Development of Genome-Wide Intron Length Polymorphism (ILP) Markers in Tea Plant (Camellia sinensis) and Related Applications for Genetics Research.

The market value of tea is largely dependent on the tea species and cultivar. Therefore, it is important to develop efficient molecular markers covering the entire tea genome that can be used for the identification of tea varieties, marker-assisted breeding, and mapping important quantitative trait loci for beneficial traits. In this study, genome-wide molecular markers based on intron length polymorphism (ILP) were developed for tea trees. A total of 479, 1393, and 1342 tea ILP markers were identified using the PCR method in silico from the 'Shuchazao' scaffold genome, the chromosome-level genome of 'Longjing 43', and the ancient tea DASZ chromosome-level genome, respectively. A total of 230 tea ILP markers were used to amplify six tea tree species. Among these, 213 pairs of primers successfully characterize products in all six species, with 112 primer pairs exhibiting polymorphism. The polymorphism rate of primer pairs increased with the improvement in reference genome assembly quality level. The cross-species transferability analysis of 35 primer pairs of tea ILP markers showed an average amplification rate of 85.17% through 11 species in 6 families, with high transferability in Camellia reticulata and tobacco. We also used 40 pairs of tea ILP primers to evaluate the genetic diversity and population structure of C. tetracocca with 176 plants from Puan County, Guizhou Province, China. These genome-wide markers will be a valuable resource for genetic diversity analysis, marker-assisted breeding, and variety identification in tea, providing important information for the tea industry.

Identification of immune-related biomarkers associated with tumorigenesis and prognosis in cutaneous melanoma patients.

BACKGROUND: Skin cutaneous melanoma (SKCM) is one of the most malignant and aggressive cancers, causing about 72% of deaths in skin carcinoma. Although extensive study has explored the mechanism of recurrence and metastasis, the tumorigenesis of cutaneous melanoma remains unclear. Exploring the tumorigenesis mechanism may help identify prognostic biomarkers that could serve to guide cancer therapy. METHOD: Integrative bioinformatics analyses, including GEO database, TCGA database, DAVID, STRING, Metascape, GEPIA, cBioPortal, TRRUST, TIMER, TISIDB and DGIdb, were performed to unveil the hub genes participating in tumor progression and cancer-associated immunology of SKCM. Furthermore, immunohistochemistry (IHC) staining was performed to validate differential expression levels of hub genes between SKCM tissue and normal tissues from the First Affiliated Hospital of Soochow University cohort. RESULTS: A total of 308 differentially expressed genes (DEGs) and 12 hub genes were found significantly differentially expressed between SKCM and normal skin tissues. Functional annotation indicated that inflammatory response, immune response was closely associated with SKCM tumorigenesis. KEGG pathways in hub genes include IL-10 signaling and chemokine receptors bind chemokine signaling. Five chemokines members (CXCL9, CXCL10, CXCL13, CCL4, CCL5) were associated with better overall survival and pathological stages. IHC results suggested that significantly elevated CXCL9, CXCL10, CXCL13, CCL4 and CCL5 proteins expressed in the SKCM than in the normal tissues. Moreover, our findings suggested that IRF7, RELA, NFKB1, IRF3 and IRF1 are key transcription factors for CCL4, CCL5, CXCL10. In addition, the expressions of CXCL9, CXCL10, CXCL13, CCL4 and CCL5 were positively correlated with infiltration of six immune cells (B cell, CD8+T cells, CD4+T cells, macrophages, neutrophils, dendritic cells) and 28 types of TILs. Among them, high levels of B cells, CD8+T cells, neutrophils and dendritic cells were significantly related to longer SKCM survival time. CONCLUSION: In summary, this study mainly identified five chemokine members (CXCL9, CXCL10, CXCL13, CCL4, CCL5) associated with SKCM tumorigenesis, progression, prognosis and immune infiltrations, which might help us evaluate several immune-related targets for cutaneous melanoma therapy.

