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MircoRNA (miRNA) are a group of small, non–coding, regulatory RNA with an average length of approximately 22 nucleotides, which mostly modulate gene expression post–transcriptionally through complementary binding to the 3ʹ‐untranslated region (3ʹ‐UTR) of multiple target genes. Emerging evidence has shown that miRNA are frequently dysregulated in a variety of human malignancies. Among them, microRNA‐145 (miR‐145) has been increasingly identified as a critical suppressor of carcinogenesis and therapeutic resistance. Resistance to tumor therapy is a challenge in cancer treatment due to the daunting range of resistance mechanisms. We reviewed the status quo of recent advancements in the knowledge of the functional role of miR‐145 in therapeutic resistance and the tumor microenvironment. It may serve as an innovative biomarker for therapeutic response and cancer prognosis., In this review, we summarize the molecular mechanisms and potential pathways involved in the regulation of miR‐145, simultaneously focusing on the roles of miR‐145 in modulating therapeutic resistance, especially the nature of their intrinsic links across diverse cancers. In addition to its diagnostic or prognostic value, miR‐145 can serve as a predictor of chemotherapeutic response.

Aroma is an important index of tea quality. The volatile C6‐compounds formed from linoleic and linolenic acids in tea leaf lipids are essential components of tea. C6‐compounds are formed and transformed during the postharvest process of tea leaves. However, the metabolic flux of these C6‐compounds, the activities of related enzymes, and the transcription of related genes during the postharvest process of oolong tea remain unclear. In this study, the chemical profiles of C6‐aldehydes and C6‐alcohols, the pattern of ADH enzyme activity, and the level of CsADH gene expression during the postharvest process of oolong tea were investigated. We found that the turnover process had a positive effect on the accumulation of C6‐alcohols and simultaneously induced ADH activity, especially during the withering stage. The expression of CsADH peaked during the turnover stage. The relative expression level of CSA019598 typically increased during the postharvest process. Correlation analysis demonstrated that CSA019598 expression increased as ADH activity increased. This finding suggests that CSA019598 may play a prominent role in regulating ADH. These results advance our understanding of C6‐compound formation during the postharvest process of oolong tea. We aim to evaluate how green leaf volatiles affect the enzymatic formation and genetic transcription of aromatic compounds in oolong tea in future studies., (a) ADH reduces C6‐Aldehydes into C6‐alcohols during the postharvest process of oolong tea under mechanical wounding conditions. (b) CSA019598 screened from tea plant genome might be an available CsADH gene which plays a major regulatory role during the postharvest process of oolong tea.

Gene set enrichment (GSE) analysis plays an essential role in extracting biological insight from genome-scale experiments. ORA (overrepresentation analysis), FCS (functional class scoring), and PT (pathway topology) approaches are three generations of GSE methods along the timeline of development. Previous versions of KOBAS provided services based on just the ORA method. Here we presented version 3.0 of KOBAS, which is named KOBAS-i (short for KOBAS intelligent version). It introduced a novel machine learning-based method we published earlier, CGPS, which incorporates seven FCS tools and two PT tools into a single ensemble score and intelligently prioritizes the relevant biological pathways. In addition, KOBAS has expanded the downstream exploratory visualization for selecting and understanding the enriched results. The tool constructs a novel view of cirFunMap, which presents different enriched terms and their correlations in a landscape. Finally, based on the previous version's framework, KOBAS increased the number of supported species from 1327 to 5944. For an easier local run, it also provides a prebuilt Docker image that requires no installation, as a supplementary to the source code version. KOBAS can be freely accessed at http://kobas.cbi.pku.edu.cn, and a mirror site is available at http://bioinfo.org/kobas., Graphical Abstract Graphical AbstractFramework of KOBAS.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a global public health threat. The efficacy of several repurposed drugs has been evaluated in clinical trials. Among these drugs, a second-generation antiandrogen agent, enzalutamide, was proposed because it reduces the expression of transmembrane serine protease 2 (TMPRSS2), a key component mediating SARS-CoV-2-driven entry, in prostate cancer cells. However, definitive evidence for the therapeutic efficacy of enzalutamide in COVID-19 is lacking. Here, we evaluated the antiviral efficacy of enzalutamide in prostate cancer cells, lung cancer cells, human lung organoids and Ad-ACE2-transduced mice. Tmprss2 knockout significantly inhibited SARS-CoV-2 infection in vivo. Enzalutamide effectively inhibited SARS-CoV-2 infection in human prostate cells, however, such antiviral efficacy was lacking in human lung cells and organoids. Accordingly, enzalutamide showed no antiviral activity due to the AR-independent TMPRSS2 expression in mouse and human lung epithelial cells. Moreover, we observed distinct AR binding patterns between prostate cells and lung cells and a lack of direct binding of AR to TMPRSS2 regulatory locus in human lung cells. Thus, our findings do not support the postulated protective role of enzalutamide in treating COVID-19 through reducing TMPRSS2 expression in lung cells., Enzalutamide, an approved drug for prostate cancer, acts on TMPRSS2 expression, a key mediator for SARS-CoV-2 infection. Here, the authors characterize the anti-SARS-CoV-2 effects of Enzalutamide in prostate cancer cells, lung cancer cells, human lung organoids and in hACE2-transduced Tmprss2 knockout mice and show lack antiviral action in human lung cells and human lung organoids, likely due to the AR-independent TMPRSS2 expression in mouse and human lung epithelial cells.

