Discovery and characterization of a novel insect-specific reovirus isolated from Psammotettix alienus.

A novel double-stranded RNA (dsRNA) virus designated Psammotettix alienus reovirus (PARV) was found in the leafhopper Psammotettix alienus in China. Spherical particles approximately 70 nm in diameter arranged in a crystalline array were observed in the salivary gland tissues of infected leafhoppers by transmission electron microscopy. Some viral particles were also encased in tubules, similar to those of previously described reoviruses. Whole-genome sequencing revealed that the dsRNA genome of PARV consists of 29 569 nucleotides (nt) divided into 10 segments ranging from 4403 to 1476 nt, with low G+C content (29.5-36.5 %). All segments contained conserved terminal sequences (5'AAC…GUCA3') and specific panhandle structures formed by inverted terminal repeats in the noncoding regions. Phylogenetic analysis based on the deduced RNA-dependent RNA polymerase (RdRp) revealed that PARV was in the fijivirus clade, but in a monophyletic lineage with an unassigned insect reovirus (Hubei insect virus 2, HBIV-2), although PARV and HBIV-2 are distinct enough to represent a new group within the genus Fijivirus. Biological assays showed that PARV infects P. alienus but not wheat plants, implying that it is a new insect-specific reovirus in the leafhopper. Given these features, PARV should be considered as a new species in the genus Fijivirus, family Reoviridae.

Transmission Characteristics of Wheat Yellow Striate Virus by its Leafhopper Vector Psammotettix alienus.

Wheat yellow striate virus (WYSV), which is found in wheat fields of Northwest China and transmitted by leafhopper vector Psammotettix alienus, is a tentative new species in the genus Nucleorhabdovirus. Although the insect vector and host range of WYSV have been characterized, many aspects of the acquisition and transmission processes by its insect vector have not been elucidated. Here, the transmission parameters of WYSV by P. alienus were determined using wheat cv. Yangmai 12 as the indicator plant under a controlled temperature (23 ± 1°C) and photoperiod (16 h of light). The results showed that the minimum periods for acquisition were 5 min and 10 min for inoculation access. The latent period for successful transmission was most commonly 16 to 20 days (minimum, 10 days; maximum, 22 days). The quantitative reverse-transcriptase PCR results indicated that the WYSV titer increased with time after acquisition, suggesting that WYSV can replicate in P. alienus. Notably, female P. alienus transovarially transmitted the virus to next generations at relatively high efficiency. Electron microscopy of the WYSV-infected leafhopper revealed bacilliform particles aggregated in the cytoplasm of the salivary gland and midgut tissues. Our present studies suggested that acquisition and transmission of WYSV by P. alienus is consistent with a propagative, circulative, and persistent mode of transmission. Details regarding transmission competencies and distribution of WYSV in P. alienus will provide a basis for designing preventive measures.

Incidence and Distribution of Insect-Transmitted Cereal Viruses in Wheat in China from 2007 to 2019.

Diseases caused by insect-transmitted viruses are the predominant constraint to wheat production worldwide. However, detailed knowledge of virus incidence and dynamics in China in recent years is very limited. Here, major wheat-growing regions of China were surveyed over 10 years for insect-transmitted viruses, and 2,143 samples were collected (in 2007 to 2015) and analyzed by molecular hybridization or multiplex reverse-transcription PCR for barley yellow dwarf viruses (BYDVs: BYDV-GAV, -GPV, and -PAV) and wheat dwarf virus (WDV). In a 4-year survey (2016 to 2019), the incidence of eight insect-transmitted viruses (BYDVs, WDV, wheat yellow striate virus [WYSV], barley yellow striate mosaic virus [BYSMV], northern cereal mosaic virus [NCMV], and rice black-streaked dwarf virus [RBSDV]) was investigated, and BYDVs and WDV were widely distributed across China. BYDV-GAV (29.0% of the tested sample) was the most abundant, followed by BYDV-PAV (23.2%) from 2007 to 2015. From 2016 to 2019, however, BYDV-PAV had become the predominant species (39.5% positive of 952 samples tested), while the incidence of BYDV-GAV (13.4%) had declined. During the entire survey, the incidence of BYDV-GPV was very low in some locations in northwestern and northern China, and all eight viruses caused only local epidemics, not large-scale outbreaks throughout China. Two new cereal-infecting rhabdoviruses, leafhopper-transmitted WYSV and planthopper-transmitted BYSMV, were also found in China in recent years.

