RNA-transfected Cells Research Articles

IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi.

Aichi virus (AiV), a small non-enveloped RNA virus, hijacks the endoplasmic reticulum (ER)-Golgi cholesterol transport machinery to form cholesterol-rich replication sites originating from Golgi membranes. Interferon-induced transmembrane proteins (IFITMs) are antiviral restriction factors, whose involvement in intracellular cholesterol transport is suggested. Here, we describe the roles of IFITM1 in cholesterol transport that affect AiV RNA replication. IFITM1 stimulated AiV RNA replication and its knockdown significantly reduced the replication. In replicon RNA-transfected or infected cells, endogenous IFITM1 localized to the viral RNA replication sites. Further, IFITM1 interacted with viral proteins and host Golgi proteins, ACBD3, PI4KB, OSBP, which constitute the replication sites. When overexpressed, IFITM1 localized to the Golgi as well as endosomes, and this phenotype was also observed for endogenous IFITM1 early in AiV RNA replication, leading to the distribution of cholesterol at the Golgi-derived replication sites. The pharmacological inhibition of ER-Golgi cholesterol transport or endosomal cholesterol export impaired AiV RNA replication and cholesterol accumulation at the replication sites. Such defects were corrected by expression of IFITM1. Overexpressed IFITM1 facilitated late endosome-Golgi cholesterol transport without any viral proteins. In summary, we propose a model in which IFITM1 enhances cholesterol transport to the Golgi to accumulate cholesterol at Golgi-derived replication sites, providing a novel mechanism by which IFITM1 enables efficient genome replication of non-enveloped RNA virus.

Associations between isoflavone exposure and reproductive damage in adult males: evidence from human and model system studies†.

It is controversial whether exposure to isoflavones exerts male reproductive toxicity. The aim of this study was to investigate whether isoflavone exposure during adulthood could have deleterious impacts on male reproductive health by the cross-sectional study, animal experiments, and in vitro tests. In the cross-sectional study, we observed that urinary isoflavones were not significantly associated with semen quality including sperm concentrations, sperm count, progressive motility, and total motility, respectively. However, negative associations were found between plasma testosterone and urinary Σisoflavones, genistein, glycitein, and dihydrodaidzein. In the animal experiments, serum and intratesticular testosterone levels were decreased in mice exposed to several dosages of genistein. Genistein administration caused upregulation of estrogen receptor alpha and downregulation of cytochrome P45017A1 protein levels in testes of mice. In vitro tests showed that genistein caused a concentration-dependent inhibition of testosterone production by TM3 Leydig cells. Elevated protein expression of estrogen receptor alpha and decreased messenger RNA/protein level of cytochrome P45017A1 were also observed in genistein-treated cells. Protein level of cytochrome P45017A1 and testosterone concentration were significantly restored in the estrogen receptor alpha small interferring RNA-transfected cells, compared to cells that treated with genistein alone. The results demonstrate that exposure to isoflavones during adulthood may be associated with alterations of reproductive hormones. Particularly, genistein, which inhibits testosterone biosynthesis through upregulation of estrogen receptor alpha in Leydig cells of mice, might induce the disruption of testosterone production in human. The present study provides novel perspective into potential targets for male reproductive compromise induced by isoflavone exposure.

Characterization of a Human Sapovirus Genotype GII.3 Strain Generated by a Reverse Genetics System: VP2 Is a Minor Structural Protein of the Virion.