The effectiveness of hypochlorites (NaClO and Ca(ClO)2) on the reduction of antibiotic resistance genes (ARGs) during waste activated sludge (WAS) fermentation was determined by the quantitative PCR. NaClO and Ca(ClO)2 exhibited distinct effects on ARGs fates. Ca(ClO)2 was effective in removing all investigated ARGs, and the efficiency was highly dose-dependent. Unexpectedly, the NaClO treatment attenuated ARGs with lower efficiency and even caused the propagation of certain ARGs (i.e., aadA1 and tetQ) at higher doses. The extracellular polymeric substances dissolution and membrane integrity suggested that unstable NaClO had acute effects on bacteria initially, while it was ineffective to further attenuate ARGs released from hosts due to the rapid consumption of oxidative ClO-. Without lasting and strong oxidative stress, the microbial activities of tolerant ARGs hosts will partially recover and then contribute to the ARGs dissemination across genera. In contrast, solid-state Ca(ClO)2 was slowly released and exhibited prolonged effects on bacteria by disrupting cell membranes and removing the susceptible ARGs released from hosts. Furthermore, bacterial taxa-ARG network analysis indicated that Ca(ClO)2 reduced the abundance of potential hosts, and the metabolic pathway and gene expression related to ARGs propagation were significantly downregulated by Ca(ClO)2, which contributed to efficient ARGs attenuation.

Rapid and low-cost methods of detecting mutations and polymorphisms are crucial for genotyping applications including mutagenesis and gene editing. S1 family endonucleases such as T7E1, EndoV and CELI can potentially be used in enzymatic mismatch detection. Among them, CELI has been shown to be effective in detecting mutations in Targeting Induced Local Lesions IN Genomes (TILLING). However, current method of CELI purification from celery is laborious, and challenging for many non-biochemical laboratories, and the presence of post-translational modifications hinders efficient production of the enzyme in E. coli. Here, we report an efficient system for bulk production of enzymatically active CELI endonuclease through transient expression in a model plant Nicotiana benthamiana. We also optimized the reaction buffer, by additions of Mn2+ and DTT, with enhanced mismatch cleavage activity. Using the new CELI production and reaction system, we were able to routinely detect mismatches in 1/32 mixed mutant and wildtype DNA samples. We believe the newly established system has many applications in characterization of mutations occurred in natural variations, mutagenized populations and gene editing.

Background The Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants. Despite this transient expression system has been successfully applied in a number of plant species, it is poorly developed in Caragana intermedia. Results In this study, we established an Agrobacterium-mediated transient expression system in C. intermedia leaves and optimized the effect of different Agrobacterial strains, several surfactants and the concentration of Silwet L-77, which would affect transient expression efficiency. Among the 5 Agrobacterial strains examined, GV3101 produced the highest GUS expression level. Besides, higher level of transient expression was observed in plants infiltrated with Silwet L-77 than with Triton X-100 or Tween-20. Silwet L-77 at a concentration of 0.001% greatly improved the level of GUS transient expression. Real-time PCR showed that expression of CiDREB1C was highly up-regulated in transiently expressed plants and reached the highest level at the 2nd day after infiltration. Based on this optimized transient transformation method, we characterized CiDREB1C function in response to drought, salt and ABA treatment. The results showed that transiently expressed CiDREB1C in C. intermedia leaves could enhance the survival rate and chlorophyll content, and reduce the lodging rate compared with the control seedlings under drought, salt and ABA treatments. Furthermore, the rate of leaf shedding of CiDREB1C transient expression seedlings was lower than that of the control under ABA treatment. Conclusions The optimized transient expression condition in C. intermedia leaves were infiltrated with Agrobacterial strains GV3101 plus Silwet L-77 at a concentration of 0.001% added into the infiltration medium. Transiently expressed CiDREB1C enhanced drought, salt and ABA stress tolerance, indicated that it was a suitable and effective tool to determine gene function involved in abiotic stress response in C. intermedia. Electronic supplementary material The online version of this article (10.1186/s12870-019-1800-4) contains supplementary material, which is available to authorized users.