A real-world disproportionality analysis of Everolimus: data mining of the public version of FDA adverse event reporting system.

Background: Everolimus is an inhibitor of the mammalian target of rapamycin and is used to treat various tumors. The presented study aimed to evaluate the Everolimus-associated adverse events (AEs) through data mining of the US Food and Drug Administration Adverse Event Reporting System (FAERS). Methods: The AE records were selected by searching the FDA Adverse Event Reporting System database from the first quarter of 2009 to the first quarter of 2022. Potential adverse event signals were mined using the disproportionality analysis, including reporting odds ratio the proportional reporting ratio the Bayesian confidence propagation neural network and the empirical Bayes geometric mean and MedDRA was used to systematically classify the results. Results: A total of 24,575 AE reports of Everolimus were obtained using data from the FAERS database, and Everolimus-induced AEs occurrence targeted 24 system organ classes after conforming to the four algorithms simultaneously. The common significant SOCs were identified, included benign, malignant and unspecified neoplasms, reproductive system and breast disorders, etc. The significant AEs were then mapped to preferred terms such as stomatitis, pneumonitis and impaired insulin secretion, which have emerged in the study usually reported in patients with Everolimus. Of note, unexpected significant AEs, including biliary ischaemia, angiofibroma, and tuberous sclerosis complex were uncovered in the label. Conclusion: This study provided novel insights into the monitoring, surveillance, and management of adverse drug reaction associated with Everolimus. The outcome of serious adverse events and the corresponding detection signals, as well as the unexpected significant adverse events signals are worthy of attention in order to improving clinical medication safety during treatment of Everolimus.

Construction of cytomegalovirus promoter-driven gene expression system in Laodelphax striatellus.

The small brown planthopper (SBPH, Laodelphax striatellus) is a significant rice pest, responsible for transmitting rice stripe virus (RSV) in a persistent and propagative manner. RSV is one of the most detrimental rice viruses, causing rice stripe disease, which results in considerable loss of rice grain yield. While RNA interference and gene knockout techniques have enabled gene downregulation in SBPH, no system currently exists for the overexpression of endogenous or exogenous genes. Consequently, the development of a protein expression system for SBPH is imperative to serve as a technical foundation for pest control and gene function investigations. This study aimed to construct an expression vector using the promoter of the constitutive-expressed tubulin gene of SBPH, and promoter of human cytomegalovirus (CMV). Fluorescence experiments demonstrated that both tubulin and CMV promoter could drive green fluorescent protein (GFP) expression in SBPH, and could also facilitate the expression of a nucleocapsid protein (NP) -GFP fusion protein containing viral NP with comparable efficiency. Through expression vector optimization, we have identified that the 3 tandem CMV promoters display a significantly higher promoter activity compared with both the 2 tandem CMV promoters and the single CMV promoter. In addition, the incorporation of Star polycation nanoparticles significantly enhanced the expression efficiency in SBPH. These results provide a promising technical platform for investigating gene functions in SBPH.

Association of virome dynamics with mosquito species and environmental factors.

BACKGROUND: The pathogenic viruses transmitted by mosquitoes cause a variety of animal and human diseases and public health concerns. Virome surveillance is important for the discovery, and control of mosquito-borne pathogenic viruses, as well as early warning systems. Virome composition in mosquitoes is affected by mosquito species, food source, and geographic region. However, the complex associations of virome composition remain largely unknown. RESULTS: Here, we profiled the high-depth RNA viromes of 15 species of field-caught adult mosquitoes, especially from Culex, Aedes, Anopheles, and Armigeres in Hainan Island from 2018 to 2020. We detected 57 known and 39 novel viruses belonging to 15 families. We established the associations of the RNA viruses with mosquito species and their foods, indicating the importance of feeding acquisition of RNA viruses in determining virome composition. A large fraction of RNA viruses were persistent in the same mosquito species across the 3 years and different locations, showing the species-specific stability of viromes in Hainan Island. In contrast, the virome compositions of single mosquito species in different geographic regions worldwide are visibly distinct. This is consistent with the differences in food sources of mosquitoes distributed broadly across continents. CONCLUSIONS: Thus, species-specific viromes in a relatively small area are limited by viral interspecific competition and food sources, whereas the viromes of mosquito species in large geographic regions may be governed by ecological interactions between mosquitoes and local environmental factors. Video Abstract.

