Dextran sulfate sodium-induced gut microbiota dysbiosis aggravates liver injury in mice with S100-induced autoimmune hepatitis.

Recently, the incidence of autoimmune hepatitis (AIH) has gradually increased, and the disease can eventually develop into cirrhosis or even hepatoma if left untreated. AIH patients are often characterized by gut microbiota dysbiosis, but whether gut microbiota dysbiosis contributes to the progression of AIH remains unclear. In this study, we investigate the role of gut microbiota dysbiosis in the occurrence and development of AIH in mice with dextran sulfate sodium salt (DSS) induced colitis. C57BL/6J mice were randomly divided into normal group, S100-induced AIH group, and DSS+S100 group (1 % DSS in the drinking water), and the experimental cycle lasted for four weeks. We demonstrate that DSS administration aggravates hepatic inflammation and disruption of the intestinal barrier, and significantly changes the composition of gut microbiota in S100-induced AIH mice, which are mainly characterized by increased abundance of pathogenic bacteria and decreased abundance of beneficial bacteria. These results suggest that DSS administration aggravates liver injury of S100-induced AIH, which may be due to DSS induced gut microbiota dysbiosis, leading to disruption of the intestinal barrier, and then, the microbiota translocate to the liver, aggravating hepatic inflammation.

In vivo CRISPR screen identifies LTN1 as a novel tumor suppressor ubiquitinating insulin-like growth factor 2 mRNA-binding protein 1 in hepatocellular carcinoma.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a frequent and aggressive kind of cancer. Although E3 ligases play important roles in HCC development, several E3 ligases remain unknown. APPROACH AND RESULTS: Through in vivo CRISPR knockout (KO) screens targeting related E3 ligase genes in HCC nude mice models, we discovered LTN1 as a novel tumor suppressor in HCC. Co-IP paired with 2D-LC-MS/MS and subsequent western blotting in HCC cells were used to identify the interactome of LTN1. Compared to matched normal tissues, the expression of LTN1 was decreased in human HCC tissues (ANT) (157/209). Clinically, patients with HCC who expressed low levels of LTN1 had a poor prognosis. Forced expression of LTN1 decreased cell growth in vitro and in vivo, whereas knockdown of LTN1 increased cell growth. Mechanistically, elevated LTN1 expression inhibited HCC cell growth by ubiquitinating and destabilizing the IGF2BP1 protein, which inhibited the c-Myc and IGF-1R signaling pathways. There was a negative correlation between the LTN1 protein expression and the IGF2BP1 protein expression in HCC tissues (R2=0.2799, P=0.0165). CONCLUSIONS: LTN1 may be a crucial tumor suppressor for determining the prognosis and a possible therapeutic target since it inhibits the proliferation of HCC cells by ubiquitinating IGF2BP1.

ß-arrestin2 deficiency ameliorates S-100-induced autoimmune hepatitis in mice by inhibiting infiltration of monocyte-derived macrophage and attenuating hepatocyte apoptosis.

Autoimmune hepatitis (AIH) is a progressive hepatitis syndrome characterized by high transaminase levels, interface hepatitis, hypergammaglobulinemia, and the presence of autoantibodies. Misdiagnosis or delayed treatment of AIH can lead to cirrhosis or liver failure, which poses a major risk to human health. ß-Arrestin2, a key scaffold protein for intracellular signaling pathways, has been found to be involved in many autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. However, whether ß-arrestin2 plays a role in AIH remains unknown. In the present study, S-100-induced AIH was established in both wild-type mice and ß-arrestin2 knockout (Arrb2 KO) mice, and the experiments identified that liver ß-arrestin2 expression was gradually increased, and positively correlated to serum ANA, ALT and AST levels during AIH progression. Furthermore, ß-arrestin2 deficiency ameliorated hepatic pathological damage, decreased serum autoantibody and inflammatory cytokine levels. ß-arrestin2 deficiency also inhibited hepatocyte apoptosis and prevented the infiltration of monocyte-derived macrophages into the damaged liver. In vitro experiments revealed that ß-arrestin2 knockdown suppressed the migration and differentiation of THP-1 cells, whereas ß-arrestin2 overexpression promoted the migration of THP-1 cells, which was regulated by the activation of the ERK and p38 MAPK pathways. In addition, ß-arrestin2 deficiency attenuated TNF-α-induced primary hepatocyte apoptosis by activating the Akt/GSK-3ß pathway. These results suggest that ß-arrestin2 deficiency ameliorates AIH by inhibiting the migration and differentiation of monocytes, decreasing the infiltration of monocyte-derived macrophages into the liver, thereby reducing inflammatory cytokines-induced hepatocytes apoptosis. Therefore, ß-arrestin2 may act as an effective therapeutic target for AIH.