We devised a reverse genetics system to generate an infectious human sapovirus (HuSaV) GII.3 virus. Capped/uncapped full-length RNAs derived from HuSaV GII.3 AK11 strain generated by in vitro transcription were used to transfect HuTu80 human duodenum carcinoma cells; infectious viruses were recovered from the capped RNA-transfected cells and passaged in the cells. Genome-wide analyses indicated no nucleotide sequence change in the virus genomes in the cell-culture supernatants recovered from the transfection or those from the subsequent infection. No virus growth was detected in the uncapped RNA-transfected cells, suggesting that the 5′-cap structure is essential for the virus’ generation and replication. Two types of virus particles were purified from the cell-culture supernatant. The complete particles were 39.2-nm-dia., at 1.350 g/cm3 density; the empty particles were 42.2-nm-dia. at 1.286 g/cm3. Two proteins (58-kDa p58 and 17-kDa p17) were detected from the purified particles; their molecular weight were similar to those of VP1 (~60-kDa) and VP2 (~16-kDa) of AK11 strain deduced from their amino acids (aa) sequences. Protein p58 interacted with HuSaV GII.3-VP1-specific antiserum, suggesting that p58 is HuSaV VP1. A total of 94 (57%) aa of p17 were identified by mass spectrometry; the sequences were identical to those of VP2, indicating that the p17 is the VP2 of AK11. Our new method produced infectious HuSaVs and demonstrated that VP2 is the minor protein of the virion, suggested to be involved in the HuSaV assembly.

HMGB1 enhances chemotherapy resistance in multiple myeloma cells by activating the nuclear factor-κB pathway.

Chemotherapy resistance is a main obstacle in the clinical chemotherapeutic treatment of multiple myeloma (MM). High-mobility group box 1 (HMGB1) has been revealed to be associated with the sensitivity of MM cells to chemotherapy, but how HMGB1 regulates chemotherapy resistance in MM has yet to be fully elucidated. In the present study, the exact molecular mechanism underlying HMGB1-mediated drug resistance in MM was explored using three chemotherapy-resistant MM cells (RPMI8226/ADR, RPMI8226/BOR and RPMI8226/DEX) that were successfully established. Reverse transcription-quantitative polymerase chain reaction revealed that the three chemotherapy-resistant MM cells exhibited a higher release of HMGB1 compared with the parental RPMI8226 cells. Interference with endogenous HMGB1 increased the sensitivity of drug-resistant MM cells to chemotherapy, which was supported by the low IC50 value and the enlargement of cell apoptosis. Furthermore, short hairpin (sh)RNA-transfected MM cells showed an obvious elevation in phosphorylated (p)-IKKα/β, p-IκBα and p-p65 in whole cell lysate and/or nucleus, and treatment of nuclear factor (NF)-κB activator reversed the effect of shHMGB1-mediated cell viability and apoptosis in MM cells. In conclusion, HMGB1 regulates drug resistance in MM cells by regulating NF-κB signaling pathway, suggesting that HMGB1 has the potential to serve as a target for MM treatment.

Phospholipase C Delta 3 inhibits apoptosis and promotes proliferation, migration, and invasion of thyroid cancer cells via Hippo pathway.

In recent decades, the incidence of thyroid cancer (TC) has rapidly increased, leading us to explore the complex underlying mechanisms. We identified the gene Phospholipase C Delta 3 (PLCD3) as a potential oncogene in TC by conducting the whole transcriptome sequencing. Our study is to understand the oncogenic role of PLCD3 in TC. We verified the overexpression of PLCD3 in TC from The Cancer Genome Atlas, Gene Expression Omnibus databases, and a locally validated cohort. Clinical correlation analysis showed that PLCD3 expression was related to histological type, T stage, lymph node metastasis (LNM), and disease stage. The high expression of PLCD3 could be a distinguishing factor for TC and its LNM. The biological function was examined using small interfering RNA-transfected TC cell lines. Silenced PLCD3 could inhibit colony formation, migration, and invasion ability and promote apoptosis of TC cell lines. PLCD3 silencing reversed the epithelial-mesenchymal transition but induced the apoptotic progress. Further exploration revealed that PLCD3 might be associated with critical genes of the Hippo pathway. The expressions of RHOA, YAP1/TAZ, and their downstream targets were decreased significantly when PLCD3 was down-regulated. YAP1 overexpression rescued the tumor-suppressive effect caused by PLCD3 silencing. This study demonstrates that PLCD3 is an oncogene that supports tumorigenesis and progression in TC, and PLCD3 may be a potential target gene for TC treatment.

Effects of Egr1 on pancreatic acinar intracellular trypsinogen activation and the associated ceRNA network.