We previously showed that Guy1, a primary signal expressed from the Y chromosome, is a strong candidate for a male-determining factor that confers female-specific lethality in Anopheles stephensi (Criscione et al., 2016). Here, we present evidence that Guyl increases X gene expression in Guy1-transgenic females from two independent lines, providing a mechanism underlying the Guy1-conferred female lethality. The median level gene expression (MGE) of X-linked genes is significantly higher than autosomal genes in Guy1-transgenic females while there is no significant difference in MGE between X and autosomal genes in wild-type females. Furthermore, Guyl significantly upregulates at least 40% of the 996 genes across the X chromosome in transgenic females. Guy1-conferred female-specific lethality is remarkably stable and completely penetrant. These findings indicate that Guyl regulates dosage compensation in An. stephensi and components of dosage compensation may be explored to develop novel strategies to control mosquito-borne diseases. National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [AI105575, AI121284] National Institutes of Health AI105575 Zhijian Jake Tu; National Institutes of Health AI121284 Zhijian Jake Tu; The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Chitin is the second abundant natural polysaccharide, and the development of chitin and its derivatives have received more and more attention. Glucosamine sulfate (GAS) obtained by the hydrolysis of chitin can promote the growth of chondrocytes. The coaxial electrospinning technology had been utilized to encapsulated GAS into the core of polycaprolactone (PCL) nanofibers. It could protect the GAS from the environment and allow it to release sustainably over time. From the results of scanning electron microscopy (SEM), PCL/GAS nanofibers performed a typical fiber scaffold surface. Transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX) results confirmed the feasibility to use coaxial electrospinning to load GAS. Tensile tests exhibited that PCL/GAS mats could be stretched more than twice its own length. Besides, the cell experiments illustrated that PCL/GAS had a significant effect on the proliferation and growth of rat articular chondrocytes, attesting its prospect for cartilage regeneration.

Tea is one of three major non-alcoholic beverages that are popular all around the world. The economic value of tea product largely depends on the post-harvest physiology of tea leaves. The utilization of quantitative reverse transcription polymerase chain reaction is a widely accepted and precise approach to determine the target gene expression of tea plants, and the reliability of results hinges on the selection of suitable reference genes. A few reliable reference genes have been documented using various treatments and different tissues of tea plants, but none has been done on post-harvest leaves during the tea manufacturing process. The present study selected and analyzed 15 candidate reference genes: Cs18SrRNA, CsGADPH, CsACT, CsEF-1α, CsUbi, CsTUA, Cs26SrRNA, CsRuBP, CsCYP, CselF-4α, CsMON1, CsPCS1, CsSAND, CsPPA2, CsTBP. This study made an assessment on the expression stability under two kinds of post-harvest treatment, turn over and withering, using three algorithms—GeNorm, Normfinder, and Bestkeeper. The results indicated that the three commonly used reference genes, CsTUA, Cs18SrRNA, CsRuBP, together with Cs26SrRNA, were the most unstable genes in both the turn over and withering treatments. CsACT, CsEF-1α, CsPPA2, and CsTBP were the top four reference genes in the turn over treatment, while CsTBP, CsPCS1, CsPPA2, CselF-4α, and CsACT were the five best reference genes in the withering group. The expression level of lipoxygenase genes, which were involved in a number of diverse aspects of plant physiology, including wounding, was evaluated to validate the findings. To conclude, we found a basis for the selection of reference genes for accurate transcription normalization in post-harvest leaves of tea plants.

Sleep disturbance can result in memory impairment, and both sleep and hippocampal memory formation are maintained by circadian clock genes. Although preoperative sleep deprivation is known to be an independent risk factor for postoperative cognitive dysfunction (POCD) after inhalation anesthesia, the circadian mechanisms involved are currently unclear. To examine this issue, we constructed models of rapid eye movement sleep deprivation (RSD) and POCD after sevoflurane inhalation, to evaluate the circadian mechanisms underlying preoperative sleep deprivation-induced POCD after sevoflurane inhalation. Morris water maze probe test performance revealed that RSD aggravated the hippocampal-dependent memory impairment induced by sevoflurane anesthesia, and the recovery period of memory impairment was prolonged for more than a week by sleep deprivation. Western blot analysis revealed that sleep deprivation inhibited hippocampal Bmal1 and Egr1 expression for more than 7 days after sevoflurane inhalation. Importantly, hippocampal Per2 expression levels were first decreased by sevoflurane inhalation then increased from the third day by sleep deprivation. Sleep deprivation enhanced the expression of hippocampal inflammatory factors IL-1β and IL-6 after sevoflurane inhalation. In addition, sevoflurane inhalation activated the plasma expression of S100β and IL-6, particularly after sleep deprivation. Sleep deprivation aggravated pathogenic impairment of pyramidal neurons and activated astrocytes in CA1 after sevoflurane inhalation. These results suggest that preoperative RSD aggravates hippocampal memory impairment by enhancing neuroinflammatory injuries after sevoflurane inhalation, which is related to hippocampal clock gene abnormalities.