Circulating TP73-AS1 and CRNDE serve as diagnostic and prognostic biomarkers for non-small cell lung cancer.

BACKGROUND: Circulating long noncoding RNAs (lncRNAs) are considered a new class of biomarkers for the diagnosis and prognosis of various malignancies. We aimed to identify circulating lncRNAs as biomarkers for the diagnosis and prognosis of non-small cell lung cancer (NSCLC). METHODS: The expression of 14 candidate lncRNAs was measured in matched cancer and ipsilateral normal lung tissues of 20 patients with NSCLC using quantitative reverse-transcription PCR. In plasma samples from training and testing sets, significantly and aberrantly expressed lncRNAs, TA73-AS1 and CRNDE, were further analyzed. Receiver operating characteristic (ROC) curves were constructed, and the areas under the ROC curves (AUC) were obtained to assess diagnostic performance. The Kaplan-Meier survival analysis was used to assess the impact of plasma TA73-AS1 and CRNDE expression on tumor-free survival (TFS) of patients with NSCLC. The effect of TP73-AS1 expression on NSCLC cells was investigated in vitro. RESULTS: AUC values of plasma TA73-AS1 and CRNDE were 0.822 and 0.815 in the training set and 0.843 and 0.804 in the testing set, respectively, to distinguish NSCLC from healthy controls. The combination of plasma TP73-AS1, CRNDE, and two classical tumor markers, carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA21-1), showed excellent diagnostic performance for NSCLC (AUC =0.927 in the training set; AUC = 0.925 in the testing set). Furthermore, the high expression of the two plasma lncRNAs correlated with worse TFS in patients with NSCLC. In vitro cell model studies revealed that TP73-AS1 overexpression facilitated NSCLC cell survival, invasion, and migration. CONCLUSION: Circulating TP73-AS1 and CRNDE could be potential biomarkers for the diagnosis and prognostic prediction of NSCLC.

Effect of folic acid supplementation on the change of plasma S-adenosylhomocysteine level in Chinese hypertensive patients: a randomized, double-blind, controlled clinical trial.

The relationship between folic acid and S-adenosylhomocysteine (SAH) is controversial. This study aims to explore the effect of different doses of folic acid supplementation on SAH levels in hypertensive patients and the modification of methylene-tetrahydrofolate reductase (MTHFR) C677T gene polymorphism. A randomized, double-blind, controlled clinical trial was conducted. Hypertensive patients aged 45-75 years without a history of stroke and cardiovascular disease were selected, who were randomly assigned to one of 8 dose groups. This trial has been registered with Trial Number: ChiCTR1800016135. In the total population, folic acid supplementation of 0.4-2.0 mg/day had no effect on SAH level (ß = 0.47, 95% CI: -0.86-1.79, p = 0.491), while folic acid supplementation of 2.4 mg/day significantly increased SAH level (ß = 1.93, 95% CI: 0.22-3.64, p = 0.027). Stratified analysis found that MTHFR C677T genotype CC supplemented with 2.4 mg/day folic acid had no effect on SAH level (ß = 0.30, 95% CI: -2.74-3.34, p = 0.847), while CT and TT genotype supplemented with 2.4 mg/day folic acid showed a significant increase in SAH level (CT: ß = 2.98, 95% CI: 0.34-5.62, p = 0.027; TT: ß = 3.00, 95% CI: -0.51-6.51, p = 0.095; CT combined with TT: ß = 2.99, 95% CI: 0.90-5.09, p = 0.005). In conclusion, supplementation of 2.4 mg/day folic acid can lead to increased SAH levels, especially in MTHFR C677T genotype CT and TT.