Acute pancreatitis (AP) is a common digestive disorder with high morbidity and mortality. The present study aimed to investigate the expression of early growth response protein 1 (Egr1), and the effect of competing endogenous (ce)RNA network on trypsinogen activation. Pancreatic acinar intracellular trypsinogen activation (PAITA) is an important event in the early stage of AP; however, the underlying mechanisms remain unclear. The present study used taurolithocholic acid 3-sulfate (TLC-S)-treated AR42J cells (pancreatic cell line) to establish a PAITA model. A gene microarray and bioinformatics analysis was performed to identify the potential key targets in PAITA. The results demonstrated that Egr1, an important transcription factor, was significantly overexpressed in PAITA. In Egr1 small interfering (si)RNA-transfected cells, Egr1 expression was decreased and trypsinogen activation was significantly decreased compared with negative control siRNA-transfected cells, indicating that in TLC-S-induced PAITA, overexpression of Egr1 enhanced trypsinogen activation. A ceRNA network [mRNA-microRNA (miRNA/miR)-long non-coding (lnc)RNA] generated using the PAITA model revealed that the effects of Egr1 on PAITA may be regulated by multiple ceRNA pairs, and the lncRNAs (including NONRATT022624 and NONRATT031002) and miRNAs [including Rattus norvegicus (rno)-miR-214-3p and rno-miR-764-5p] included in the ceRNA pairs may serve roles in PAITA by regulating the expression of Egr1. The results of the present study may provide novel targets for researching the underlying mechanisms of, and developing treatments for AP.

Knockdown of Mg2+/Mn2+ dependent protein phosphatase 1A promotes apoptosis in BV2 cells infected with Brucella suis strain 2 vaccine.

The ability to inhibit host macrophage apoptosis is one of the survival strategies of intracellular bacteria, including Brucella. In the present study the role of Mg2+/Mn2+ dependent protein phosphatase 1A (PPM1A) in the apoptosis of Brucella suis (B. suis) strain 2 vaccine-infected BV2 cells was investigated. Compared with control cells, the protein expression levels of cleaved caspase-3 were markedly increased in PPM1A short hairpin (sh)RNA-transfected BV2 cells. Flow cytometry analysis showed that treatment with JNK activator anisomycin significantly increased the rate of apoptosis in BV2 cells in comparison with the control cells. Furthermore, PPM1A shRNA significantly increased the levels of JNK phosphorylation and the levels of cleaved caspase-3 in BV2 cells infected with B. suis strain 2 in comparison with the control cells. DAPI staining showed nuclear condensation in B. suis infected BV2 cells transfected with PPM1A shRNA in comparison with the control shRNA cells. Flow cytometry analysis showed that PPM1A shRNA significantly increased the percentage of apoptotic BV2 cells infected with B. suis strain 2 compared with those transfected with control shRNA. Taken together, these data suggested that knockdown of PPM1A promotes apoptosis in B. suis strain 2-infected BV2 cells and that PPM1A may be a potential target in the development of treatments to inhibit intracellular growth of B. suis.

Aligned Nanofibrillar Scaffolds as a Platform for the Treatment of Volumetric Muscle Loss Injury