Despite modest sequence conservation and rapid evolution, long non-coding RNAs (lncRNAs) appear to be conserved in expression pattern and function. However, analysis of lncRNAs across tissues and developmental stages remains largely uncharacterized in mammals. Here, we systematically investigated the lncRNAs of the Guizhou miniature pig (Sus scrofa), which was widely used as biomedical model. We performed RNA sequencing across 9 organs and 3 developmental skeletal muscle, and developed a filtering pipeline to identify 10,813 lncRNAs (9,075 novel). Conservation patterns analysis revealed that 57% of pig lncRNAs showed homology to humans and mice based on genome alignment. 5,455 lncRNAs exhibited typical hallmarks of regulatory molecules, such as high spatio-temporal specificity. Notably, conserved lncRNAs exhibited higher tissue specificity than pig-specific lncRNAs and were significantly enriched in testis and ovary. Weighted co-expression network analysis revealed a set of conserved lncRNAs that are likely involved in postnatal muscle development. Based on the high degree of similarity in the structure, organization, and dynamic expression of pig lncRNAs compared with human and mouse lncRNAs, we propose that these lncRNAs play an important role in organ physiology and development in mammals. Our results provide a resource for studying animal evolution, morphological complexity, breeding, and biomedical research.

Glucocorticoid receptor DNA binding factor 1 (GRF-1) is an important Rho family GTPase-activating protein, and the dysregulation of GRF-1 expression maybe involved in tumor progression. However, the role of GRF-1 expression in the osteosarcoma prognosis has been well less elaborated. Here, we conducted a hospital-based case study, including 247 patients with pathologically confirmed osteosarcoma to evaluate the associations between GRF-1 expression and osteosarcoma prognosis. We found that high GRF-1 expression was correlated with poor outcome of osteosarcoma compared with low GRF-1 expression (the median recurrence-free survival times, 11 months vs 56 months; the median overall survival times, 18 months vs 53 months). Like tumor stage, the GRF-1 expression was an independent prognostic factor influencing the survival of osteosarcoma [hazard ratio values (95 % confidence interval) were 5.39 (3.54-8.20) for recurrence-free survival (RFS) and 6.58 (4.44-9.74) for overall survival (OS), respectively]. Furthermore, the high expression of GRF-1 was significantly associated with larger tumor size, tumor dedifferentiation, and increasing metastasis risk. Functionally, the knockdown of GRF-1 expression inhibited tumor cells proliferation and induced cell apoptosis. These results indicate for the first time that GRF-1 expression may modify osteosarcoma prognosis and may be a potential tumor therapeutic target.

Background In the present study, a well-defined glucose and guanidine based copolymer, galactosylated 2-hydroxypropyl methacrylamide-s-3-guanidinopropyl methacrylamide (HPMA-s-GPMA) abbreviated as GGH was prepared and self-assembled with small hairpin RNA (shRNA) to inhibit human telomerase reverse transcriptase (hTERT) gene expression in vitro to develop a shRNA carrier. Methods First, HPMA-s-APMA copolymers were synthesized by aqueous reversible addition-fragmentation chain transfer polymerization, followed by galactosylation and guanidinylation. Then, three target shRNAs containing green fluorescent protein gene as a reporter were combined with GGH to form shRNA/GGH polyplexes. Results GGH copolymers could condense shRNA to form shRNA/GGH polyplex particles with a diameter in the range 122.8–331.6 nm in phosphate-buffered saline, and zeta potential values ranging from +3.7 to +16.5 mV at various charge ratios (N/P). That the cytotoxicity of GGH copolymers was significantly lower than that of PEI in human hepatocellular liver carcinoma cells (HepG2) and human cervix epithelial carcinoma cells. The transfection efficiency of shRNA/GGH polyplexes was higher than that of PEI at a charge ratio of 12 in the HepG2 cell line. Furthermore, shRNA/GGH polyplexes could effectively silence hTERT mRNA expression in serum-free medium (p

Quantitative real-time PCR (qRT-PCR) has becoming a widely used method for detection and characterization of gene expression in recent years. However, the accuracy of the qPCR output strongly depends on the performance of selected internal reference genes. In this study, we used periwinkle as material to select the most stably expressed reference genes during Wheat Blue Dwarf (WBD) phytoplasma infection in a spatial and temporal way. Ten candidate reference genes were selected, and their expression stability were evaluated using the algorithms geNorm and NormFinder program. Our results demonstrated that all of selected reference genes did not express stably enough across all of the experimental groups, the suitable reference genes should be selected according to the experiment conditions. In the present study, CrL23 performed well when all samples were considered. CrTUB and CrACT, CrEF1α and CrACT were the most stable reference genes for samples from different tissues of periwinkle according to the outcome of geNorm and NormFinder, respectively. In addition, CrTUB and CrTUA were the most stable reference genes for flower malformation during WBD phytoplasma infection. Furthermore, to illustrate the usefulness of the most stable reference genes, the expression pattern of CrDEF involving in flower abnormality during WBD phytoplasma infection was analyzed. Taken together, the present study is valuable for future research on gene expression of periwinkle infected by WBD phytoplasma and maybe other phytoplasmas.