Chronic restraint stress exacerbates neurological deficits and disrupts the remodeling of the neurovascular unit in a mouse intracerebral hemorrhage model.

Growing evidences have shown that patients recovering from stroke experience high and unremitting stress. Chronic restraint stress (CRS) has been found to exacerbate neurological impairments in an experimental focal cortical ischemia model. However, there have been no studies reporting the effect and mechanism of CRS on intracerebral hemorrhage (ICH). This study aimed to evaluate the effect of CRS on a mouse ICH model. Adult male C57BL mice were subjected to infusion of collagenase IV (to induce ICH) or saline (for sham) into the left striatum. After ICH, animals were stressed with application of CRS protocol for 21 days. Our results showed that CRS significantly exacerbated neurological deficits (Garcia test, corner turn test, and wire grip test) and the ipsilateral brain atrophy and reduced body weight gain after ICH. Immunofluorescence staining indicated that CRS exerted significant suppressive effects on neuron, astrocyte, vascular endothelial cell and pericyte and excessively activated microglia post ICH. All of the key cellular components mentioned above are involved in the neurovascular unit (NVU) remodeling in the peri-hemorrhagic region after ICH. Western blot results showed that matrix metalloproteinase (MMP)-9 and tight junction (TJ) proteins including zonula occludens-1, occludin and claudin-5 were increased after ICH, but MMP-9 protein was further up-regulated and TJ-related proteins were down-regulated by CRS. In addition, ICH-induced activation of endoplasmic reticulum stress and apoptosis were further strengthened by CRS. Collectively, CRS exacerbates neurological deficits and disrupts the remodeling of the peri-hemorrhagic NVU after ICH, which may be associated with TJ proteins degradation and excessive activation of MMP-9 and endoplasmic reticulum stress-apoptosis.LAY SUMMARYCRS exacerbates neurological deficits and disrupts the remodeling of the NVU in the recovery stage after ICH, which suggest that monitoring chronic stress levels in patients recovering from ICH may merit consideration in the future.

Salubrinal offers neuroprotection through suppressing endoplasmic reticulum stress, autophagy and apoptosis in a mouse traumatic brain injury model.

Traumatic brain injury (TBI) is a complex injury that can cause severe disabilities and even death. TBI can induce secondary injury cascades, including but not limited to endoplasmic reticulum (ER) stress, apoptosis and autophagy. Although the investigators has previously shown that salubrinal, the selective phosphatase inhibitor of p-eIF2α, ameliorated neurologic deficits in murine TBI model, the neuroprotective mechanisms of salubrinal need further research to warrant the preclinical value. This study was undertaken to characterize the effects of salubrinal on cell death and neurological outcomes following TBI in mice and the potential mechanisms. In the current study, ER stress-related proteins including p-eIF2α, GRP78 and CHOP showed peak expressions both in the cortex and hippocampus from day 2 to day 3 after TBI, indicating ER stress was activated in our TBI model. Immunofluorescence staining showed that CHOP co-located NeuN-positive neuron, GFAP-positive astrocyte, Iba-1-positive microglia, CD31-positive vascular endothelial cell and PDGFR-ß-positive pericyte in the cortex on day 2 after TBI, and these cells mentioned above constitute the neurovascular unit (NVU). We also found TBI-induced plasmalemma permeability, motor dysfunction, spatial learning and memory deficits and brain lesion volume were alleviated by continuous intraperitoneal administration of salubrinal post TBI. To investigate the underlying mechanisms further, we determined that salubrinal suppressed the expression of ER stress, autophagy and apoptosis related proteins on day 2 after TBI. In addition, salubrinal administration decreased the number of CHOP+/TUNEL+ and CHOP+/LC3+ cells on day 2 after TBI, detected by immunofluorescence. In conclusion, these data imply that salubrinal treatment improves morphological and functional outcomes caused by TBI in mice and these neuroprotective effects may be associated with inhibiting apoptosis, at least in part by suppressing ER stress-autophagy pathway.