Volumetric muscle loss (VML) injury is the traumatic loss of skeletal muscle fibers, along with associated vascular and neural structures, resulting in long‐term functional disability. To date, several strategies have been developed to target skeletal muscle and vascular regeneration and recovery of muscle function, with only limited improvements reported. Our objective, using a standardized unilateral VML injury to the rat tibialis anterior (TA) muscle, was two‐fold: (1) examine the rate of cell population and distribution of cell phenotypes in a bioengineered aligned collagen scaffold; and (2) evaluate the in vivo transfection efficiency of aligned collagen scaffolds loaded with firefly luciferase (Fluc) using bioluminescence imaging. We hypothesize that a modified mRNA (mmRNA)‐incorporated aligned collagen scaffold could be an ideal platform to stimulate vascular and muscle fiber regeneration following VML injury. In total, 25 adult male Lewis rats were used across studies. In Study 1, following unilateral VML injury to the TA muscle (~82mg removed), rats received implantation of an aligned collagen scaffold (6mm in diameter, 0.5–3.0mm thickness), consisting of two interconnected bundles of BioBridge threads that were cross‐linked, lyophilized, and sterilized. The TA muscle was evaluated histologically at 1, 2, and 4 weeks post‐VML to examine cell population and distribution, fibrotic content, and extent of scaffold degradation within the remaining TA muscle and VML defect area. In Study 2, in vivo bioluminescence imaging quantified localized RNA‐transfected cell expression in rats up to 2 weeks post‐Fluc injection within the TA muscle, with or without VML injury, or implantation of an aligned collagen scaffold loaded with Fluc‐RNA following VML injury to evaluate transfection efficiency for potential mmRNA cargo. Scaffolds exhibited minimal degradation through 4 weeks following implantation. Quantification of cellular distributions within the VML defect area indicated significant macrophage infiltration and inflammation up to 4 weeks post‐VML. Intramuscular Fluc injections indicated peak bioluminescence signal (3.0x105 p/s/cm2/sr) occurs at 7 days post‐injection. These results serve as a reference for our ongoing development of a mmRNA‐loaded scaffold delivery system. Our long‐term goal is to evaluate candidate growth factors to co‐deliver in the mmRNA‐loaded aligned collagen scaffolds to facilitate organized, functional skeletal muscle and vascular regeneration following VML injury.Support or Funding InformationThis work is supported by: US Army Medical Research and Materiel Command under Contract Number W81XWH19C0021; T32AR050938; P217A170051.

Therapeutic Cancer Vaccination with Ex Vivo RNA-Transfected Dendritic Cells-An Update.

Over the last two decades, dendritic cell (DC) vaccination has been studied extensively as active immunotherapy in cancer treatment and has been proven safe in all clinical trials both with respect to short and long-term side effects. For antigen-loading of dendritic cells (DCs) one method is to introduce mRNA coding for the desired antigens. To target the whole antigenic repertoire of a tumor, even the total tumor mRNA of a macrodissected biopsy sample can be used. To date, reports have been published on a total of 781 patients suffering from different tumor entities and HIV-infection, who have been treated with DCs loaded with mRNA. The majority of those were melanoma patients, followed by HIV-infected patients, but leukemias, brain tumors, prostate cancer, renal cell carcinomas, pancreatic cancers and several others have also been treated. Next to antigen-loading, mRNA-electroporation allows a purposeful manipulation of the DCs’ phenotype and function to enhance their immunogenicity. In this review, we intend to give a comprehensive summary of what has been published regarding clinical testing of ex vivo generated mRNA-transfected DCs, with respect to safety and risk/benefit evaluations, choice of tumor antigens and RNA-source, and the design of better DCs for vaccination by transfection of mRNA-encoded functional proteins.

Establishment of infectious genotype 4 cell culture-derived hepatitis C virus.

To establish infectious genotype 4a (GT4a) cell culture-derived hepatitis C virus (HCVcc), we constructed full-length ED43 and 12 mutants possessing single or double mutations that increase ED43 replicon replication, and performed cell culture after RNA transfection. Sequential long-term culture of full-length ED43 RNA-transfected cells showed increased viral production in two ED43 mutants named ED43 QK/SI and TR/SI among the tested clones. These ED43 mutants possessed a common mutation, R1405G, in the NS3 helicase region and another mutation, D2413G or V2414A, in the NS5a-NS5b cleavage site. Furthermore, serial reinfection of naïve Huh7.5.1 cells accelerated peak HCV production at an earlier time point after every infection. After the fourth infection, we found a common mutation, R1405G, and six additional mutations in both ED43 QK/SI and TR/SI mutants. All seven mutations supported continuous viral production for more than 40 days in both ED43 QS-7M (QK/SI with seven mutations) and ED43 TS-7M (TR/SI with seven mutations). In addition, ED43 TS-7M did not require additional mutations for continuous virus culture up to 124 days. Both ED43 QS-7M and TS-7M were sensitive to the neutralizing E2 antibodies HCV1 and AR3A and the direct-acting antivirals, simeprevir, ledipasvir and sofosbuvir. In conclusion, we established an infectious ED43 strain containing adaptive mutations, which is important for the analysis of HCV genotype-specific pathogenesis, development of pan-genotypic agents and analysis of drug resistance.