Purpose tumor-infiltrating immune cells are highly relevant to the progression and prognosis of colorectal cancer (CRC). The aim of this study is to explore the immune cells and immune-related gene expression in tumor microenvironment of CRC. Methods CIBERSORT, a deconvolution algorithm, was used to analyze the infiltration of 22 immune cell types in the tumor microenvironment and immune-related gene expression in 404 CRC and 40 adjacent non-tumorous tissues. Results a wide heterogeneity of immune cells among different paired tissues and in tumor stages was uncovered. M0 macrophages, M1 macrophages and CD4 memory activated T cells were infiltrated significantly more in CRC compared with normal tissues in both TCGA and GEO cohorts. CRC with T1–2 tumor stage showed increased CD4 memory activated T cells compared with T3–4 tumors. M0 macrophages were the highest in stage N1 tumors. Significant immune-related genes were identified to build prognostic models by Cox regression analysis. The concordance index of the prognostic model for TNM stage I–II was 0.69, and 0.71 for stage III–IV. The AUC values for 1-, 3-, and 5-year survivals were 0.674, 0.773, 0.812 for TNM stage I–II, respectively, and 0.764, 0.782, 0.803 for stage III–IV, respectively. Conclusion these results could assist clinicians in selecting targets for immunotherapies and individualize treatment strategies for patients with CRC.

BACKGROUND: The relationship between survivin and extranodal, nasal-type natural killer/T cell lymphoma (ENKTCL) was unclearly established yet. We here studied the potential prognostic roles of survivin and its implication as a target in ENKTCL therapy. METHODS: ENKTCL patients’ peripheral blood were collected and tested by ELISA. ENKTCL cell lines were cultured with or without survivin inhibitor and tested by MTT and Flow cytometry. According to the gene expression profiles from the ArrayExpress Archive under E-TABM-702, survivin co-regulated cluster was established by Coupled Two-way Clustering Algorithm. RESULTS: Seventeen point six percent of total 17 ENKTCL patients were serum survivin-positive. These patients had poorer outcome than that of negative cases (P

Aberrant expression of microRNAs (miRNAs) has been involved in the development and progression of malignancy. MicroRNA-9 (miR-9) has been confirmed to be underexpressed in many types of cancers. However, the relationship between miR-9 and the Wnt/β-catenin signaling pathway in oral squamous cell carcinoma (OSCC) remains largely unknown. Here we showed that the miR-9 was underexpressed in patients with OSCC and several OSCC cell lines. Lentivirus-mediated miR-9 overexpression in highly aggressive (Tca8113 and SCC-9) tumor cells significantly inhibited proliferation of the two cell lines in vitro and in vivo. Furthermore, we found that the CXC chemokine receptor 4 (CXCR4) gene was a direct target of miR-9. RNA interference silencing of CXCR4 proved that miR-9 underexpression led to constitutive activation of β-catenin through activation of CXCR4 expression in OSCC cells. Finally, we also analyzed the possible relationship between miR-9 and the genes downstream of the Wnt/β-catenin pathway in OSCC development and progression. These results provide new evidence of miR-9 as a promising tumor gene therapeutic target for OSCC patients.

Auxin, a vital plant hormone, regulates a variety of physiological and developmental processes. It is involved in fruit abscission through transcriptional regulation of many auxin-related genes, including early auxin responsive genes (i.e., auxin/indole-3-acetic acid (AUX/IAA), Gretchen Hagen3 (GH3) and small auxin upregulated (SAUR)) and auxin response factors (ARF), which have been well characterized in many plants. In this study, totally five auxin-related genes, including one AUX/IAA (LcAUX/IAA1), one GH3 (LcGH3.1), one SAUR (LcSAUR1) and two ARFs (LcARF1 and LcARF2), were isolated and characterized from litchi fruit. LcAUX/IAA1, LcGH3.1, LcSAUR1, LcARF1 and LcARF2 contain open reading frames (ORFs) encoding polypeptides of 203, 613, 142, 792 and 832 amino acids, respectively, with their corresponding molecular weights of 22.67, 69.20, 11.40, 88.20 and 93.16 kDa. Expression of these genes was investigated under the treatment of girdling plus defoliation which aggravated litchi fruitlet abscission due to the blockage of carbohydrates transport and the reduction of endogenous IAA content. Results showed that transcript levels of LcAUX/IAA1, LcGH3.1 and LcSAUR1 mRNAs were increased after the treatment in abscission zone (AZ) and other tissues, in contrast to the decreasing accumulation of LcARF1 mRNA, suggesting that LcAUX/IAA1, LcSAUR1 and LcARF1 may play more important roles in abscission. Our results provide new insight into the process of fruitlet abscission induced by carbohydrate stress and broaden our understanding of the auxin signal transduction pathway in this process at the molecular level.