Comparative Transcriptome Analysis of Developing Seeds and Silique Wall Reveals Dynamic Transcription Networks for Effective Oil Production in Brassica napus L.

Vegetable oil is an essential constituent of the human diet and renewable raw material for industrial applications. Enhancing oil production by increasing seed oil content in oil crops is the most viable, environmentally friendly, and sustainable approach to meet the continuous demand for the supply of vegetable oil globally. An in-depth understanding of the gene networks involved in oil biosynthesis during seed development is a prerequisite for breeding high-oil-content varieties. Rapeseed (Brassica napus) is one of the most important oil crops cultivated on multiple continents, contributing more than 15% of the world's edible oil supply. To understand the phasic nature of oil biosynthesis and the dynamic regulation of key pathways for effective oil accumulation in B. napus, comparative transcriptomic profiling was performed with developing seeds and silique wall (SW) tissues of two contrasting inbred lines with ~13% difference in seed oil content. Differentially expressed genes (DEGs) between high- and low-oil content lines were identified across six key developmental stages, and gene enrichment analysis revealed that genes related to photosynthesis, metabolism, carbohydrates, lipids, phytohormones, transporters, and triacylglycerol and fatty acid synthesis tended to be upregulated in the high-oil-content line. Differentially regulated DEG patterns were revealed for the control of metabolite and photosynthate production in SW and oil biosynthesis and accumulation in seeds. Quantitative assays of carbohydrates and hormones during seed development together with gene expression profiling of relevant pathways revealed their fundamental effects on effective oil accumulation. Our results thus provide insights into the molecular basis of high seed oil content (SOC) and a new direction for developing high-SOC rapeseed and other oil crops.

Synthesis of Highly Functionalized Indoles and Indolones via Selectivity-Switchable Olefinations.

Highly functionalized indoles and indolones were prepared via selectivity-switchable mono- or diolefinations. The Julia olefination of the products followed by a Brønsted acid-prompted cyclization afforded indolones, whereas the indoles were obtained by a sequential Wittig olefination and electrocyclization. This method opens divergent access to highly functionalized nitrogen-containing bicyclic or tricyclic heterocycles.

Transition-metal-free oxychlorination of alkenyl oximes: in situ generated radicals with tert-butyl nitrite.

Oxychlorination of alkenyl oximes is harder compared to the analogous oxybromination or oxyiodination because of the difficulty associated with the formation of chlorine cations or radicals. A transition-metal-free oxychlorination of alkenyl oximes has been developed, using t-BuONO as a dual oxidant and AlCl3 as a chlorine source. This convenient and practical method has been used to construct chloroisoxazolines in moderate to good yields, whereas N-chlorosuccinimide (NCS) failed to promote this reaction.

Charge transport dependent high open circuit voltage tandem organic photovoltaic cells with low temperature deposited HATCN-based charge recombination layers.

Mechanisms of charge transport between the interconnector and its neighboring layers in tandem organic photovoltaic cells have been systematically investigated by studying electronic properties of the involving interfaces with photoelectron spectroscopies and performance of the corresponding devices. The results show that charge recombination occurs at HATCN and its neighboring hole transport layers which can be deposited at low temperature. The hole transport layer plays an equal role to the interconnector itself. These insights provide guidance for the identification of new materials and the device architecture for high performance devices.

Effects of linagliptin and liraglutide on glucose- and angiotensin II-induced collagen formation and cytoskeleton degradation in cardiac fibroblasts in vitro.

