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The aim of this study was to evaluate the nutrition and metabolism status alteration during immunotherapy in advanced hepatocellular carcinoma (HCC) patients. Patients with advanced HCC who participated in the clinical trials of single-agent anti-PD-1 immunotherapy or sorafenib were retrospectively included. We analyzed self-comparison of the nutritional and metabolic indices of patients in the anti-PD-1 and sorafenib treatment group. We conducted mutual-comparison of the mentioned indices between the disease progression group and disease control group among anti-PD-1 treatment patients. We further analyzed those indices with statistical differences by partial correlation and survival analysis. Both self-comparison before and after treatment in the anti-PD-1 group and mutual-comparison of disease progression and the control group showed significant differences in multiple indices, but we did not observe significant differences in the sorafenib group. Strikingly, albumin (ALB)/prognostic nutritional index (PNI, calculated by serum albumin and lymphocyte count) decreased distinctly in the immunotherapy disease progression group patients. However, changes in ALB/PNI were not significant in disease progression patients from the sorafenib group or in the disease control patients with immunotherapy. Partial correlation analysis suggested that ALB and PNI were positively correlated with the efficacy of immunotherapy. Furthermore, survival analysis showed that the median progression-free survival and median overall survival of patients in the ALB/PNI decreased group were significantly shorter than those of patients from the ALB/PNI increased group. Anti-PD-1 immunotherapy might alter the nutritional and metabolic status in advanced HCC patients. We also should pay attention to the nutritional and metabolic status of patients when drug resistance is detected.

Background: Acquired hydrocephalus (AH) is a common complication in patients with severe brain injury. Brain tissue injury has been proposed to induce a neuroinflammatory reaction reflected by cytokines release, particularly interleukin-6 (IL-6), which associates with early brain damage. The present study measured IL-6 in the cerebrospinal fluid (CSF) of AH patients and determined its relationship to functional outcome following shunt operation.Methods: The study included a total of 32 patients with a shunt operation due to hydrocephalus. CSF samples from 26 AH subjects and 6 iNPH patients were collected via lumbar puncture before surgery. IL-6 level was measured using the micro ELISA immunoassay method. AH subjects were dichotomized into good versus poor outcomes based on modified Rankin Scale (mRS) at 3 months after shunting.Results: CSF analysis demonstrated that IL-6 was significantly elevated in the CSF of the AH group compared to controls (p = 0.023). Within the AH group, eighteen (69.2%) had a good outcome while eight (30.8%) patients had a poor outcome. Mean IL-6 level in the good outcome group was approximately four-times higher than the poor outcome group (p = 0.004). Glasgow Coma Scale (GCS) on admission was significantly different between the two groups (p = 0.014). IL-6 level and admission GCS were significantly correlated with improvement of mRS score (r = 0.473, p = 0.015 and r = 0.691, p＜0.0001, respectively). Receiver operating characteristic curve analysis showed that both factors can accurately differentiate between patients with good versus poor functional outcome (AUC = 0.861, p = 0.0039 and AUC = 0.823, p = 0.0098, respectively). Conclusions: The CSF level of IL-6 is elevated in AH patients and higher levels correlate with improvement of post-shunt functional outcome. Therefore, IL-6 CSF level might serve as a complementary surrogate parameter for operative indication. A possible IL-6 threshold in clinical routine might be a 6.98-pg/ml cutoff value to rule out unresponsive and poor outcome AH patients that are under the 6.98-pg/ml threshold.

Shoot branching is an important agronomic trait that directly determines plant architecture and affects crop productivity. To promote crop yield and quality, axillary branches need to be manually removed during cucumber production for fresh market and thus are undesirable. Auxin is well known as the primary signal imposing for apical dominance and acts as a repressor for lateral bud outgrowth indirectly. The TEOSINTE BRANCHED1 / CYCLOIDEA/PCF ( TCP ) family gene BRANCHED1 ( BRC1 ) has been shown to be the central integrator for multiple environmental and developmental factors that functions locally to inhibit shoot branching. However, the direct molecular link between auxin and BRC1 remains elusive. Here we find that cucumber BRANCHED1 ( CsBRC1 ) is expressed in axillary buds and displays a higher expression level in cultivated cucumber than in its wild ancestor. Knockdown of CsBRC1 by RNAi leads to increased bud outgrowth and reduced auxin accumulation in buds. We further show that CsBRC1 directly binds to the auxin efflux carrier PIN-FORMED ( CsPIN3 ) and negatively regulates its expression in vitro and in vivo. Elevated expression of CsPIN3 driven by the CsBRC1 promoter results in highly branched cucumber with decreased auxin levels in lateral buds. Therefore, our data suggest that CsBRC1 inhibits lateral bud outgrowth by direct suppression of CsPIN3 functioning and thus auxin accumulation in axillary buds in cucumber, providing a strategy to breed for cultivars with varying degrees of shoot branching grown in different cucumber production systems.