Tumour necrosis factor alpha (TNF-α) is one kind of cytokines which is related to inflammation and lipid metabolism. TNF-α cDNA was cloned from the liver of blunt snout bream (Megalobrama amblycephala) through real-time polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. The full-length cDNA of TNF-α covered 1467 bp, with an open reading frame (ORF) of 723 bp, which encodes 240 amino acids. It possessed the TNF family signature IIIPDDGIYFVYSQ. After the lipopolysaccharide (LPS) challenge test, a graded tissue-specific expression pattern of TNF-α was observed and there was high expression abundance in the kidney, brain and liver. After 8 weeks feeding trial, liver samples, two groups fed with 6% and 11% lipid levels, were collected. The results showed that, for fish fed with high-fat diet, the triglyceride of serum and lipid content of liver were elevated. Furthermore, TNF-α and peroxisome proliferator-activated receptors (PPARα, β) mRNA expression of fish fed 11% lipid diet were significantly up-regulated (p

Background and Objective Periodontitis is an infectious disease in which the host immune and inflammatory responses play essential roles in resistance to bacterial infection, as well as the induction of tissue destruction if the immune response is dysregulated. The triggering receptor expressed on myeloid cells (TREMs) modulates inflammatory and innate immune signaling. TREM-1 is considered as an amplifier of the immune response, while TREM-2 is a negative regulator that has yet to be explored in periodontal disease before. We hypothesized that TREMs participated in the innate immune responses during the pathogenesis of periodontitis. Therefore, the aim of this study was to evaluate TREM-1 and TREM-2 expression in the gingival tissues from patients with chronic periodontitis and healthy subjects as well as their correlation with clinical periodontal parameters. This study is the first to identify TREM-2 in periodontal tissue, as well as the protein expression changes of TREM-1 and TREM-2 in periodontal tissues. Material and Methods Gingival tissue sections were collected from 31 healthy subjects and 53 patients with chronic periodontitis. Immunohistochemistry and quantitative real-time polymerase chain reaction were employed to evaluate the protein and mRNA expression of these receptors in gingival tissues. The recorded clinical parameters were probing depth, clinical attachment loss, plaque index and bleeding on probing. Results In addition to myeloid cells in gingival connective tissues, TREM-1 and TREM-2 were also found expressed in gingival epithelial cells. In particular, TREM-1 was detected in almost all gingival epithelium from both healthy and inflamed biopsies. The expression levels of TREM-1 and TREM-2 were significantly increased in the periodontitis group compared to the healthy group. Increased levels of these receptors are to be positively correlated with site-specific periodontal parameters. Conclusion The increased expression of TREM-1 and TREM-2 levels in periodontitis may confer diagnostic and potential therapeutic targets as well as indicating their association with the clinical severity of the disease.

Reverse transcription quantitative PCR (RT-qPCR) as the accurate and sensitive method is use for gene expression analysis, but the veracity and reliability result depends on whether select appropriate reference gene or not. To date, several reliable reference gene validations have been reported in fruits trees, but none have been done on preharvest and postharvest longan fruits. In this study, 12 candidate reference genes, namely, CYP, RPL, GAPDH, TUA, TUB, Fe-SOD, Mn-SOD, Cu/Zn-SOD, 18SrRNA, Actin, Histone H3, and EF-1a, were selected. Expression stability of these genes in 150 longan samples was evaluated and analyzed using geNorm and NormFinder algorithms. Preharvest samples consisted of seven experimental sets, including different developmental stages, organs, hormone stimuli (NAA, 2,4-D, and ethephon) and abiotic stresses (bagging and girdling with defoliation). Postharvest samples consisted of different temperature treatments (4 and 22°C) and varieties. Our findings indicate that appropriate reference gene(s) should be picked for each experimental condition. Our data further showed that the commonly used reference gene Actin does not exhibit stable expression across experimental conditions in longan. Expression levels of the DlACO gene, which is a key gene involved in regulating fruit abscission under girdling with defoliation treatment, was evaluated to validate our findings. In conclusion, our data provide a useful framework for choice of suitable reference genes across different experimental conditions for RT-qPCR analysis of preharvest and postharvest longan fruits.

Syndecan-4 (Syn4), a single-pass transmembrane heparin sulphate proteoglycan (HSPG), plays significant role in the formation of focal adhesions and interacts with many growth factors to regulate cell migration and neural induction. Here, we show the new roles of syndecan-4(syn4) in zebrafish embryonic neurogenesis. Syn4 is broadly and dynamically expressed throughout the early stages of embryonic development. Knockdown of syn4 increases the expression of the marker genes of multiple types of neural cells. The increased expression of the marker genes is resulted from excessive proliferation of the neural cells. In addition, disrupting syn4 expression results in truncated and multiple aberrant branching of caudal primary (CaP) axons. Collectively, these data indicate that Syn4 suppresses the cellular proliferation during neurogenesis and is crucial for the formation of CaP axons during zebrafish embryogenesis.

Diabetes mellitus is associated with decreased NO bioavailability in the myocardium. Ginsenoside Rb1 has been shown to confer cardioprotection against ischemia reperfusion injury. The aim of this study was to investigate whether Ginsenoside Rb1 exerts cardioprotective effects during myocardial ischemia-reperfusion in diabetic rats and whether this effect is related to increase the production of NO via enhancing eNOS expression in the myocardium. The myocardial I/R injury were induced by occluding the left anterior descending artery for 30 min followed by 120 min reperfusion. An eNOS inhibitor L-NAME or Rb1 were respectively administered 25 min or 10 min before inducing ischemia. Ginsenoside Rb1 preconditioning reduced myocardial infarct size when compared with I/R group. Ginsenoside Rb1 induced myocardial protection was accompanied with increased eNOS expression and NO concentration and reduced plasma CK and LDH ( ). Moreover, the myocardial oxidative stress and tissue histological damage was attenuated by Ginsenoside Rb1 ( ). L-NAME abolished the protective effects of Ginsenoside Rb1. It is concluded that Ginsenoside Rb1 protects against myocardium ischemia/reperfusion injury in diabetic rat by enhancing the expression of eNOS and increasing the content of NO as well as inhibiting oxidative stress.