[Study on effect of heavy metal cadmium ions on the ultramicrostructure damage of Pheretima aspergillum gastrointestinal epithelial cells].

OBJECTIVE: To study the effect of cadmium ions of different concentrations on gastrointestinal epithelial cells structure of Pheretima aspegillum (PA). METHODS: PA were contaminated with cadmium ions of different concentrations,and the structure of the body skin was observed, under light microscope and transmission electron microscope. RESULTS: With the increasing of cadmium ions concentration, a large number of lysosomal hyperplasia could be seen in the PA intestinal epithelial cells, the Golgi complex distributed around, and some Golgi complex hyperplasiaed, extended to a large bubble, microvilli, cilia arranged in irregular, disordered. While in the group contaminated with the high concentration cadmium ions, such as 30 mg/kg, the microvilli of the PA intestinal epithelial cells contracted, necrotic ulcer lesions occurred in the ciliated cells. CONCLUSION: The ultrastructure damage extent of PA gastrointestinal epithelial cells is dependent on the amount of the heavy metal contamination. PA with lower concentration Cd contamination shows mainly lysosomal proliferation, indicating heavy metal accumulation in lysosomes to eliminate toxic substances as a responsible reaction, this kind of damage is reversible. However, PA with higher concentration Cd contamination shows mainly microvilli and mitochondrial damage, nuclear membrane disintegration, nucleoplasm spillover, leading to necrosis, irreversible damage, indicating heavy metal accumulation of PA is related to this trait of intestinal epithelial cells.

Regulation of RNA Interference Pathways in the Insect Vector Laodelphax striatellus by Viral Proteins of Rice Stripe Virus.

RNA interference (RNAi), especially the small interfering RNA (siRNA) and microRNA (miRNA) pathways, plays an important role in defending against viruses in plants and insects. However, how insect-transmitted phytoviruses regulate the RNAi-mediated antiviral response in vector insects has barely been uncovered. In this study, we explored the interaction between rice stripe virus (RSV) and the miRNA and siRNA pathways of the small brown planthopper, which is a vector insect. The transcript and protein levels of key genes in the two RNAi pathways did not change during the RSV infection process. When the expression of insect Ago1, Ago2, or Translin was silenced by the injection of double-stranded RNAs targeting these genes, viral replication was promoted with Ago2 silencing but inhibited with Translin silencing. Protein-protein binding assays showed that viral NS2 and RNA-dependent RNA polymerase interacted with insect Ago2 and Translin, respectively. When NS2 was knocked down, the transcript level of Ago2 increased and viral replication was inhibited. Therefore, viral NS2 behaved like an siRNA suppressor in vector insects. This protein-binding regulation of insect RNAi systems reflects a complicated and diverse coevolution of viruses with their vector insects.

The Origin and Molecular Epidemiology of Dengue Fever in Hainan Province, China, 2019.

There was an outbreak of Dengue fever on September 5, 2019, in Hainan Province, which has not been endemic for 28 years. We aim to describe the clinical and epidemiological features of the 2019 outbreak in Hainan Province and identify the cause. All type 1 Dengue fever cases that occurred in this outbreak of Hainan exhibited mild clinical symptoms. The epidemiological investigations indicate that the outbreak might originate from workers in the Xiuying area, Haikou City, form a concentrated outbreak, and then spread out. Bayesian phylogenies results and epidemiological data were used to infer a likely series of events for the dengue virus's potential spread and trace the possible sources. The strains' sequences were close to a sequence from the nearby Guangdong province, supporting the hypothesis that the dengue virus was imported from Guangdong province and then spread across Hainan province. Furthermore, it is interesting that two other strains didn't group with this cluster, suggesting that additional introduction pathways might exist. The study indicated that the dengue fever epidemic presented two important modes in Hainan. Firstly, epidemics prevalence was caused by imported cases, and then endogenous epidemics broke out in the natural epidemic focus.

Serum hepatitis B viral (HBV) DNA is a predictive biomarker for survival in non-small cell lung cancer patients with chronic HBV infection.