Background: Wax gourd (Benincasa hispida Cong., 2n=2x=24) is one of the most important winter vegetables of the Cucurbitaceae family. There are only limited markers available for this crop and the draft genome of wax gourd provides a powerful tool for SSR marker development.Results: In this study, we developed genome-wide SSR markers from wax gourd genome and characterized their distribution and frequency of different motifs and repeats. A total of 52,431 microsatellites from wax gourd genome were identified, of which 39,319 SSR markers were developed. 1,152 non-wax gourd SSR markers were selected from cucumber, melon, watermelon and pumpkin to test their transferability in wax gourd. 580 SSR markers could be transferable in wax gourd, and 42 of them were detected with polymorphic in 11 tested accessions of wax gourd. In addition, 11 good polymorphic transferrable SSR markers and 21 SSR markers of wax gourd were selected to investigate the genetic diversity and population structure of 129 wax gourd accessions. 112 alleles were detected by these 32 SSR markers. The result of population structure showed that the 129 wax gourd accessions were divided into two main populations, and the genetic diversity analysis separated them into two clusters. Conclusions: The large number of wax gourd SSR markers developed in this study provides a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection (MAS) in wax gourd.

The fruit trichomes of Cucurbitaceae are widely desired in many Asian countries and have been a key determinant of cucumber (Cucumis sativus L.) cultivar selection for commercial production and breeding. However, our understanding of the initiation and development of cucumber trichomes is still limited. Here, we found that the cucumber TINY BRANCHED HAIR (TBH) gene is preferentially expressed in multicellular trichomes. Overexpression of CsTBH in tbh mutants restored the trichome phenotype and increased the percentage of female flowers, whereas silencing of CsTBH in wild-type plants resulted in stunted trichomes with a lower rate of female flowers. Furthermore, we provide evidence that CsTBH can directly bind to the promoters of cucumber 1-Aminocyclopropane-1-Carboxylate Synthase (CsACS) genes and regulate their expression, which affects multicellular trichome development, ethylene accumulation, and sex expression. Two cucumber acs mutants with different trichome morphology and sex morphs compared with their near-isogenic line further support our findings. Collectively, our study provides new information on the molecular mechanism of CsTBH in regulating multicellular trichome development and sex expression through an ethylene pathway.

Background Breast carcinoma is one of the most common tumors in women. The immune microenvironment, especially T cell infiltration, is related to the occurrence and prognosis of breast carcinoma. Objective This study investigated the gene expression patterns associated with tumor-infiltrating CD4+ and CD8+ T cells in invasive breast carcinomas. Methods The gene expression data and corresponding clinical phenotype data from the Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) were downloaded. The stromal and immune score were calculated using ESTIMATE. The differentially expressed genes (DEGs) with a high vs. low stromal score and a high vs. low immune score were screened and then functionally enriched. The tumor-infiltrating immune cells were investigated using the Cibersort algorithm, and the CD4+ and CD8+ T cell-related genes were identified using a Spearman correlation test of infiltrating abundance with the DEGs. Moreover, the miRNA-mRNA pairs and lncRNA-miRNA pairs were predicted to construct the competing endogenous RNAs (ceRNA) network. Kaplan-Meier (K-M) survival curves were also plotted. Results In total, 478 DEGs with a high vs. low stromal score and 796 DEGs with a high vs. low immune score were identified. In addition, 39 CD4+ T cell-related genes and 78 CD8+ T cell-related genes were identified; of these, 14 genes were significantly associated with the prognosis of BRCA patients. Moreover, for CD4+ T cell-related genes, the chr22-38_28785274-29006793.1–miR-34a/c-5p–CAPN6 axis was identified from the ceRNA network, whereas the chr22-38_28785274-29006793.1–miR-494-3p–SLC9A7 axis was identified for CD8+ T cell-related genes. Conclusions The chr22-38_28785274-29006793.1–miR-34a/c-5p–CAPN6 axis and the chr22-38_28785274-29006793.1–miR-494-3p–SLC9A7 axis might regulate cellular activities associated with CD4+ and CD8+ T cell infiltration, respectively, in BRCA.