Antigen-presenting cells including dendritic cells (DCs) express mannan receptors (MR) on their surface, which can be exploited in cancer therapy by designing immune-stimulatory viruses coated with mannan-modified capsids that then bind to DCs and initiate a potent immune response. Although the combination of anti-angiogenesis and cancer immunotherapy agents has a synergistic antitumor effect, more effective strategies for delivering such combinations are still required. Here we report the design and application of mannan-modified adenovirus that expresses both telomerase reverse transcriptase (TERT) and vascular endothelial growth factor receptor-2 (VEGFR-2). Cytotoxic T lymphocytes that are reactive to TERT and VEGFR-2 are capable of mounting an anti-tumour response in murine breast and colon tumour models and in a lung metastatic model. Compared with mannan-modified TERT adenovirus vaccine or mannan-modified VEGFR-2 adenovirus vaccine alone, the combined vaccine showed remarkably synergistic anti-tumour immunity in these models. Both TERT- and VEGFR-2-specific cytotoxic T lymphocytes (CTL) were identified in an in vitro cytotoxicity assay and the CTL activity against tumour cells was significantly elevated in the combined vaccine group. Furthermore, CTL-mediated toxicity was blocked by anti-CD8 monoclonal antibodies. Thus, the combined mannan-modified TERT and VEGFR-2 adenovirus confers potent anti-tumour immunity by targeting both tumour cells and intratumoural angiogenesis.

Fibrinogen-like protein A (FGLA), a member of the fibrinogen-related protein superfamily, exists in different tissues of vertebrates and invertebrates. FGLA plays crucial roles including innate immune response, blood clotting and regeneration. In this study, the fibrinogen-like protein A (fglA) was cloned from Apostichopus japonicus using rapid amplification of cDNA ends PCR techniques. The cDNA sequence of fglA is 1,524 bp with a 849 bp open reading frame encoding a polypeptide of 282 amino acids, with an N-terminal signal peptide and a conserved C-terminal domain. Bioinformatic analysis revealed that the predicted molecular weight of the whole protein is 31.9 kDa and it has an isoelectric point of 5.64. In-situ hybridization demonstrated that fglA is widely distributed in body wall, intestines, longitudinal muscles and respiratory tree. The expression levels of fglA during different regeneration stages in the body wall, intestine and respiratory trees were analyzed by real-time PCR. The expression of fglA gradually increased within 1 h in body wall, and reached a plateau before decreasing to the basal level. This indicates that fglA is associated with the regeneration of Apostichopus japonicus.

In the present study, a well-defined glucose and guanidine based copolymer, galactosylated 2-hydroxypropyl methacrylamide-s-3-guanidinopropyl methacrylamide (HPMA-s-GPMA) abbreviated as GGH was prepared and self-assembled with small hairpin RNA (shRNA) to inhibit human telomerase reverse transcriptase (hTERT) gene expression in vitro to develop a shRNA carrier.First, HPMA-s-APMA copolymers were synthesized by aqueous reversible addition-fragmentation chain transfer polymerization, followed by galactosylation and guanidinylation. Then, three target shRNAs containing green fluorescent protein gene as a reporter were combined with GGH to form shRNA/GGH polyplexes.GGH copolymers could condense shRNA to form shRNA/GGH polyplex particles with a diameter in the range 122.8-331.6 nm in phosphate-buffered saline, and zeta potential values ranging from +3.7 to +16.5 mV at various charge ratios (N/P). That the cytotoxicity of GGH copolymers was significantly lower than that of PEI in human hepatocellular liver carcinoma cells (HepG2) and human cervix epithelial carcinoma cells. The transfection efficiency of shRNA/GGH polyplexes was higher than that of PEI at a charge ratio of 12 in the HepG2 cell line. Furthermore, shRNA/GGH polyplexes could effectively silence hTERT mRNA expression in serum-free medium (p 0.01) and decrease the aggregation of protein in the medium with the presence of 10% serum. In addition, hTERT mRNA expression in HepG2 cells demosntrate a significant difference between siRNA/GGH polyplexes and blank samples (p 0.05).GGH copolymers could integrate advantages relating to galactose content for hepatocyte targeting, guanidino groups for cell penetration and HPMA component for shielding, showing great potential for effective hepatocyte targeting gene delivery.