The mechanism of carbon source utilization by microalgae when co-cultivated with photosynthetic bacteria.

Microalgae-photosynthetic bacteria (PSB) co-culture, which is promising for wastewater treatment and lipid production, is lacking of study. In this work, the combinations of 3 microalgae and 3 PSB strains were firstly screened and then different inoculation ratios of the co-cultures were investigated. It was found the best promotion was Chlorella pyrenoidosa/Rhodobacter capsulatus co-culture (1:1), where the biomass productivity, acetate assimilation rate and lipid productivity were 1.64, 1.61 and 2.79 times than that of the sum of pure microalgae and PSB cultures, respectively. Meanwhile, the inoculation ratio significantly affected the growth rate and lipid productivity of co-culture systems. iTRAQ analysis showed that PSB played a positive effect on acetate assimilation, TCA cycle and glyoxylate cycle of microalgae, but decreased the carbon dioxide utilization and photosynthesis, indicating PSB promoted the microalgae metabolism of organic carbon utilization and weakened inorganic carbon utilization. These findings provide in-depth understanding of carbon utilization in microalgae-PSB co-culture.

Role of eIF3C Overexpression in Predicting Prognosis of Intrahepatic Cholangiocarcinoma.

BACKGROUND: Elevated expression of eukaryotic initiation factor 3c (eIF3C) was recently uncovered to promote several types of cancer progression by inducing cell proliferation. Here, we aimed to assess the expression and prognostic value of eIF3C in intrahepatic cholangiocarcinoma (ICC) patients. METHODS: Expression of eIF3C was analyzed by immunohistochemistry in tissue microarrays (TMAs) containing 138 ICC and paired peritumoral tissues from ICC patients. Then, the roles of eIF3C in ICC cells were investigated by RNA interference, and the relationship between the eIF3C and KI67 expression was explored in ICC cells and tissues. Finally, the relation between the eIF3C level and clinicopathologic features of ICC was probed, and Kaplan-Meier and Cox's analyses were performed to assess the prognostic merit of eIF3C and KI67 in ICC patients. RESULTS: The expression of eIF3C was elevated in ICC tissues compared to paired peritumoral tissues, which was consistent with the result from the GEPIA database. The downregulation of eIF3C in ICC cells impaired the cellular invasion, metastasis, colony formation, and proliferation. Moreover, we further found a positive relationship between the eIF3C and KI67 expression in ICC cells and tissues. The expression of eIF3C in ICC tissues was positively correlated with lymphatic metastasis (p = 0.049), and the high level of KI67 was frequently found in ICC patients with the large tumor (p = 0.028), high serum AFP (p = 0.019), or lymphatic metastasis (p = 0.039). Notably, patients with the eIF3C or KI67 overexpression had shorter overall survival and higher disease-free survival rates than those with low expression of eIF3C or KI67, and the combination of eIF3C or KI67 expression was an independent parameter for predicting the prognosis and recurrence of ICC patients. CONCLUSIONS: Elevated eIF3C expression promotes ICC development, and combination of eIF3C and KI67 is a valuable predictor of the survival and recurrence of ICC patient.

Four plasma miRNAs act as biomarkers for diagnosis and prognosis of non-small cell lung cancer.

Previous studies have reported that the aberrant expression of circulating microRNAs (miRNAs/miRs) can be used as diagnostic and prognostic markers in non-small cell lung cancer (NSCLC). The present study aimed to assess the diagnostic and prognostic predictive values of four plasma miRNAs for NSCLC. A total of 12 candidate miRNAs were selected that have previously been reported to be aberrantly expressed in NSCLC, and their plasma levels in the training set were detected via reverse transcription-quantitative PCR analysis. The screened out miRNAs were further validated in the testing set. The area under the curve (AUC) of the receiver operating characteristic curve was constructed to evaluate diagnostic performance. Kaplan-Meier survival analysis was performed to assess the association between the plasma miRNA levels and disease-free survival (DFS) time. The results demonstrated that 4/12 plasma miRNAs (miR-210, miR-1290, miR-150 and miR-21-5p) were highly expressed in patients with NSCLC compared with their expression levels in patients with benign lung disease (BLD) and healthy controls in the training and testing sets, respectively. The AUC values of the four-miRNA panel were 0.96 and 0.93 in the training and testing sets, respectively, for distinguishing patients with NSCLC from healthy controls, which were similar to the AUC values for distinguishing patients with NSCLC from patients with BLD (0.96 and 0.94). The AUC values of the four-miRNA panel in patients with stage I NSCLC were comparable to that of patients with stage II-III NSCLC (0.942 and 0.965). Patients with high plasma levels of miR-210 and miR-150 had worse DFS than those with low plasma levels of these miRNAs. In addition, patients whose plasma levels of the four miRNAs decreased by >50% after surgery exhibited a good DFS. Taken together, the results of the present study suggest that these four miRNAs (miR-210, miR-1290, miR-150 and miR-21-5p) act as useful biomarkers for early diagnosis and prognosis of NSCLC.