AIM: Glucagon-like peptide-1 (GLP-1) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors can not only lower blood glucose levels, but also alleviate cardiac remodeling after myocardial ischemia and hypertension. In the present study, we investigated the effects of a DPP-4 inhibitor (linagliptin) and a GLP-1 activator (liraglutide) on glucose- and angiotensin II (Ang II)-induced collagen formation and cytoskeleton reorganization in cardiac fibroblasts in vitro, and elucidated the related mechanisms. METHODS: Cardiac fibroblasts were isolated from the hearts of 6-week-old C57BL/6 mice, and then exposed to different concentrations of glucose or Ang II for 24 h. The expression of fibrotic signals (fibronectin, collagen-1, -3 and -4), as well as ERK1/2 and NF-κB-p65 in the fibroblasts was examined using Western blotting assays. F-actin degradation was detected under inverted laser confocal microscope in fibroblasts stained with Rhodamine phalloidin. RESULTS: Glucose (1-40 mmol/L) and Ang II (10-8-10-5 mol/L) dose-dependently increased the expression of fibronectin, collagens, phospho-ERK1/2 and phospho-NF-κB-p65 in cardiac fibroblasts. High concentrations of glucose (≥40 mmol/L) and Ang II (≥10-6 mol/L) caused a significant degradation of F-actin (less assembly F-actin fibers and more disassembly fibers). ERK1/2 inhibitor U0126 (10 µmol/L) and NF-κB inhibitor JSH-23 (10 µmol/L) both markedly suppressed glucose- and angiotensin II-induced fibronectin and collagen expressions in cardiac fibroblasts. Furthermore, pretreatment with liraglutide (10-100 nmol/L) or linagliptin (3 and 30 nmol/L) significantly decreased glucose- and Ang II-induced expression of fibrotic signals, phospho-ERK1/2 and phospho-NF-κB-p65 in cardiac fibroblasts. Moreover, pretreatment with liraglutide (30 nmol/L) or liraglutide (100 nmol/L) markedly inhibited glucose-induced F-actin degradation, however, only liraglutide inhibited Ang II-induced F-actin degradation. CONCLUSION: Linagliptin and liraglutide inhibit glucose- and Ang II-induced collagen formation in cardiac fibroblasts via activation of the ERK/NF-κB/pathway. Linagliptin and liraglutide also markedly inhibit glucose-induced F-actin degradation in cardiac fibroblasts, but only liraglutide inhibits Ang II-induced F-actin degradation.

Thermal spin filtering, thermal spin switching and negative-differential-resistance in thermal spin currents in zigzag SiC nanoribbons.

Spin caloritronics with a combination of spintronics and thermoelectrics has potential applications in future information science and opens a new direction in the development of multi-functional materials. Based on density functional theory and the nonequilibrium Green's function method, we calculate thermal spin-dependent transport through a zigzag silicon carbide nanoribbon (ZSiCNR), which is a heterojunction consisting of a left electrode (ZSiC-2H1H) and right electrode terminated (ZSiC-1H1H) by hydrogen. Our results show that when the temperature in the left contact increases over a critical value, the thermal spin-down current increases remarkably from zero, while the thermal spin-up current remains zero in the total-temperature region, indicating that a perfect thermal spin filter together with a perfect spin switcher is obtained. Furthermore, the thermal spin current shows a negative differential resistance effect and quantum oscillation behaviors. These results suggest that the zigzag SiC nanoribbon proposed by us can be designed as a highly-efficient spin caloritronics device with multiple functionalities.

Salidroside protects pulmonary artery endothelial cells against hypoxia-induced apoptosis via the AhR/NF-κB and Nrf2/HO-1 pathways.