Aquaporin 4 regulation by ginsenoside Rb1 intervenes with oxygen-glucose deprivation/reoxygenation-induced astrocyte injury.

Background:Spinal cord ischemia-reperfusion injury (SCII) is a common complication of spinal surgery as well as thoracic and abdominal surgery. Acute cytotoxic edema is the key pathogenic alteration. Therefore, avoiding or decreasing cellular edema has become the major target for SCII treatment.Methods:The antiedema activity of ginsenoside Rb1 on aquaporin (AQP) 4, nerve growth factor (NGF), and brain-derived neurotrophic factor expression was detected by western blot and real-time polymerase chain reaction under conditions of oxygen-glucose deprivation/reoxygenation (OGD/R) in a rat astrocyte model in vitro. In addition, the cellular membrane permeability of AQP4 overexpressing cells or AQP4 small interfering RNA-transfected cells was detected.Results:Ginsenoside Rb1 significantly prevented OGD/R-induced AQP4 downregulation in rat astrocytes. In addition, ginsenoside Rb1 treatment or AQP4 overexpression in rat astrocytes significantly attenuated the OGD/R-induced increase of cellular membrane permeability. Moreover, ginsenoside Rb1 obviously prevented the OGD/R-induced decrease of NGF and BDNT expression in rat astrocytes.Conclusion:These findings demonstrate that ginsenoside Rb1 can relieve spinal cord edema and improve neurological function by increasing AQP4 expression.

The expression of microRNA-675 in gastric cancer and its effect on biological behavior

Objective To investigate the association between microRNA5 (miRNA, miR)-675 expression level in tumor tissues and adjacent tissues from patients with gastric cancer and prognosis. Meanwhile, to explore the influence of miR-675 in the gastric carcinoma (SGC-7901) cells. Methods The miRNA data of gastric cancer in TCGA databases were collected, and the survival time was analyzed according to the expression level of miR-675 in tumor tissues. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-675 in 30 gastric tumor tissues and adjacent non-tumor tissues. Cell counting assay kit-8 (CCK-8) was used to analyze the effect of miR-675 on the proliferation of SGC-7901. Flow cyometry was used to analyze the role of miR-675 in the apoptosis of SGC-7901. Results The expression levels of miR-675 in tumor tissues was significantly increased in the patients with gastric carcinoma form TCGA data and clinical samples (3.78±0.10 vs. 2.66±0.23, t=3.236, P<0.01), the difference was statistically significant. More importantly, the medial survival time was lower in the patients with high expression levels of miR-675 than that of lower expression of miR-675 group (675 d vs. 1 294 d, χ2=7.711, P<0.01), the difference was statistically significant. The results of CCK-8 proliferation assay showed that cell proliferation rate was reduced in miR-675 inhibitor transfected cells compared with the control and scrambled RNA-transfected cells (t=3.875, P<0.01), the difference was statistically significant. It was also found that the apoptosis of miR-675 inhibitor transfected cells was significantly increased (18.13±3.39)%. Conclusion MiR-675 may play an important role in the development of gastric cancer. Key words: Gastric cancer; MicroRNA-675; Prognosis

Expression of macrophage migration inhibitory factor in footpad skin lesions with diabetic neuropathy.