circHMGCS1-016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma.

BACKGROUND: Accumulating evidence indicates that circRNAs may serve as essential regulators in the progression of several human cancers, but the function and mechanism of circRNAs in intrahepatic cholangiocarcinoma (ICC) are largely unknown. METHODS: RNA-seq was used to assess differentially expressed circRNAs between 4 ICC and peritumor tissues. Quantitative RT-PCR and in situ hybridization were used to determine the circHMGCS1-016 expression in ICC tissues. The function and mechanism of circHMGCS1-016 were further identified via in vivo experiments. The clinical characteristics and prognostic significance of circHMGCS1-016 were analyzed by a retrospective study. The functions of circHMGCS1-016 were assessed via modifying circRNA expression in ICC cells. Moreover, the molecular mechanisms of circHMGCS1-016 in ICC cells were explored by circRNA precipitation, miRNA immunoprecipitation, SILAC and luciferase reporter assays. RESULTS: We identified that compared with peritumor tissues, ICC tissues expressed hsa_circ_0008621 (circHMGCS1-016) high by RNA-seq, which was further identified by qRT-PCR and in situ hybridization. Moreover, the expression of circHMGCS1-016 was revealed to be associated with survival and recurrence of ICC patients. By regulating circHMGCS1-016 expression, we found that elevated circHMGCS1-016 promoted ICC development both in vitro and in vivo. By SILAC and circRNA-pull down, we demonstrated that circHMGCS1-016 induced ICC cell invasion and reshaped the tumor immune microenvironment via the miR-1236-3p/CD73 and GAL-8 axis. In ICC tissues, we uncovered that a high level of circHMGCS1-016 was positively associated with CD73 and GAL-8 expression and negatively related to the CD8+ T cells infiltration, which was further validated by establishing a humanized mouse tumor model. Importantly, we displayed that ICC patients with high levels of circHMGCS1-016 in tumor tissues benefited less from anti-PD1 treatment compared to those with low levels of circHMGCS1-016. CONCLUSIONS: CircHMGCS1-016 is a forceful contributor in ICC development and immune tolerance via miR-1236-3p/CD73 and GAL-8 axis. CircHMGCS1-016 can be explored as a new potential biomarker and therapeutic target for PD1-resistant ICC.

The effect of care transition pathway implementation on patients undergoing joint replacement during the COVID-19 pandemic: a quasi-experimental study from a tertiary care hospital orthopedic department in Beijing, China.

BACKGROUND: The coronavirus disease (COVID-19) pandemic has had a massive impact on individuals globally. The Chinese government has formulated effective response measures, and medical personnel have been actively responding to challenges associated with the epidemic prevention and control strategies. This study aimed to evaluate the effect of the implementation of a care transition pathway on patients that underwent joint replacement during the COVID-19 pandemic. METHODS: A quasi-experimental study was designed to evaluate the effect of implementing a care transition pathway for patients who underwent joint replacement during the COVID-19 pandemic in the orthopedic department of a tertiary care hospital in Beijing, China. Using a convenient sampling method, a total of 96 patients were selected. Of these, 51 patients who had undergone joint replacement in 2019 and received treatment via the routine nursing path were included in the control group. The remaining 45 patients who underwent joint replacement during the COVID-19 epidemic in 2020 and received therapy via the care transition pathway due to the implementation of epidemic prevention and control measures were included in the observation group. The quality of care transition was assessed by the Care Transition Measure (CTM), and patients were followed up 1 week after discharge. RESULTS: The observation group was determined to have better general self-care preparation, written planning materials, doctor-patient communication, health monitoring, and quality of care transition than the control group. CONCLUSIONS: A care transition pathway was developed to provide patients with care while transitioning through periods of treatment. It improved the patient perceptions of nursing quality. The COVID-19 pandemic is a huge challenge for health professionals, but we have the ability to improve features of workflows to provide the best possible patient care.