BACKGROUND: The apoptosis of pulmonary artery endothelial cells (PAECs) is an important factor contributing to the development of pulmonary hypertension (PH), a serious cardio-pulmonary vascular disorder. Salidroside (SAL) is a bioactive compound derived from an herb Rhodiola, but the potential protective effects of SAL on PAECs and the underlying mechanisms remain elusive. PURPOSE: The objective of this study was to determine the role of SAL in the hypoxia-induced apoptosis of PAECs and to dissect the underlying mechanisms. STUDY DESIGN: Male Sprague-Dawley (SD) rats were subjected to hypoxia (10% O2) for 4 weeks to establish a model of PH. Rats were intraperitoneally injected daily with SAL (2, 8, and 32 mg/kg/d) or vehicle. To define the molecular mechanisms of SAL in PAECs, an in vitro model of hypoxic cell injury was also generated by exposed PAECs to 1% O2 for 48 h. METHODS: Various techniques including hematoxylin and eosin (HE) staining, immunofluorescence, flow cytometry, CCK-8, Western blot, qPCR, molecular docking, and surface plasmon resonance (SPR) were used to determine the role of SAL in rats and in PAECs in vitro. RESULTS: Hypoxia stimulation increases AhR nuclear translocation and activates the NF-κB signaling pathway, as evidenced by upregulated expression of CYP1A1, CYP1B1, IL-1ß, and IL-6, resulting in oxidative stress and inflammatory response and ultimately apoptosis of PAECs. SAL inhibited the activation of AhR and NF-κB, while promoted the nuclear translocation of Nrf2 and increased the expression of its downstream antioxidant proteins HO-1 and NQO1 in PAECs, ameliorating the hypoxia-induced oxidative stress in PAECs. Furthermore, SAL lowered right ventricular systolic pressure, and decreased pulmonary vascular remodeling and right ventricular hypertrophy in hypoxia-exposed rats. CONCLUSIONS: SAL may attenuate the apoptosis of PAECs by suppressing NF-κB and activating Nrf2/HO-1 pathways, thereby delaying the progressive pathology of PH.

Large negative differential resistance and rectifying behaviors in isolated thiophene nanowire devices.

We design isolated molecular nanowires composed of thiophene oligomers sandwiched between two one-dimensional gold electrodes. Electronic transport through the molecular junctions with two interface geometries is studied by performing the first principles calculations based on density functional theory and nonequilibrium Green's function. The current-voltage (I-V) curves of the molecular wires display an unexpected negative differential resistance and rectifying behaviors along with the oscillation effects, different from other theoretical and experimental studies about the analogous thiophene devices. The significant difference is attributed to the design of the one-dimensional gold electrodes with large enough vacuum layer in transverse direction in order to suppress the interaction between wires. Such transport behaviors indicate that the thiophene molecular device would be an important candidate in future molecular electronics.

Mapping of BnMs4 and BnRf to a common microsyntenic region of Arabidopsis thaliana chromosome 3 using intron polymorphism markers.

A recessive epistatic genic male sterile two-type line, 7365AB (Bnms3ms3ms4msRrfRf/BnMs3ms3ms4ms4RfRf), combined with the fertile interim-maintainer 7365C (Bnms3ms3ms4ms4rfrf) is an effective pollination control system in hybrid rapeseed production. We report an effective strategy used to fine map BnMs4 and BnRf. The two genes were both defined to a common microsyntenic region with Arabidopsis chromosome 3 using intron polymorphism (IP) markers developed according to Arabidopsis genome information and published genome organization of the A genome. The near-isogenic lines 7365AC (Bnms3ms3ms4ms4Rfrf/Bnms3ms3ms4ms4rfrf) of BnRf and 736512AB (Bnms3ms3Ms4ms4RfRf/Bnms3ms3ms4ms4RfRf) of BnMs4 were constructed to screen developed markers and create genetic linkage maps. Nine polymorphic IP markers (P1-P9) were identified. Of these, P2, P3, P4, and P6 were linked to both BnMs4 and BnRf with genetic distances <0.6 cM. Three simple sequence repeat markers, SR2, SR3, and SR5, were also identified by using public information. Subsequently, all markers linked to the two genes were used to compare the micro-collinearity of the regions flanking the two genes with Brassica rapa and Arabidopsis. The flanking regions showed rearrangements and inversion with fragments of different Arabidopsis chromosomes, but a high collinearity with B. rapa. This collinearity provided extremely valuable reference for map-based cloning in polyploid Brassica species. These IP markers could be exploited for comparative genomic studies within and between Brassica species, providing an economically feasible approach for molecular marker-assisted selection breeding, accelerating the process of gene cloning, and providing more direct evidence for the presence of multiple alleles between BnMs4 and BnRf.