Oms66 is aBorrelia burgdorferiouter membrane porin protein whose role in Lyme disease pathogenesis and immunity has not been well established. Oms66 was solubilized from whole-cell lysates of strain B313 (which is derived from B31 but lacks OspA, -B, -C, and -D) and purified to homogeneity by fast-protein liquid chromatography. Purified native Oms66 (nOms66), which retained the ability to form large channels in a planar lipid bilayer model membrane system, and denatured Oms66 (hOms66) were used to immunize New Zealand White rabbits. The resulting Oms66 antisera were tested in a complement-dependent borreliacidal assay in parallel with basal serum and with serum from rabbits immune to reinfection withB. burgdorferi(IRS). IRS showed high-titer complement-dependent killing of both strains B31 and B313. Sera from animals immunized with nOms66 showed high-titer complement-dependent killing activity against strain B313 but exhibited no killing of B31. By comparison, serum generated from immunizations with hOms66 showed no killing activity against either strain. Following adsorption of antiserum to nOms66 with recombinant Oms66 (rOms66), the serum antibodies no longer bound to rOms66 or to nOms66 that had been denatured with 8 M urea. However, the antibodies still bound to nOms66 and killing activity against B313 was retained, thus suggesting that native, conformational epitopes are targets of this bactericidal activity. Six C3H HeJ mice were immunized with nOms66 and were challenged using “host-adapted”B. burgdorferiB31 by skin implantation of infected mouse ear tissue. Four of the six mice were protected against both localized and disseminated infection. These findings indicate that native Oms66 can elicit potent bactericidal activity and significant protective immunity against host-adapted organisms.

During embryonic development of Artemia sinica, environmental stresses induce the embryo diapause phenomenon, required to resist apoptosis and regulate cell cycle activity. The small ubiquitin-related modifier-1 (SUMO), a reversible post-translational protein modifier, plays an important role in embryo development. SUMO regulates multiple cellular processes, including development and other biological processes. The molecular mechanism of diapause, diapause termination and the role of As-sumo-1 in this processes and in early embryo development of Artemia sinica still remains unknown. In this study, the complete cDNA sequences of the sumo-1 homolog, sumo ligase homolog, caspase-1 homolog and cyclin B homolog from Artemia sinica were cloned. The mRNA expression patterns of As-sumo-1, sumo ligase, caspase-1, cyclin B and the location of As-sumo-1 were investigated. SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E proteins were analyzed during different developmental stages of the embryo of A. sinica. Small interfering RNA (siRNA) was used to verify the function of sumo-1 in A. sinica. The full-length cDNA of As-sumo-1 was 476 bp, encoding a 92 amino acid protein. The As-caspases-1 cDNA was 966 bp, encoding a 245 amino-acid protein. The As-sumo ligase cDNA was 1556 bp encoding, a 343 amino acid protein, and the cyclin B cDNA was 739 bp, encoding a 133 amino acid protein. The expressions of As-sumo-1, As-caspase-1 and As-cyclin B were highest at the 10 h stage of embryonic development, and As-sumo ligase showed its highest expression at 0 h. The expression of As-SUMO-1 showed no tissue or organ specificity. Western blotting showed high expression of As-SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E at the 10 h stage. The siRNA caused abnormal development of the embryo, with increased malformation and mortality. As-SUMO-1 is a crucial regulation and modification protein resumption of embryonic diapause and early embryo development of A. sinica.

P21-activated protein kinase (Pak1), a main downstream effector of small Rho GTPases, plays an important role in the regulation of cell morphogenesis, motility, mitosis and angiogenesis. However, the role of Pak1 in gastric cancer metastasis remains unclear. Here, we showed that Pak1 is overexpressed in gastric cancer tissues from 74 patients by immunohistochemistry. Overexpression of Pak1 was associated with metastasis and prognosis of gastric cancer. In addition, overexpression of Pak1 increased gastric cancer cell motility and invasion, whereas downregulation of Pak1 expression reduced gastric cancer cell migration and invasion. In further study, data showed that activated Pak1 inhibited stress fiber and focal adhesion complex formation in gastric cancer cells and led to the formation of motile phenotypes. Importantly, activated Pak1 elicited phosphorylation of the ERK and JNK-dependent pathway in gastric cancer cell lines. In conclusion, our results suggest that Pak1 is overexpressed in gastric cancer and plays an important role in the metastasis of gastric cancer. The mechanism by which Pak1 induces cancer metastasis may involve activation of ERK and JNK.

In the present paper, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) was used to examine and identify differentially expressed genes in Capsicum annuum exposed to UV-B irradiation. Around 4000 transcript derived fragments (TDFs) were visualized and in total 183 TDFs were isolated, sequenced and analyzed by Blast 2 go. Among these TDFs, 84 of them showed homology to known genes. There were 43 TDFs showing up-regulated expression, 24 TDFs showing down-regulated expression and 29 TDFs showing both up-regulated and down-regulated expression, respectively. Some of these TDFs were found to be in response/related to UV-B stress, including carbonic anhydrase, calcium-dependent protein, thionin-like protein, bzip protein and so on. In particular, chlorophyll a/b binding protein (Capcab) responding to UV-B stress was cloned. It was concluded that Capcab could play a protective role in plant anti-UV-B and maintaining photosynthetic rate under UV-B stress.