BackgroundDiabetic neuropathy originating in distal lower extremities is associated with pain early in the disease course, overwhelming in the feet. However, the pathogenesis of diabetic neuropathy remains unclear. Macrophage migration inhibitory factor has been implicated in the onset of neuropathic pain and the development of diabetes. Objective of this study was to observe pain syndromes elicited in the footpad of diabetic neuropathy rat model and to assess the contributory role of migration inhibitory factor in the pathogenesis of diabetic neuropathy.MethodsDiabetic neuropathy was made in Sprague Dawley rats by streptozotocin. Pain threshold was evaluated using von Frey monofilaments for 24 weeks. On comparable experiment time after streptozotocin injection, all footpads were prepared for following procedures; glutathione assay, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining, immunohistochemistry staining, real-time reverse transcription polymerase chain reaction, and Western blot. Additionally, human HaCaT skin keratinocytes were treated with methylglyoxal, transfected with migration inhibitory factor/control small interfering RNA, and prepared for real-time reverse transcription polymerase chain reaction and Western blot.ResultsAs compared to sham group, pain threshold was significantly reduced in diabetic neuropathy group, and glutathione was decreased in footpad skin, simultaneously, cell death was increased. Over-expression of migration inhibitory factor, accompanied by low expression of glyoxalase-I and intraepidermal nerve fibers, was shown on the footpad skin lesions of diabetic neuropathy. But, there was no significance in expression of neurotransmitters and inflammatory mediators such as transient receptor potential vanilloid 1, mas-related G protein coupled receptor D, nuclear factor kappa B, tumor necrosis factor-alpha, and interleukin-6 between diabetic neuropathy group and sham group. Intriguingly, small interfering RNA-transfected knockdown of the migration inhibitory factor gene in methylglyoxal-treated skin keratinocytes increased expression of glyoxalase-I and intraepidermal nerve fibers in comparison with control small interfering RNA-transfected cells, which was decreased by induction of methylglyoxal.ConclusionsOur findings suggest that migration inhibitory factor can aggravate diabetic neuropathy by suppressing glyoxalase-I and intraepidermal nerve fibers on the footpad skin lesions and provoke pain. Taken together, migration inhibitory factor might offer a pharmacological approach to alleviate pain syndromes in diabetic neuropathy.

Activation of hepatic Nogo-B receptor expression—A new anti-liver steatosis mechanism of statins

Deficiency of hepatic Nogo-B receptor (NgBR) expression activates liver X receptor α (LXRα) in an adenosine monophosphate-activated protein kinase α (AMPKα)-dependent manner, thereby inducing severe hepatic lipid accumulation and hypertriglyceridemia. Statins have been demonstrated non-cholesterol lowering effects including anti-nonalcoholic fatty liver disease (NAFLD). Herein, we investigated if the anti-NAFLD function of statins depends on activation of NgBR expression. In vivo, atorvastatin protected apoE deficient or NgBR floxed, but not hepatic NgBR deficient mice, against Western diet (WD)-increased triglyceride levels in liver and serum. In vitro, statins reduced lipid accumulation in nonsilencing small hairpin RNA-transfected (shNSi), but not in NgBR small hairpin RNA-transfected (shNgBRi) HepG2 cells. Inhibition of cellular lipid accumulation by atorvastatin is related to activation of AMPKα, and inactivation of LXRα and lipogenic genes. Statin also inhibited expression of oxysterol producing enzymes. Associated with changes of hepatic lipid levels by WD or atorvastatin, NgBR expression was inversely regulated. At cellular levels, statins increased NgBR mRNA and protein expression, and NgBR protein stability. In contrast to reduced cellular cholesterol levels by statin or β-cyclodextrin, increased cellular cholesterol levels decreased NgBR expression suggesting cholesterol or its synthesis intermediates inhibit NgBR expression. Indeed, mevalonate, geranylgeraniol or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate or farnesol, blocked atorvastatin-induced NgBR expression. Furthermore, we determined that induction of hepatic NgBR expression by atorvastatin mainly depended on inactivation of extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (Akt). Taken together, our study demonstrates that statins inhibit NAFLD mainly through activation of NgBR expression.

Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.