Identification of Hub Genes and Immune-Related Pathways for Membranous Nephropathy by Bioinformatics Analysis.

OBJECTIVE: We aim to explore the detailed molecular mechanisms of membrane nephropathy (MN) related genes by bioinformatics analysis. METHODS: Two microarray datasets (GSE108109 and GSE104948) with glomerular gene expression data from 65 MN patients and 9 healthy donors were obtained from the Gene Expression Omnibus (GEO) database. After processing the raw data, DEGs screening was conducted using the LIMMA (linear model for microarray data) package and Gene set enrichment analysis (GSEA) was performed with GSEA software (v. 3.0), followed by gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. The protein-protein interaction (PPI) network analysis was carried out to determine the hub genes, by applying the maximal clique centrality (MCC) method, which was visualized by Cytoscape. Finally, utilizing the Nephroseq v5 online platform, we analyzed subgroups associated with hub genes. The findings were further validated by immunohistochemistry (IHC) staining in renal tissues from MN or control patients. RESULTS: A sum of 370 DEGs (188 up-regulated genes, 182 down-regulated genes) and 20 hub genes were ascertained. GO and KEGG enrichment analysis demonstrated that DEGs of MN were preponderantly associated with cell damage and complement cascade-related immune responses. Combined with literature data and hub gene-related MN subset analysis, CTSS, ITGB2, and HCK may play important roles in the pathological process of MN. CONCLUSION: This study identified novel hub genes in MN using bioinformatics. We found that some hub genes such as CTSS, ITGB2, and HCK might contribute to MN immunopathological process, providing new insights for further study of the molecular mechanisms underlying glomerular injury of MN.

Early Menopause May Associate With a Higher Risk of CKD and All-Cause Mortality in Postmenopausal Women: An Analysis of NHANES, 1999-2014.

Background: Chronic kidney disease (CKD) in women is often accompanied by hormone disorders such as sex hormones, and most women with CKD are in the post-menopausal age group. Due to the close relationship between menopause and sex hormones, we aimed to explore the association between early menopause and CKD in post-menopausal women, and the influence of early menopause on longevity in the CKD population. Methods: Information regarding 4,945 post-menopausal women was extracted from the database of the National Health and Nutrition Examination Survey (NHANES) 1999-2014, and then divided into 4 groups according to the type of menopause (natural or surgical) and early menopause (menopause at age <45) or not. The association between early menopause and CKD prevalence was examined using multivariable logistic regression, while we used multivariable Cox proportional hazards models to investigate the possible relationship between early menopause and all-cause mortality in CKD and non-CKD populations. The differences in the levels of sex hormones between women with and without CKD were also explored. Results: Compared with women with natural menopause at age ≥45, women experiencing early natural menopause had a higher risk of CKD [OR = 1.26 (1.01-1.56)]. Similarly, as compared to women with surgical menopause at age ≥ 45, women in the early surgical menopause group were more likely to have CKD [OR = 1.38 (1.05-1.81)]. In addition, early surgical menopause was associated with higher mortality in the non-CKD group [HR = 1.62 (1.06-2.49)], but not in the CKD group. Women with CKD had a higher level of luteinizing hormone and follicle-stimulating hormone, combined with a lower level of testosterone and estradiol than the non-CKD women. Conclusion: Both early natural and surgical menopause were associated with a higher risk of CKD. Early surgical menopause was a hazard factor for survival in the non-CKD group, but not in the CKD group. Further research is required to understand the mechanisms.

Analysis of influencing factors of phenanthrene adsorption by different soils in Guanzhong basin based on response surface method.