Purpose: To study the association between pretreatment serum hepatitis B viral (HBV) DNA copy numbers and clinical outcome of non-small cell lung cancer (NSCLC) patients with chronic HBV infection. Patients and methods: We retrospectively evaluated the medical records of NSCLC HBV (+) patients between January 2008 and December 2010. The HBV DNA copy numbers and other prognostic factors including albumin (ALB), C-reactive protein (CRP), platelet, neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and Glasgow Prognostic Score (GPS) were obtained before any antitumor treatment. Kaplan-Meier curves and the log-rank test were used to calculate prognostic significance. A multivariable Cox proportional hazard regression was modeled to analyze the independent prognostic factors for NSCLC HBV (+) patients. All independent prognostic factors in the Cox multivariable analysis were used to build a nomogram. The predictive accuracy of HBV DNA, TNM stage and nomogram was evaluated with the concordance index (C-index) and time-dependent receiver operating characteristics (ROC) curves, and simultaneously compared with traditional TNM staging system respectively. Results: A total of 188 patients were recruited in this study; the median age was 56 years, and the median overall survival (OS) was 34 months. Cox multivariate analysis results showed independent factors for OS including TNM stage (P=0.028), treatment (P=0.002), HBV DNA (P<0.001), and GPS (P=0.026). The nomogram model for survival was built based on four prognostic factors. The C-index for HBV DNA was 0.67, 0.69 for TNM stage, and 0.76 for the nomogram model. There was no statistical difference between HBV DNA and TNM stage (P=0.48). However, the C-index values of nomogram model were statistically higher both than HBV DNA (0.76 vs 0.67, P<0.001), and TNM stage (0.76 vs 0.69, P<0.001). Conclusion: Pretreatment serum HBV DNA copy numbers can act as a prognostic marker of survival for NSCLC patients with chronic HBV infection.

miR-206 regulates alveolar type II epithelial cell Cx43 expression in sepsis-induced acute lung injury.

The present study investigated the relationship between connexin 43 (Cx43) expression in alveolar type II epithelial cells (ATII) and alveolar air-blood barrier permeability, and the effect of microRNA-206 (miR-206) on the expression of Cx43 in sepsis-induced acute lung injury. For the in vivo study, rats were divided into the sham, caecum ligation and perforation (CLP), CLP+Cx43 inhibitors (Cx43-In) and CLP+miR-206 analogs (miR-206-Mi) groups. CLP method was used to prepare an acute lung injury model of sepsis. Following successful modeling, lung tissue was collected for hematoxylin and eosin (HE) staining, and the wet to dry weight ratio (W/D) and protein content in bronchoalveolar lavage fluid (BALF) were detected. Cx43 expression in lung tissue was determined by immunohistochemistry and western blot analysis. Additionally, miR-206 and Cx43 expression levels in lung tissue were detected by reverse transcription-quantitative polymerase chain reaction. Rat ATII cells were cultured in Transwells plates to form monolayers, then treated with Cx43 mRNA inhibitors or miR-206 analogs. The cell monolayers were then stimulated with lipopolysaccharide and their permeability was evaluated by detecting fluorescein-labeled dextran at the lower chamber of the Transwells. The dual luciferase reporter gene assay was used to investigate whether miR-206 targeted the 3' untranslated region of Cx43 mRNA to regulate Cx43 expression, thereby regulating the permeability of the alveolar air-blood barrier. Results demonstrated that the CLP method induced damage to the alveolar structure, thickened the alveolar wall, caused hyperemia and hemorrhage in the pulmonary interstitium and caused infiltration of inflammatory cells. Edema in the pulmonary interstitium and alveolar space, exudation of neutrophilic granulocyte and pink edema fluid in alveolar cavities were also observed. W/D ratio, the BALF protein content, and expression of Cx43mRNA and Cx43 were increased significantly, whilst miR-206 expression decreased compared with the control group. The lung tissue inflammatory response was attenuated, and the W/D ratio and BALF protein content decreased in the Cx43-In and miR-206-Mi groups compared with the CLP group. Moreover, Cx43 mRNA and protein expression were decreased significantly in the Cx43-In and miR-206-Mi groups. In addition, the dual luciferase reporter gene assay determined that the untranslated region of Cx43 mRNA had a complementary sequence to miR-206. Of note, Cx43 mRNA expression in the miR-206-Mi group was not significantly decreased in vitro. In conclusion, the increase in ATII cell Cx43 expression during sepsis-induced acute lung injury resulted in an increase in the permeability of the alveolar air-blood barrier. miR-206 targeted the Cx43 mRNA 3'untranslated region to downregulate Cx43 expression, which further improved the permeability of the alveolar air-blood barrier.