Endogenous circadian clocks regulate 24-h rhythms of physiology and behavior. Circadian rhythm disruption (CRD) is suggested as a risk factor for inflammatory bowel disease. However, the underlying molecular mechanisms remain unknown. Intestinal biopsies from Per1/2 mutant and wild-type (WT) mice were investigated by electron microscopy, immunohistochemistry, and bromodeoxyuridine pulse-chase experiments. TNF-α was injected intraperitoneally, with or without necrostatin-1, into Per1/2 mice or rhythmic and externally desynchronized WT mice to study intestinal epithelial cell death. Experimental chronic colitis was induced by oral administration of dextran sodium sulfate. In vitro, caspase activity was assayed in Per1/2-specific small interfering RNA-transfected cells. Wee1 was overexpressed to study antiapoptosis and the cell cycle. Genetic ablation of circadian clock function or environmental CRD in mice increased susceptibility to severe intestinal inflammation and epithelial dysregulation, accompanied by excessive necroptotic cell death and a reduced number of secretory epithelial cells. Receptor-interacting serine/threonine-protein kinase (RIP)-3-mediated intestinal necroptosis was linked to increased mitotic cell cycle arrest via Per1/2-controlled Wee1, resulting in increased antiapoptosis via cellular inhibitor of apoptosis-2. Together, our data suggest that circadian rhythm stability is pivotal for the maintenance of mucosal barrier function. CRD increases intestinal necroptosis, thus rendering the gut epithelium more susceptible to inflammatory processes.-Pagel, R., Bär, F., Schröder, T., Sünderhauf, A., Künstner, A., Ibrahim, S. M., Autenrieth, S. E., Kalies, K., König, P., Tsang, A. H., Bettenworth, D., Divanovic, S., Lehnert, H., Fellermann, K., Oster, H., Derer, S., Sina, C. Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.

The effect of microRNA-449 on the proliferation and apoptosis in papillary thyroid carcinoma

Objective To investigate the microRNA (miRNA, miR)-449 expression level and significance in papillary thyroid carcinoma (PTC). Meanwhile, to explore the influence of miR-449 in the thyroid papillary carcinoma TPC1 cell. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect the expression of miR-449 in papillary thyroid carcinoma tissues and adjacent non-tumor tissues. Cell counting assay kit-8 (CCK-8) was used to analyze the influence of miR-449 in the proliferation of TPC1. Flow cyometry was used to analyze the influence of miR-449 in the apoptosis of TPC1. Results The relative expression levels of miR-449 in PTC tissues (0.665 5±0.049 8) were significantly(P=0.002) lower than those in adjacent non-tumor tissues (1.405 0±0.079 4). More importantly, the expression levels of miR-449 were significantly associated with the higher clinical stage (P=0.036). The results of CCK-8 proliferation assay showed that cell proliferation rate was reduced in miR-449 transfected cells compared with the negative control (NC) and scrambled RNA-transfected cells (P=0.000). In the flow cytometry analysis, it was found that the apoptosis rate of miR-449 transfected cells [(15.13±0.52)%] was significantly increased (P=0.009). Conclusion MiR-449 function as a tumor suppressor miRNA in PTC, indicating that miR-449 could act as an important role in the molecular pathomechanism of PTC. Meanwhile, these results could provide some novel clues for the individual precision medicine. Key words: Papillary thyroid carcinoma; MicroRNA-449; Apoptosis

Amino Acid Mutations in the NS4A Region of Hepatitis C Virus Contribute to Viral Replication and Infectious Virus Production.

The HCV cell culture system using the JFH-1 strain and HuH-7 cells can be used to assess the complete HCV life cycle in cultured cells. This cell culture system has been used to develop direct-acting antivirals against HCV, and the ability to use various HCV strains within this system is important for future studies. In this study, we aimed to establish a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified an effective cell culture-adaptive mutation in NS4A and established a replication-competent J6CF strain in cultured cells with minimum essential modifications. The described strategy can be used in establishing a novel HCV cell culture system, and the replication-competent J6CF clone composed of the minimum essential modifications needed for cell culture adaptation will be valuable as another representative of genotype 2a strains.

Treatment of colon cancer cells with 5-fluorouracil can improve the effectiveness of RNA-transfected antitumor dendritic cell vaccine.

Non-cytotoxic concentrations of selected chemotherapeutic agents amplify the antigen presentation capacity of dendritic cells (DCs) and are able to increase the immunogenicity of the colon cancer cell lineage HCT‑116, as previously demonstrated by our group. Since this functional alteration was associated with changes in gene expression, we aimed to evaluate whether transcriptional changes of tumor cells can be transferred to DCs, increasing their ability to induce a specific antitumor response. Therefore, HCT‑116 cells were treated with two different concentrations of 5-fluorouracil (5-FU), and their total RNA was transfected into human monocyte-derived DC, which function was evaluated through their ability to stimulate the proliferation of normal allogeneic T lymphocytes (MLR), and to generate cytolytic Tcells. The transfected DCs demonstrated an increased percentage of CD83+, HLA-DR+, CD80+ and CD86+ cells. These phenotypical changes were followed by functional improvement demonstrated by the increased capacity of these DC to induce allogeneic Tcell proliferation and to generate specific anti-HCT‑116 cytolytic Tcells, as demonstrated by IFN-γ production following in vitro challenge with tumor cells. Our results allow us to conclude that treatment of tumor cells with a non-toxic concentration of 5-FU induces immunogenic changes that are transferred to DC by transfection of total RNA.