Cucumber is an important vegetable crop bearing fleshy pepo fruit harvested immature. Fruits left unpicked in time during summer production, as well as unfavorable environmental conditions during post-harvest shelf, will cause cucumber fruits to turn yellow and ripen, and thus impair the market value. Identification of maturity-related genes is of great agricultural and economic importance for cucumber production. Here, we isolated and characterized a MADS-box gene, Cucumis sativus SHATTERPROOF (CsSHP) in cucumber. Expression analysis indicated that CsSHP was specifically enriched in reproductive organs including stamens and carpels. Ectopic expression of CsSHP was unable to rescue the indehiscence silique phenotype of shp1 shp2 mutant plant in Arabidopsis. Instead, overexpression of CsSHP resulted in early flowering, precocious phenotypes, and capelloid organs in wild-type Arabidopsis. Biochemical analysis indicated that CsSHP directly interacted with cucumber SEPALLATA (SEP) proteins. CsSHP expression increased significantly during the yellowing stage of cucumber ripening, and was induced by exogenous application of abscisic acid (ABA). Therefore, CsSHP may participate in fruit maturation through the ABA pathway and floral organ specification via interaction with CsSEPs to form protein complex in cucumber.

The WUSCHEL-CLAVATA3 pathway genes play essential role in shoot apical meristem maintenance and floral organ development, and under intense selection during crop domestication. The carpel number is an important fruit trait affecting fruit shape, size, and internal quality in cucumber, but the molecular mechanism remains elusive. Here, we found that CsCLV3 expression was negatively correlated with carpel number in cucumber cultivars. CsCLV3-RNAi led to increased number of petals and carpels, whereas overexpression of CsWUS resulted in more sepals, petals and carpels, suggesting that CsCLV3 and CsWUS function as a negative and a positive regulator for carpel number variation, respectively. Biochemical analyses indicated that CsWUS directly binds to the promoter of CsCLV3 and activates its expression. Overexpression of CsFUL1A, a FRUITFULL-like MADS-box gene, resulted in more petals and carpels. CsFUL1A can directly bind to CsWUS promoter to stimulate its expression. Further, we found that auxin participates in carpel number variation in cucumber through interaction of CsARF14 with CsWUS. Therefore, we identified a gene regulatory pathway involving CsCLV3, CsWUS, CsFUL1A and CsARF14 in determining carpel number variation in an important vegetable crop-cucumber.

L-securinine is a natural product extracted and isolated from the leaf of dried Securinega suffruticosa. The aim of the present study was to explore the effects of L-securinine on proliferation, and the methylation profile of the dickkopf-related protein 1 (DKK1) gene in human lung cancer cells and fibroblasts. L-securinine was extracted, isolated and the structure was identified. The cytotoxicity of L-securinine in A549 cells was evaluated by Cell Counting Kit-8 assays. The expression and DNA methylation profile of DKK genes was analyzed by reverse transcription-quantitative polymerase chain reaction and bisulfite sequencing polymerase chain reaction, respectively. L-securinine inhibited the proliferation of lung cancer cells; the half-maximal inhibitory concentration values were 8.92, 4.73 and 3.81 µg/ml, at 24, 36 and 48 h post-treatment, respectively. DKK1, 2 and 3 expression was significantly increased in A549 cells compared with HLF-a cells. L-securinine induced the downregulation of DKK1 in A549 cells in a dose-dependent manner and induced methylation changes at CpG sites in the DKK1 promoter region. L-securinine may be a potential anticancer drug that mediates its effects by altering DKK1 gene methylation.

TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes, as plant-specific transcription factors, play vital roles in flower pattern, leaf development and plant architecture. Our recent study shows that the TCP gene BRANCHED1 (CsBRC1) specifically regulates shoot branching in cucumber. Here, we found CsBRC1 had a closely related paralogous gene CsBRC1-like. The synteny analysis revealed that these two genes originated from a segmental duplication. CsBRC1-like displayed different expression patterns in cucumber compared with CsBRC1, indicating that they may have functional differentiation. Ectopic expression of CsBRC1-like in Arabidopsis brc1-1 mutant resulted in reduced rosette branches and rosette leaves, whereas silencing CsBRC1-like in cucumber only led to a deformed true leaf of seedling rather than affecting the shoot branching. RNA-seq analysis of wild-type and CsBRC1-like-RNAi plants implicated that CsBRC1-like might regulate early leaf development through affecting the transcripts of auxin and cytokinin related genes in cucumber. Moreover, CsBRC1-like directly interacts with CsTCP10a and CsBRC1 in vivo. Our results demonstrated that CsBRC1-like has a specific role in regulating leaf development, and CsBRC1-like and CsBRC1 may have overlapping roles in shoot branching.