High level of CD73 predicts poor prognosis of intrahepatic cholangiocarcinoma.

Background: Despite recent improvements in the diagnosis and therapy of intrahepatic cholangiocarcinoma (ICC), the prognosis for ICC patients remains poor. Therefore, it is needed to identify new biological indicators for ICC progression. Methods: Immunohistochemistry was engaged to inspect the ecto-5'-nucleotidase (CD73) and CD8 expressions in tissue microarrays including tissues from 140 ICC patients. Then, the association between the level of CD73/CD8 and clinicopathologic characteristics of ICC was analysed. Finally, the prognostic value of CD73 and CD8 levels in ICC patients was assessed by Kaplan-Meier and multivariate and univariate analyses. Results: The CD73 expression was evidently upregulated in ICC tissues compared to the corresponding peritumoral tissues. The elevated CD73 expression was positively related to the lymphatic metastasis (p=0.049). While the level of tumour-infiltrating CD8 T+ cells in tumour tissues was negatively associated with serum AFP (p=0.019), tumor size (p=0.028), and lymphatic metastasis (p=0.039). Additionally, patients with elevated CD73 expression or low tumour-infiltrating CD8+ T cells exhibited shorter overall survival (OS) and higher disease-free survival (DFS) rates than patients with low CD73 expression and/or high tumour-infiltrating CD8+ T cells. Notably, the overexpression of CD73 or low tumour-infiltrating CD8+ T cells was an independent indicator for predicting the OS and DFS of ICC patients. Conclusions: We revealed that CD73 expression and low tumour-infiltrating CD8+T cells are valuable predictors of survival and recurrence in patients with ICC.

A propensity-matched analysis of stereotactic body radiotherapy and sublobar resection for stage I non-small cell lung cancer in patients at high risk for lobectomy: the results in a Chinese population.

BACKGROUND: To investigate the comparative effectiveness of stereotactic body radiotherapy (SBRT) and sublobar resection (SLR) in patients with stage I non-small cell lung cancer (NSCLC) considered to be high-risk lobectomy patients. METHODS: From January 2012 to December 2015, patients who underwent SBRT or SLR for clinical stage I NSCLC were examined retrospectively. Propensity score matching (PSM) was performed to reduce selection bias in SBRT and SLR patients. RESULTS: Data from 86 SBRT and 79 SLR patients was collected. Median follow-up periods of the SBRT and SLR groups were 32 and 37 months, respectively. Patients treated with SBRT exhibited significantly higher age, higher likelihood of being male, larger tumor diameter, lower forced expiratory volume in 1 second (FEV1), and poorer performance status compared with SLR patients. There were no significant differences between SBRT and SLR patients for 3-year overall survival (OS) (80.3% and 82.3%, P=0.405), cause-specific survival (CSS) (81.3% and 83.4%, P=0.383), and local control (LC) (89.7% and 86.0%, P=0.501). Forty-nine patients were identified from each group after performing PSM. After patients were matched for age, gender, performance status, tumor characteristics and pulmonary function, no significant differences were observed in 3-year OS (85.4% and 73.3%, P=0.649), CSS (87.2% and 74.9%, P=0.637) and LC (95.6% and 82.1%, P=0.055). Prevalence of significant adverse events (grade 3 or worse) was 0% and 10.2% in the matched SBRT and SLR groups (P=0.056), respectively. CONCLUSIONS: Disease control and survival in the SBRT patients was equivalent to that seen in SLR patients with stage I NSCLC considered high-risk lobectomy candidates. SBRT could therefore be an alternative option to SLR in treating patients with a high operative risk.

Functional differences in the products of two TRAF3 genes in antiviral responses in the Chinese giant salamander, Andrias davidianus.