Porphyromonas gingivalis-induced IL-33 down-regulates hCAP-18/LL-37 production in human gingival epithelial cells.

hCAP-18/LL-37 is an antimicrobial peptide that is mainly expressed in epithelial cells. Gingival epithelial cells play pivotal roles in antimicrobial defense by expressing hCAP-18/LL-37. Porphyromonas gingivalis is a primary pathogen for chronic periodontitis and produces cysteine proteinase gingipains, which induce proinflammatory cytokines production, leading to enhance inflammatory responses. In contrast, gingipains attenuate immune responses, leading to induce anti-inflammatory responses. In this study, we investigated the ability of gingipains to attenuate P. gingivalis-induced hCAP-18/LL-37 production by human gingival epithelial Ca9-22 cells. The expression of LL-37 mRNA was increased by the infection of Ca9-22 cells with a P. gingivalis gingipains-null mutant KDP136 compared with P. gingivalis wild-type strain ATCC 33277. Interleukin (IL)-33 is involved in the development of chronic inflammatory diseases, and P. gingivalis infection increases IL-33 production by human gingival epithelial cells. P. gingivalis-induced LL-37 mRNA expression was augmented in IL-33 small interfering RNA-transfected Ca9-22 cells. Maxacalcitol (22-oxacalcitriol: OCT) is a biologically active metabolite of vitamin D3 analog, and OCT increases hCAP-18/LL-37 production by human gingival epithelial cells. The increasing expression of LL-37 mRNA by OCT was down-regulated by infection of the cells with P. gingivalis ATCC 33277 in Ca9-22 cells. Furthermore, P. gingivalis infection induced IL-33 mRNA expression in Ca9-22 cells; therefore, P. gingivalis-induced endogenous IL-33 down-regulated hCAP-18/LL-37 production by the bacterium. These findings suggested that endogenous IL-33 down-regulates the induction of hCAP-18/LL-37 production in human gingival epithelial cells.

URAT1 inhibition by ALPK1 is associated with uric acid homeostasis.

The aim of this study was to identify a protein for urate transporter 1 (URAT1) regulation. The clinical dataset consisted of 492 case-control samples of Han Chinese (104 gout and 388 controls). Three alpha kinase 1 ( ALPK1 ) and SLC22A12 loci associated with high gout risk and uric acid levels were genotyped. The overexpression of ALPK1 on URAT1 protein expression was evaluated in vivo in h ALPK1 transgenic mice. The in vitro protein levels of ALPK1 and URAT1 in ALPK1 small interfering RNA-transfected human kidney-2 cells with MSU crystal stimulation were examined. ALPK1 , which is a single nucleotide polymorphism (SNP) of rs11726117 (M861T; T), reduced the risk of gout via the SLC22A12 gene SNPs rs3825016 and rs475688, as compared with the subject of ALPK1 rs11726117 (C) allele {rs11726117 [CT + TT] vs rs3825016, odds ratio [OR] 0.39 [95% confidence interval (CI) 0.23, 0.67]; rs11726117 [CT + TT] vs rs475688, OR 0.39 [95% CI 0.23, 0.67]}. ALPK1-overexpressed mice demonstrated lower levels of URAT1 protein ( P = 0.0045). Mouse endogenous ALPK1 proteins were detected in renal proximal tubule cells. MSU crystals inhibited URAT1 expressions through an upregulation of ALPK1 in human kidney-2 cells. Elevated ALPK1 expression decreased URAT1 expression. ALPK1 might prevent the impact of urate reuptake via SLC22A12 and appeared to be negatively associated with gout. ALPK1 is a potential repressor of URAT1 protein expression.