Inhibition of histone lysine-specific demethylase 1 elicits breast tumor immunity and enhances antitumor efficacy of immune checkpoint blockade.

Immunotherapy strategies have been emerging as powerful weapons against cancer. Early clinical trials reveal that overall response to immunotherapy is low in breast cancer patients, suggesting that effective strategies to overcome resistance to immunotherapy are urgently needed. In this study, we investigated whether epigenetic reprograming by modulating histone methylation could enhance effector T lymphocyte trafficking and improve therapeutic efficacy of immune checkpoint blockade in breast cancer with focus on triple-negative breast cancer (TNBC) subtype. In silico analysis of The Cancer Genome Atlas (TCGA) data shows that expression of histone lysine-specific demethylase 1 (LSD1) is inversely associated with the levels of cytotoxic T cell-attracting chemokines (C-C motif chemokine ligand 5 (CCL5), C-X-C motif chemokine ligand 9 and 10 (CXCL9, CXCL10)) and programmed death-ligand 1 (PD-L1) in clinical TNBC specimens. Tiling chromatin immunoprecipitation study showed that re-expression of chemokines by LSD1 inhibition is associated with increased H3K4me2 levels at proximal promoter regions. Rescue experiments using concurrent treatment with small interfering RNA or inhibitor of chemokine receptors blocked LSD1 inhibitor-enhanced CD8+ T cell migration, indicating a critical role of key T cell chemokines in LSD1-mediated CD8+ lymphocyte trafficking to the tumor microenvironment. In mice bearing TNBC xenograft tumors, anti-PD-1 antibody alone failed to elicit obvious therapeutic effect. However, combining LSD1 inhibitors with PD-1 antibody significantly suppressed tumor growth and pulmonary metastasis, which was associated with reduced Ki-67 level and augmented CD8+ T cell infiltration in xenograft tumors. Overall, these results suggest that LSD1 inhibition may be an effective adjuvant treatment with immunotherapy as a novel management strategy for poorly immunogenic breast tumors.

Epigallocatechin-3-gallate inhibits growth of activated hepatic stellate cells by enhancing the capacity of glutathione synthesis.

Activation of hepatic stellate cells (HSC), the key effectors in hepatic fibrogenesis, is characterized by enhanced cell proliferation and overproduction of extracellular matrix. Oxidative stress promotes HSC activation. Glutathione (GSH) is the most important intracellular antioxidant, whose synthesis is mainly regulated by glutamate-cysteine ligase (GCL). We reported previously that (-)-epigallocatechin-3-gallate (EGCG), the major and most active component in green tea extracts, inhibited HSC activation. The aim of this study is to elucidate the underlying mechanisms. We hypothesize that this inhibitory effect of EGCG might mainly result from its antioxidant capability by increasing de novo synthesis of GSH. In this report, we observe that EGCG enhances the levels of cytoplasmic and mitochondrial GSH and increases GCL activity by inducing gene expression of the catalytic subunit GCLc, leading to de novo synthesis of GSH. Real-time polymerase chain reaction and Western blotting analyses show that de novo synthesis of GSH is required for EGCG to regulate the expression of genes relevant to apoptosis and to cell proliferation. Additional experiments demonstrate that exogenous transforming growth factor (TGF)-beta1 suppresses GCLc gene expression and reduces the level of GSH in cultured HSC. Transient transfection assays and Western blotting analyses further display that EGCG interrupts TGF-beta signaling by reducing gene expression of TGF-beta receptors and Smad4, leading to increased expression of GCLc. These results support our hypothesis and collectively demonstrate that EGCG increases the level of cellular GSH in HSC by stimulating gene expression of GCLc, leading to the inhibition of cell proliferation of activated HSC in vitro.

Curcumin protects the rat liver from CCl4-caused injury and fibrogenesis by attenuating oxidative stress and suppressing inflammation.