Adsorption desorption is an important behavior affecting the migration of phenanthrene in soil. In this study, three typical soils of loess, silts and silty sand in Guanzhong Basin, Shaanxi Province, China were used as adsorbents. Batch equilibrium experiments were carried out to study the adsorption desorption kinetics and isotherm of phenanthrene in different soils. Response surface method (RSM) was used to study the effects of temperature, pH, phenanthrene concentration and organic matter content on soil adsorption of phenanthrene. The results showed that after adsorption, the outline of soil particles became more blurred and the degree of cementation increased. The kinetic adsorption of phenanthrene by soil conforms to the quasi second-order kinetic model, and the adsorption desorption isotherm is nonlinear and conforms to the Freundlich model. Due to the difference of soil properties, the adsorption amount of phenanthrene by soil is loess > silty sand > silts. The thermodynamic results show that the adsorption of phenanthrene by soil is spontaneous and endothermic, and the desorption is spontaneous and exothermic. Through RSM, the interaction between phenanthrene concentration and soil organic matter in Loess and silts is significant, and the interaction between temperature and soil organic matter in silty sand is significant. Among the four factors affecting the adsorption rate of loess, silts and silty sand, soil organic matter is the most significant. The theoretical optimum adsorption rates of loess, silts and silty sand are 98.89%, 96.59% and 93.37% respectively.

Stabilization of the second oxyanion intermediate by 1,4-dihydroxy-2-naphthoyl-coenzyme A synthase of the menaquinone pathway: spectroscopic evidence of the involvement of a conserved aspartic acid.

1,4-Dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) synthase, or MenB, catalyzes an intramolecular Claisen condensation involving two oxyanion intermediates in the biosynthetic pathway of menaquinone, an essential respiration electron transporter in many microorganisms. Here we report the finding that the DHNA-CoA product and its analogues bind and inhibit the synthase from Escherichia coli with significant ultraviolet--visible spectral changes, which are similar to the changes induced by deprotonation of the free inhibitors in a basic solution. Dissection of the structure--affinity relationships of the inhibitors identifies the hydroxyl groups at positions 1 (C1-OH) and 4 (C4-OH) of DHNA-CoA or their equivalents as the dominant and minor sites, respectively, for the enzyme--ligand interaction that polarizes or deprotonates the bound ligands to cause the observed spectral changes. In the meantime, spectroscopic studies with active site mutants indicate that C4-OH of the enzyme-bound DHNA-CoA interacts with conserved polar residues Arg-91, Tyr-97, and Tyr-258 likely through a hydrogen bonding network that also includes Ser-161. In addition, site-directed mutation of the conserved Asp-163 to alanine causes a complete loss of the ligand binding ability of the protein, suggesting that the Asp-163 side chain is most likely hydrogen-bonded to C1-OH of DHNA-CoA to provide the dominant polarizing effect. Moreover, this mutation also completely eliminates the enzyme activity, strongly supporting the possibility that the Asp-163 side chain provides a strong stabilizing hydrogen bond to the tetrahedral oxyanion, which takes a position similar to that of C1-OH of the enzyme-bound DHNA-CoA and is the second high-energy intermediate in the intracellular Claisen condensation reaction. Interestingly, both Arg-91 and Tyr-97 are located in a disordered loop forming part of the active site of all available DHNA-CoA synthase structures. Their involvement in the interaction with the small molecule ligands suggests that the disordered loop is folded in interaction with the substrates or reaction intermediates, supporting an induced-fit catalytic mechanism for the enzyme.

A bicarbonate cofactor modulates 1,4-dihydroxy-2-naphthoyl-coenzyme a synthase in menaquinone biosynthesis of Escherichia coli.