Hyperlipidemia occurs very often in modern society along with a high calorie intake and is regarded as one of the greatest risk factors for the prevalence of cardiac vascular disease (CVD). In this study, we investigated the anti-hyperlipidemic effect of the rice bran polysaccharides (RBP) and its mechanism in a high fat diet animal model. 60 ICR mice were randomly divided into 3 groups, which included Control, HFD (high fat diet) and HFD + RBP, and each group included 20 mice. The control group was fed with a standard diet while the other two groups were fed with HFD. In addition, the HFD + RBP group was fed with 500 mg kg-1 of rice bran polysaccharides by intragastric administration while the other two groups were intragastrically administered with water. The results showed that RBP treatment for 10 weeks obviously decreased the body weight, liver weight and adipose tissues of mice; and it decreased the levels of total cholesterol (TC), triglycerides (TG) and low density lipoprotein-cholesterol (LDL-c) in the plasma. H&E staining of the liver tissues showed that RBP treatment decreased the size of fat droplets compared with the HFD group. Microarray analysis revealed that RBP treatment results in 80 genes being up-regulated while 72 genes were down-regulated in the tissues of liver. IPA software analysis suggested that NF-κB may play a vital role in the lipid-lowering effect of RBP. Real-time quantitative PCR confirmed that the mRNA levels of PPAR-α, PPAR-γ, PPAR-δ, SREBP-1C, FASN, ACC, SIRT and CD36, which are related to lipid metabolism, were significantly regulated by RBP supplementation compared to HFD. The western blot analysis further confirmed these altered expressions after RBP treatment. Taken together, these results suggest that the oral administration of RBP exerts lipid-lowering in high fat diet mice via regulating the lipid metabolism-related gene expression.

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of domestic and wild cattle responsible for major economic losses in dairy and beef industries throughout the world. Inhibition of viral entry plays a crucial role in the control of BoHV-1 infection and aptamers have been reported to inhibit viral replication. In this study, nine DNA aptamers that target BoHV-1 were generated using systemic evolution of ligands by exponential enrichment. Of the nine candidates, aptamer IBRV-A4 exhibited the highest affinity and specificity for BoHV-1, which bound to BoHV-1 with a Kd value of 3.519 nM and demonstrated the greatest virus binding as shown by fluorescence imaging. The neutralizing ability of aptamer IBRV-A4 was determined using neutralization assays and real time PCR in BoHV-1 infected Madin-darby bovine kidney cells. Virus titration, immunofluorescence and confocal laser scanning microscopy showed virus replication significantly decreased when aptamer IBRV-A4 was added to BoHV-1 infected MDBK cells at 0 and 0.5 hours post-infection, whereas no change was seen when IBRV-A4 was added 2 hours post-infection. This concludes that aptamer IBRV-A4 efficiently inhibits viral entry of BoHV-1 in MDBK cells and is therefore a novel tool for diagnosis and treatment of BoHV-1 infection in cattle.

In Western countries and China, the dietary habit of high calories usually results in hyperlipidemia, which is closely associated with cardiovascular diseases. In the study, we investigated the antihyperlipidemic effect of oryzanol and its molecular mechanism in the high fat diet (HFD) mouse model. In total, 60 ICR mice were randomly divided into control group, HFD group, and HFD+Ory group. The mice from the HFD+Ory group were additionally fed with 100 mg/kg of oryzanol by intragastric administration. Our data indicated that oryzanol treatment for 10 weeks significantly reduced bodyweight, liver weight, and adipose tissues weight of the mice; lowered the contents of total cholesterol (TC), triglycerides (TG), and low density lipoprotein-cholesterol (LDL-C); and elevated high density lipoprotein-cholesterol (HDL-C) in the plasma of HFD mice. Compared with the HFD group, HE staining showed that oryzanol treatment decreased the size of fat droplets of liver tissues and the size of adipocytes. Gene chip data found that oryzanol administration caused 32 genes to increase expressions while 60 genes had reduced expressions in the liver tissues of HFD mice. IPA software was used to analyze the protein interaction network and found that transcript factor NF-κB located in the central role of network, meaning NF-κB may have important function in the lipid-lowering effect of oryzanol. Western blotting and RT-qPCR confirmed that lipid metabolism-related gene expressions were obviously regulated by oryzanol administration. Oryzanol also inhibited expressions of inflammatory factor in the liver tissues of HDF mice. Taken together, our data indicate that oryzanol treatment can regulate lipid metabolism-related gene expressions and inhibit HDF-caused obesity in mice.