We previously demonstrated that curcumin, a polyphenolic antioxidant purified from turmeric, up-regulated peroxisome proliferator-activated receptor (PPAR)-gamma gene expression and stimulated its signaling, leading to the inhibition of activation of hepatic stellate cells (HSC) in vitro. The current study evaluates the in vivo role of curcumin in protecting the liver against injury and fibrogenesis caused by carbon tetrachloride (CCl(4)) in rats and further explores the underlying mechanisms. We hypothesize that curcumin might protect the liver from CCl(4)-caused injury and fibrogenesis by attenuating oxidative stress, suppressing inflammation, and inhibiting activation of HSC. This report demonstrates that curcumin significantly protects the liver from injury by reducing the activities of serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, and by improving the histological architecture of the liver. In addition, curcumin attenuates oxidative stress by increasing the content of hepatic glutathione, leading to the reduction in the level of lipid hydroperoxide. Curcumin dramatically suppresses inflammation by reducing levels of inflammatory cytokines, including interferon-gamma, tumor necrosis factor-alpha, and interleukin-6. Furthermore, curcumin inhibits HSC activation by elevating the level of PPARgamma and reducing the abundance of platelet-derived growth factor, transforming growth factor-beta, their receptors, and type I collagen. This study demonstrates that curcumin protects the rat liver from CCl(4)-caused injury and fibrogenesis by suppressing hepatic inflammation, attenuating hepatic oxidative stress and inhibiting HSC activation. These results confirm and extend our prior in vitro observations and provide novel insights into the mechanisms of curcumin in the protection of the liver. Our results suggest that curcumin might be a therapeutic antifibrotic agent for the treatment of hepatic fibrosis.

Iron chelators reduce chromosomal breaks in ataxia-telangiectasia cells.

Ataxia-telangiectasia (A-T) is characterized by ataxia, genomic instability, and increased cancer incidence. Previously, iron chelator concentrations which suppressed normal cell colony formation increased A-T cell colony formation. Similarly, iron chelators preferentially increased A-T cell colony formation following peroxide exposure compared to normal cells. Last, A-T cells exhibited increased short-term sensitivity to labile iron exposure compared to normal cells, an event corrected by recombinant ATM (rATM) expression. Since chromosomal damage is important in A-T pathology and iron chelators exert beneficial effects on A-T cells, we hypothesized that iron chelators would reduce A-T cell chromosomal breaks. We treated A-T, normal, and A-T cells expressing rATM with labile iron, iron chelators, antioxidants, and t-butyl hydroperoxide, and examined chromosomal breaks and ATM activation. Additionally, the effect of ATM-deficiency on transferrin receptor (TfR) expression and TfR activity blockage in A-T and syngeneic A-T cells expressing rATM was examined. We report that (1) iron chelators and iron-free media reduce spontaneous and t-butyl hydroperoxide-induced chromosomal breaks in A-T, but not normal, or A-T cells expressing rATM; (2) labile iron exposure induces A-T cell chromosomal breaks, an event lessened with rATM expression; (3) desferal, labile iron, and copper activate ATM; (4) A-T cell TfR expression is lowered with rATM expression and (5) blocking TfR activity with anti-TfR antibodies increases A-T cell colony formation, while lowering chromosomal breaks. ATM therefore functions in iron responses and the maintenance of genomic stability following labile iron exposure.

RNAi Knockdown of Ape1 Gene in the Differentiation of Mouse Embryonic Stem Cells.

Murine embryonic stem cells (ES) are pluripotent cells and have the potential to become a wide variety of specialized cell types. Mouse ES cell differentiation can be regarded as a valuable biological tool that has led to major advances in our understanding of cell and developmental biology. In vitro differentiation of mouse ES cells can be directed to a specific lineage formation, such as hematopoietic lineage, by appropriate cytokine and/or growth factor stimulation. To study specific gene function in early developmental events, gene knockout approaches have been traditionally used, however, this is a time-consuming and expensive approach. Recently, we have shown that siRNA is an effective strategy to knock down target gene expression, such as Ape1, during ES cell differentiation, and consequently, one can alter cell fates in ES-derived differentiated cells. This approach will be applicable to test the function of a wide variety of gene products using the ES cell differentiation system.