Tumour necrosis factor receptor associated factor 3 (TRAF3) is a crucial transducing protein for linking upstream receptor signals and downstream antiviral signalling pathways. Previous studies mostly clarified the functions of TRAF3 in mammals, birds and fish, but little is known about the characterization and function of TRAF3 in amphibians. In this study, the molecular and functional identification of two TRAF3 genes, AdTRAF3A and AdTRAF3B, were investigated in the Chinese giant salamander Andrias davidianus. The complete open reading frames (ORFs) of AdTRAF3A and AdTRAF3B were 1698 bp and 1743 bp in length, encoding 565 and 580 amino acids, respectively. Both AdTRAF3A and AdTRAF3B deduced proteins contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Phylogenetic analysis showed that the AdTRAF3 protein clustered together with other known TRAF3 proteins. Gene expression analysis showed that AdTRAF3s were broadly distributed in all examined tissues with similar distribution patterns. AdTRAF3s in the blood or spleen positively responded to Giant salamander iridovirus (GSIV) and poly (I:C) induction but exhibited distinct response patterns. Silencing AdTRAF3A/B remarkably suppressed the expression of IFN signalling pathway-related genes when leukocytes were treated with DNA virus and the viral RNA analogue. Moreover, overexpression of AdTRAF3A may induce the activation of the IFN-ß promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-ß promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-ß promoter activity, and its inhibitory effect was enhanced when the RING finger or MATH domain was deleted. Furthermore, AdTRAF3A rather than AdTRAF3B significantly induced NF-κB activation, implying that AdTRAF3A may function as an enhancer in both the IFN and NF-κB signalling pathways. Taken together, our results suggest that the two TRAF3 genes play different crucial regulatory roles in innate antiviral immunity in Chinese giant salamanders.

Oct4 Regulates the Transition of Cancer Stem-Like Cells to Tumor Endothelial-Like Cells in Human Liver Cancer.

Octamer-binding transcription factor 4 (Oct4) has been recently implicated as a proangiogenic regulator in several induced pluripotent stem cells (iPSCs), however, its role in cancer stem-like cells (CSCs) remain unclear. We report here that Oct4 participates in tumor vasculogenesis in liver CSCs (LCSCs). We identify that LCSCs possess the potential of endothelial trans-differentiation under endothelial induction, present endothelial specific markers and their functions in vitro, and participate in neovasculogenesis in vivo. The knockdown of the Oct4A by short hairpin RNA (shRNA) in LCSCs represses endothelial trans-differentiation potential, but induces endothelial lineage-restricted differentiation, the latter is positively regulated by Oct4B1. Furthermore, Oct4 regulates vasculogenesis in LCSCs may be via the AKT-NF-κB-p65 signaling pathway. This work reveals Oct4, which is a crucial regulator, plays a critical role in tumor endothelial-like cells transition of LCSCs through Oct4A and Oct4B1 by different ways. The simultaneous inhibition of both the isoforms of Oct4 is hence expected to help regress neovascularization derived from CSCs. Our findings may provide insights to the possible new mechanisms of tumor vasculogenesis for primary liver cancer.

Identification and functional analysis of transforming growth factor-ß type III receptor (TßR3) from Scylla paramamosain: The first evidence of TßR3 involved in development and innate immunity in invertebrates.

Transforming growth factor-ß type III receptor (TßR3), as a co-receptor of TGF-ß superfamily, plays critical roles in development and growth as well as some disease pathogeneses by presenting ligands to other receptors in vertebrates. However, the identification and functional characterization of TßR3 had not been reported yet in invertebrates. In the present study, TßR3 was first identified and characterized in mud crab Scylla paramamosain. The obtained cDNA length of SpTßR3 was 2, 424 bp with a 1, 854 bp open reading frame, which encoded a putative peptide of 617 amino acids containing a typical transmembrane region and a Zona pellucida (ZP) domain. Real-time PCR results showed that SpTßR3 was predominantly expressed at early embryonic development stage and early postmolt stage, suggesting its participation in development and growth. We report, for the first time in invertebrates, the challenge of both Vibro alginolyticus and Poly (I:C) could alter the expression patterns of SpTßR3. Notably, the expression levels of SpIKK, two NF-κB members (SpRelish and SpDorsal), and five antimicrobial peptide genes (SpCrustin and SpALF1-4) were significantly suppressed when SpTßR3 was interfered in vivo. Secondly, the overexpression of SpTßR3 in vitro could activate NF-κB signaling through the dual-luciferase reporter assays. Furthermore, the bacterial clearance assay after SpTßR3 was silenced in vivo highlighted the potential of SpTßR3 in activating the innate immune responses. These results implied the involvement of SpTßR3 in the innate immune responses by regulating the NF-κB pathway. This study first indicated that TßR3 was present in invertebrate, and it participated in not only the development and growth but also the innate immunity of S. paramamosain. It also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.