Mycoplasma bovis (M. bovis) is a major, but often overlooked, pathogen that causes respiratory disease, mastitis, and arthritis in cattle. It has been widespread in China since 2008. In this study, single-stranded DNA (ssDNA) aptamers with high affinity and specificity against the P48 protein of M. bovis were selected using microplates as the matrix. Of nine candidates, aptamer WKB-14 showed the best affinity in an indirect enzyme-linked aptamer assay (ELAA) and good specificity by dot blotting. To the best of our knowledge, this is the first time that an aptamer has been used in a competitive ELAA for the serological detection of M. bovis. The percent inhibition (PI) cutoff value of the indirect competitive ELAA (ic-ELAA) was 40%, assessed using 20 negative sera. In a comparative study of different detection methods, ic-ELAA with dc-ELISA and dot blotting had a higher positive detection rate than the other two commercial indirect ELISA kits.

Mycoplasma gallisepticum can infect a wide variety of birds including the commercial poultry. M. gallisepticum MGA_0676 is a putative lipoprotein, which is similar to bacterial thermostable nucleases. But the possible pathogenic effect of M. gallisepticum MGA_0676 has not been investigated so far. In the present study, we cloned the MGA_0676 gene after deletion of the amino-terminal signal sequence and mutagenesis of the Mycoplasma TGA tryptophan codons to TGG and expressed recombinant MGA_0676 protein in Escherichia coli. We identified and characterized MGA_0676 as a Ca(2+)-dependent cytotoxic nuclease of M. gallisepticum with a staphylococcal nuclease (SNc) region that displays the hallmarks of nucleases. Membrane protein immunoblot analysis and immunogold electron microscopy revealed that MGA_0676 locates on the membrane surface of M. gallisepticum. Furthermore, apoptosis assay using annexin V-FITC and propidium iodide (annexin V/PI) indicated that MGA_0676 played significant roles in apoptosis induction and pathological damages in chicken cells. Moreover, confocal microscopy showed that MGA_0676 localizes in the nuclei of host cells. Besides, after the SNc region was deleted, MGA_0676 lost its ability of nuclear localization, nuclease activity, and cytotoxicity, which revealed that the SNc region is essential for nuclear translocation and induction of apoptosis in chicken cells. The above results suggest that MGA_0676 is an important virulence factor in cellular pathology and may play a unique role in the life cycle events of M. gallisepticum.

Fatty acid desaturases are enzymes that introduce double bonds into the hydrocarbon chains of fatty acids. The fatty acid desaturases from 37 cyanobacterial genomes were identified and classified based upon their conserved histidine-rich motifs and phylogenetic analysis, which help to determine the amounts and distributions of desaturases in cyanobacterial species. The filamentous orN2-fixing cyanobacteria usually possess more types of fatty acid desaturases than that of unicellular species. The pathway of acyl-lipid desaturation for unicellular marine cyanobacteriaSynechococcusandProchlorococcusdiffers from that of other cyanobacteria, indicating different phylogenetic histories of the two genera from other cyanobacteria isolated from freshwater, soil, or symbiont. StrainGloeobacter violaceusPCC 7421 was isolated from calcareous rock and lacks thylakoid membranes. The types and amounts of desaturases of this strain are distinct to those of other cyanobacteria, reflecting the earliest divergence of it from the cyanobacterial line. Three thermophilic unicellular strains,Thermosynechococcus elongatusBP-1 and twoSynechococcusYellowstone species, lack highly unsaturated fatty acids in lipids and contain only oneΔ9desaturase in contrast with mesophilic strains, which is probably due to their thermic habitats. Thus, the amounts and types of fatty acid desaturases are various among different cyanobacterial species, which may result from the adaption to environments in evolution.

New antiviral therapy for pandemic influenza mediated by the H9N2 avian influenza virus (AIV) is increasingly in demand not only for the poultry industry but also for public health. Aptamers are confirmed to be promising candidates for treatment and prevention of influenza viral infections. Thus, we studied two DNA aptamers, A9 and B4, selected by capillary electrophoresis-based systemic evolution of ligands by exponential enrichment (CE-SELEX) procedure using H9N2 AIV purified haemagglutinin (HA) as target. Both aptamers had whole-virus binding affinity. Also, an enzyme-linked aptamer assay (ELAA) confirmed binding affinity and specificity against other AIV subtypes. Finally, we studied aptamer-inhibitory effects on H9N2 AIV infection in Madin-Darby canine kidney (MDCK) cells and quantified viral load in supernatant and in cell with quantitative PCR (qPCR). Our data provide a foundation for future development of innovative anti-influenza drugs.