1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase-fold protein catalyzing an intramolecular Claisen condensation in the menaquinone biosynthetic pathway. We have characterized this enzyme from Escherichia coli and found that it is activated by bicarbonate in a concentration-dependent manner. The bicarbonate binding site has been identified in the crystal structure of a virtually identical ortholog (96.8% sequence identity) from Salmonella typhimurium through comparison with a bicarbonate-insensitive orthologue. Kinetic properties of the enzyme and its site-directed mutants of the bicarbonate binding site indicate that the exogenous bicarbonate anion is essential to the enzyme activity. With this essential catalytic role, the simple bicarbonate anion is an enzyme cofactor, which is usually a small organic molecule derived from vitamins, a metal ion, or a metal-containing polyatomic anionic complex. This finding leads to classification of the DHNA-CoA synthases into two evolutionarily conserved subfamilies: type I enzymes that are bicarbonate-dependent and contain a conserved glycine at the bicarbonate binding site; and type II enzymes that are bicarbonate-independent and contain a conserved aspartate at the position similar to the enzyme-bound bicarbonate. In addition, the unique location of the enzyme-bound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the α-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases.

Trends of incidence and prognosis of primary adenocarcinoma of the bladder.

BACKGROUND: Primary adenocarcinoma of the bladder (ACB) is a rare malignant tumor of the bladder with limited understanding of its incidence and prognosis. METHODS: Patients diagnosed with ACB between 2004 and 2015 were obtained from the SEER database. The incidence changes of ACB patients between 1975 and 2016 were detected by Joinpoint software. Nomograms were constructed based on the results of multivariate Cox regression analysis to predict overall survival (OS) and cancer-specific survival (CSS) in patients with ACB, and the constructed nomograms were validated. RESULTS: The incidence of ACB was trending down from 1991 to 2016. A total of 1039 patients were included in the study and randomly assigned to the training cohort (727) and validation cohort (312). In the training cohort, multivariate Cox regression showed that age, marital status, primary site, histology type, grade, AJCC stage, T stage, SEER stage, surgery, radiotherapy, and chemotherapy were independent prognostic factors for OS, whereas these were age, marital status, primary site, histology type, grade, AJCC stage, T/N stage, SEER stage, surgery, and radiotherapy for CSS. Based on the above Cox regression results, we constructed prognostic nomograms for OS and CSS in ACB patients. The C-index of the nomogram OS was 0.773 and the C-index of CSS was 0.785, which was significantly better than the C-index of the TNM staging prediction model. The area under the curve (AUC) and net benefit of the prediction model were higher than those of the TNM staging system. In addition, the calibration curves were very close to the ideal curve, suggesting appreciable reliability of the nomograms. CONCLUSION: The incidence of ACB patients showed a decreasing trend in the past 25 years. We constructed a clinically useful prognostic nomogram for calculating OS and CSS of ACB patients, which can provide a personalized risk assessment for ACB patient survival.

Effect of Marital Status on Depression and Mortality among Patients with Chronic Kidney Disease from National Health and Nutrition Examination Survey 2005-2014.

BACKGROUND: The relationship between marital status and CKD is rarely studied. We aimed to explore the effect of marital status on the depression and mortality of patients with CKD. METHODS: The data sources came from the NHANES database during 2005-2014 and 3,865 participants were included in this study. We used logistic regression models to examine the relationship between marital status and depression of CKD patients. The Cox proportional hazard models were used to evaluate the association between marital status and mortality of CKD patients. RESULTS: In terms of depression in CKD patients, unmarried patients had a worse situation than married patients. Meanwhile, after adjusting the covariables, unmarried patients had increased risk of depression (OR = 1.26, 95% CI: 1.01-1.57) compared with married CKD patients, especially in males (OR = 1.45, 95% CI: 1.02-2.06) and patients with more than college education level (OR = 12.4, 95% CI: 3.75-41.02). There was a significant relationship between marital status and mortality of general CKD patients (HR = 1.36, 95% CI: 1.17-1.58). Moreover, marriage showed a protective effect against death among male patients, patients with school graduate or less and more than college educational level, patients with high income, and patients in different estimated glomerular filtration rate groups. CONCLUSIONS: The use of large numbers of participants has revealed the effect of marital status on CKD patients. Unmarried ones had a higher risk of depression than married ones among CKD patients. Meanwhile, the risk of death was higher in unmarried ones than married ones among CKD patients in this study.