Identification and functional analysis of two interferon regulatory factor 3 genes and their involvement in antiviral immune responses in the Chinese giant salamander Andrias davidianus.

Interferon regulatory factor 3 (IRF3), a crucial member of interferon regulatory factor (IRF) family, plays an important role in innate immunity in vertebrates. However, there are no reports on the characterization and especially their respective functional analysis of two IRF3 genes in some species. In this study, two IRF3 genes as well as their roles in the immune response were identified and investigated in Chinese giant salamander, Andrias davidianus. The complete open reading frames of AdIRF3A and AdIRF3B were 1, 113 bp and 1, 380 bp in length, encoding 370 and 459 amino acids, respectively. Both AdIRF3A and AdIRF3B protein contain an IRF and an IRF3 domain. Phylogenetic analysis indicated that AdIRF3s clustered together with other IRF3 proteins. Tissue distribution analysis showed that AdIRF3s were expressed in all tissues tested, with highest expression levels in blood. Both AdIRF3s actively responded to Chinese giant salamander iridovirus (GSIV) and poly (I:C) challenge in A. davidianus. AdIRF3A/B silencing significantly suppressed the DNA virus and viral RNA analog-induced expression of IFN-inducible genes. Luciferase reporter assay further confirmed the regulatory role of AdIRF3s in IFN signaling. These results provide new insights into the origin or evolution of IRF3 in amphibians and even in vertebrates.

The first evidence of four transcripts from two Interleukin 18 genes in animal and their involvement in immune responses in the largest amphibian Andrias davidianus.

Interleukin 18 (IL-18), a member of IL-1 cytokine superfamily, is an important proinflammatory cytokine with multiple functions in both innate immunity and acquired immunity. However, the characteristics and functional roles of IL-18 remain largely unknown in amphibians, which were classed as major group of vertebrates. In the present study, two IL-18 genes (AdIL-18A and AdIL-18B) and four transcripts (AdIL-18A1, AdIL-18A2, AdIL-18B1 and AdIL-18B2) were firstly identified and characterized from Chinese giant salamander (Andrias davidianus). To the best of our knowledge, this is the first report on the presence of more than one gene copy or two transcripts of IL-18 in one species. The complete open reading frames of AdIL-18A1, AdIL-18A2, AdIL-18B1 and AdIL-18B2 were 588 bp, 603 bp, 591 bp and 606 bp, respectively. The putative AdIL-18 proteins possessed the typical IL-1 domains and phylogenetic analysis indicated that AdIL-18s grouped together with other vertebrate IL-18 proteins. The expression profiles of AdIL-18s were investigated under the challenges of Aeromonas hydrophila, Staphylococcus ureae and Poly (I:C) respectively, and the results suggested that AdIL-18s were involved in the immune responses against both bacterial and viral infections. Moreover, the expression levels of two NF-κBs (P100 and P105) and four proinflammatory cytokines (IL-1ß, IL-6, TNF-α and IFN-γ) were inhibited in AdIL-18A1/A2-silenced cells when treated with bacteria and viral RNA analog. Additionally, the transcription levels of these immune-related cytokine genes were markedly induced when the lymphocytes were treated with recombinant AdIL-18A1 or AdIL-18A2 proteins, implying the involvement of AdIL-18s in triggering NF-κB signaling and proinflammatory responses. These results might provide new insights into the origin or evolution of IL-18 in amphibians and even in vertebrates.

Analysis of multidrug resistance in Streptococcus suis ATCC 700794 under tylosin stress.

