of [C5+ + C1] and [C1+ + C5] are energetically less favorable than those of [C4+ + C2] from C6H3F˙+ and C6H2F2˙+. In most cases, the calculated results agree well with the experimental observations.

Complexes of magnesium cation with cytosine were produced by laser ablation of a magnesia and cytosine mixture. Photo-induced reactions in the complexes of Mg+–cytosine have been studied at 230–440 nm. The evaporation product, Mg+, as well as other four and five reactive products were observed in the long and short wavelength regions, respectively. The ground state structures of the complexes composed of magnesium cation and the low-lying tautomers of cytosine were fully optimized at the B3LYP/6-31+G** level. The bond dissociation energies (BDEs) of the complexes for the channels we observed were calculated at the same theoretical level.

Mg + -nucleobase complexes have been generated for the first time by a laser-ablation technique. Mg + -uracil and Mg + -thymine are studied by photodissociation and density functional theory calculations. The evaporative loss of Mg + is found to be the dominant channel after photoexcitation. A number of photoproducts due to the nucleobase lesions have also been detected in the photon energy region of 3.4 eV, whereas only one of them is identified around 5.0 eV. The site of photoinduced C–N and/or C–C bond rupture is found to be at the carbonyl group where the metal is bound. A charge-transfer photoproduct is observed only from Mg + -thymine.

Ethyl isocyanate-solvated magnesium cation complexes Mg+(OCNC2H5)n were produced in a laser-ablation supersonic expansion nozzle source. Photoinduced reactions in the complexes of Mg+(OCNC2H5)n (n ...

Photoinduced reactions have been systematically studied on mass-selected complexes of Mg•+ with tri- and tetrasubstituted benzene by F and/or CH3. The complexes fall into two groups. The Group I complexes involve bidentate coordination of the o-F atoms to Mg•+, whereas the Group II complexes feature the linkage of Mg•+ to only one of the F atoms. The o-benzyne radical cations were found to be the predominant photolysis products from the Group I complexes, but the yields of the benzyne radical cation products were low for Group II. For each group of complexes, different substituents, including CH3 (medium σ-electron donor) and F (strong σ-electron acceptor and weak π-electron donor), affect the branching ratios significantly. For example, they result in different decomposition thresholds of the o-benzyne radical cations. Through the studies of aromatic substitution, the pathways for the formation and decomposition of o-benzyne radical cations have been brought to light. With the help of quantum mechanics c...

The photoinduced reactions of the complexes Mg+-SCNC2H5 and Mg+-NCSC2H5 are studied comparatively in the spectral range of 230-440 nm. One-photon excitation of the complexes through the Mg+ chromophore (3 2P-- 3 2S) gives rise to the evaporative fragment as well as the molecular activation and charge transfer products. The action spectra of the complexes consist of three broad peaks for Mg+-SCNC2H5 and two for Mg+-NCSC2H5, which accord with the structures obtained from quantum mechanics calculations. These calculations reveal two association isomers for Mg+-SCNC2H5: one is with Mg+ being linked to the S atom and the other to the N atom. The former is more stable than the latter by only 0.23 eV. Both of the isomers have been shown to exist in the complex source employed in our experiments. On the other hand, only one stable structure is found for the complex Mg+-NCSC2H5 characterized by the Mg+-N linkage. In general, the photofragments are dominated by Mg+ at lambda400 nm, which decreases with decreasing wavelength accompanied by the increase in other photoproducts. In addition, the branching ratios of Mg+ to other photoproducts are nearly constant in the short wavelength region but decrease with decreasing wavelength. The observed photoreactions have been reasonably explained.

Gas-phase complexes Mg+−[HCON(CH3)2]1,2 are mass-selected and studied by ultraviolet laser photodissociation in a time-of-flight mass spectrometer. Beside the dominant evaporation photofragments, some photoreaction products have also been detected from both complexes. Quantum mechanics calculations and deuterium-substitution experiments are employed to facilitate the structural, energetic, and mechanistic analysis of the photoreactions. For the most stable structures of the complexes, Mg+ is linked to the carbonyl oxygen atoms and situated trans to the −N(CH3)2 groups. Starting from these complex structures, photoproducts are generated through (1) H abstraction next to the carbonyl group by the photoexcited Mg+* (to form (CH3)2NCO+) and/or (2) the subsequent CO loss (to form CH3NH+CH2). For Mg+−[HCON(CH3)2]2, the further solvation tends to hold back the photoreactions because of the even more facile evaporation channels.

The photochemistry of a gas-phase complex, Mg*(+)(2-fluoropyridine), has been studied in the spectral range of approximately 230-440 nm with a molecular beam coupled with a time-of-flight mass spectrometer. Surprisingly rich chemistry has been observed. Aside from the evaporative photofragment, Mg*(+), an abundant photoproduct, C(4)H(4)*(+), is observed after the electronic excitation of Mg(+). The formation of this photoproduct is associated with the loss of a stable species, CN[bond]Mg[bond]F. Also identified in this work are reactive pathways that occur with the elimination of HCN, HF, or MgF from the complex. The observed photoreactions have been examined in detail using quantum mechanics methods. A distinct structural feature of the complex is the direct attachment of Mg*(+) to the N atom of fluoropyridine due to the strong electrostatic interaction. The key to the rich photochemistry is the formation of the FMg(+)(C(5)H(4)N) intermediate, through facile fluorine migration. Plausible photoreaction mechanisms have been proposed. These mechanisms account for the evolution of the energized complex with the pre-defined structure en route to the target photoproducts that we have detected.

Photoinduced reactions of bidentate metal complexes Mg+(2-methoxyethanol), Mg+(1,2-dimethoxyethane), and Mg+(1,2-diaminoethane) have been studied combining mass-selective photodissociation spectroscopy and quantum mechanical calculations. Besides the evaporative fragment cation Mg+, copious reactive channel photoproduct cations have been identified, which are propagated from the well-defined bidentate chelation structures upon photoexcitation of Mg+. For Mg+(2-methoxyethanol) and Mg+(1,2-dimethoxyethane), most reactive channel photoproducts are actually Mg+−molecule complexes, such as Mg+(H2O), Mg+(HCHO), Mg+(CH3OH), Mg+(C2H6O), etc. with an exception of Mg+OH. The reactive channel photoproducts of Mg+(1,2-diaminoethane) consist of a cationic complex Mg+(H2NCH3) and two other cations CH2NH2+ and Mg+NH2, which are formed from the cleavage of the C−C and C−N bonds, respectively. Action spectra and branching fractions of all of the observed photofragments have been measured in a broad spectral range of 230−4...

Ion–molecule complexes of magnesium cation with ethyl isocyanate were produced in a laser-ablation supersonic expansion nozzle source. Photo-induced reactions in the 1:1 complexes have been studied in the spectral range of 230–410 nm. Photodissociation mass spectrometry revealed the persistent product Mg+ from nonreactive quenching throughout the entire wavelength range. As for the reactive channels, the photoproducts, Mg+OCN and C2H5+, were produced only in the blue absorption band of the complex with low yields. The action spectrum of Mg+(OCNC2H5) consists of two pronounced peaks on the red and blue sides of the Mg+ 32P←32S atomic transition. The ground state geometry of Mg+–OCNC2H5 was fully optimized at B3LYP/6-31+G** level by using GAUSSIAN 98 package. The calculated absorption spectrum of the complex using the optimized structure of its ground state agrees well with the observed action spectrum. Photofragment branching fractions of the products are almost independent of the photolysis photon energy ...