Streptococcus suis is an important zoonotic pathogen. The massive use of tylosin and other antibiotics in swine production has led to the emergence of resistant phenotypes of S. suis. However, there are no adequate measures available to address the problem of bacterial resistance. This study involved the use of 1/4 MIC (0.125 µg/mL) of tylosin to investigate resistance-related proteins by S. suis ATCC 700794. Our results showed that 171 proteins were differentially expressed in S. suis tested with 1/4 MIC (0.125 µg/mL) of tylosin using iTRAQ-based quantitative proteomic methods. TCS, heat shock protein and elongation factors were differentially expressed at 1/4 MIC (0.125 µg/mL) of tylosin compared to non treated, control cells. Using quantitative RT-PCR analysis, we verified the relationship between the differentially expressed proteins in S. suis with different MIC values. The data showed that expression profile for elongation factor G (fusA), elongation factor Ts (tsf), elongation factor Tu (tuf), putative histidine kinase of the competence regulon, ComD (comD), putative competence-damage inducible protein (cinA) and protein GrpE (grpE), observed in tylosin-resistant S. suis, correlated with that of S. suis ATCC 700794 at 1/4 MIC (0.125 µg/mL). The MIC of tylosin-resistant showed high-level resistance in terramycin, chlortetracycline, ofloxacin and enrofloxacin. Our findings demonstrated the importance of elongation factors, TCS and heat shock protein during development of tylosin resistance in S. suis. Thus, our study will provide insight into new drug targets and help reduce bacterial multidrug resistance through development of corresponding inhibitors.

The Activin-like ligand Dawdle regulates innate immune responses through modulating NF-κB signaling in mud crab Scylla paramamosain.

Activins, members of transforming growth factor ß (TGF-ß) superfamily, are pleiotropic cytokines with critical roles in mediating cell proliferation, differentiation, homeostasis, apoptosis and immune response. However, the structural characteristics and specific functions of Activins remain largely unknown in invertebrates. In the present study, an Activin-like ligand Dawdle (Daw) was firstly identified and characterized from mud crab Scylla paramamosain. The obtained cDNA sequence of SpDaw was 2, 196 bp long with a 1, 149 bp open reading fame, which encoded a putative protein of 382 amino acids. The putative SpDaw protein contained a signal peptide, a TGF-ß propeptide region and a TGF-ß domain. Real-time PCR analysis demonstrated that SpDaw was predominantly expressed at early embryonic development stage and premolt stages, implying its participation in development and growth. Furthermore, SpDaw responded to both Vibro alginolyticus and Poly (I:C) challenges, suggesting the involvement of SpDaw in innate immune responses. Knockdown of SpDaw in vivo dramatically increased the expressions of NF-κB signaling genes and anti-lipopolysaccharide factor (ALF) genes, and the bacteria clearance efficiency was also markedly enhanced in SpDaw-silenced crabs. Moreover, the in vitro experiment further demonstrated that recombinant SpDaw protein could block the increased transcription of IKKs, NF-κBs and ALFs induced by pathogen challenges. Taken together, these results indicated that SpDaw not only participated in development and growth processes but also played an immune-regulatory role in crabs' innate immunity, which may pave the way for a better understanding of TGF-ß superfamily members in crustacean species.

Transcriptomic model-based lncRNAs and mRNAs serve as independent prognostic indicators in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSC) is one of most common types of cancer worldwide, and mRNAs and long non-coding RNAs (lncRNAs) have been identified as prognostic biomarkers in HNSC. In the present study, using gene expression datasets from multiple platforms, survival-associated genes in HNSC were identified. Subsequently, a combination of 17 genes (14 mRNAs and 3 lncRNA) was optimized using random forest variable hunting and a risk score model for HNSC prognosis was developed using a cohort from The Cancer Genome Atlas. Patients with high-risk scores tend to have earlier disease recurrence and lower survival rates, compared with those with low-risk scores. This observation was further validated in three independent datasets (GSE41613, GSE10300 and E-MTAB-302). Association analysis revealed that the risk score is independent of other clinicopathological observations. On the basis of the results depicted in the nomogram, the risk score performs better in 3-year survival rate prediction than other clinical observations. In summary, the lncRNA-mRNA signature-based risk score successfully predicts the survival of HNSC and serves as an indicator of prognosis.