Paralemmin-3 contributes to lipopolysaccharide-induced inflammatory response and is involved in lipopolysaccharide-Toll-like receptor-4 signaling in alveolar macrophages.

Alveolar macrophages (AMs) are the first line of defense against foreign stimulation in alveoli, and they participate in inflammatory responses during acute lung injury (ALI). Previous studies indicated that paralemmin-3 (PALM3) expression is induced by lipopolysaccharides (LPS) and may be involved in LPS-Toll-like receptor 4 (TLR4) signaling in alveolar epithelial cells. The aim of the present study was to investigate the effect of PALM3 on LPS-induced inflammation and its underlying mechanisms in rat AMs. For this purpose, the authors detected the expression of PALM3 in AMs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting following LPS stimulation. Following this, a recombinant adenovirus expressing short hairpin RNA (shRNA) for PALM3 was constructed, as well as a recombinant adenovirus carrying the rat PALM3 gene to modulate the expression of PALM3 in rat AMs. At 48 h after transfection, the PALM3 expression in AMs was detected by RT-qPCR and western blotting. The levels of several cytokines and the activity of nuclear factor-κB and interferon regulatory factor 3 in AMs were measured after LPS stimulation. The localization of PALM3 and LPS-TLR4 signaling adaptor molecules in AMs was analyzed by confocal microscopy, and the physical interactions of PALM3 with these adaptors were assessed by co-immunoprecipitation assays. LPS induced PALM3 expression in AMs and that PALM3 expression promoted the LPS-induced inflammatory response, while PALM3 downregulation suppressed the LPS-induced inflammatory response in AMs. In addition, the results demonstrated that PALM3 could interact with TLR4, myeloid differentiation factor 88, interleukin (IL)-1 receptor associated kinase-1, tumor necrosis factor receptor associated factor-6, and Toll-IL-1 receptor containing adapter molecule-2 in AMs after LPS stimulation. These results suggested that PALM3 contributes to the LPS-induced inflammatory response and participates in LPS-TLR4 signaling in AMs. These data may provide the basis for the development of novel targeted therapeutic strategies of treating ALI.

Uterine sarcoma with ambiguous histomorphology: A case report.

BACKGROUND: Leiomyosarcomas (LMS) and endometrial stromal sarcomas (ESS) may display overlapping histomorphology, which may challenge diagnostic accuracy. Since LMS and ESS have vastly different clinical behavior and adjuvant therapy recommendations, accurate diagnosis is critical. CASE: We present the case of an 83-year-old female with postmenopausal bleeding who underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy for clinically atypical appearing leiomyomata. Histologically, dual populations of cells with morphologic features of low-grade ESS and high-grade spindle cell sarcoma were seen. Immunohistochemistry and molecular studies revealed the cells to be of smooth muscle derivation, rendering a diagnosis of high-grade LMS with heterogeneous morphology (stage IB). The patient received adjuvant gemcitabine plus docetaxel. She recurred 8 months after completion of chemotherapy and was transferred to hospice care. CONCLUSION: Ancillary studies, such as immunohistochemistry and molecular testing, aid in accurate subcategorization of uterine sarcomas with ambiguous histomorphology.

The phyto-chemical (-)-epigallocatechin gallate suppresses gene expression of epidermal growth factor receptor in rat hepatic stellate cells in vitro by reducing the activity of Egr-1.

Hepatic stellate cells (HSC) are the major effectors in hepatic fibrogenesis. During liver injury, HSC become activated and proliferative. Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) are the potent mitogens for many cell types. We previously demonstrated that (-)-epigallocatechin gallate (EGCG), the major and active component in green tea extracts, inhibited HSC growth, including reducing cell proliferation, and inducing apoptosis. We have reported that EGCG interrupts PDGF signaling by reducing receptor tyrosine phosphorylation and gene expression of PDGF-beta receptor. Additional experiments are necessary to elucidate the effect of EGCG on EGF signaling in activated HSC. The aims of this study are to evaluate the effect of EGCG on the expression of EGFR and to elucidate the underlying molecular mechanisms in activated HSC. We hypothesize that EGCG might interrupt EGF signaling by suppressing gene expression of EGF receptor (EGFR) in activated HSC, which, together with the interruption of PDGF signaling, might collectively result in the inhibition of HSC growth. The present report demonstrates that the phyto-chemical dose-dependently suppresses gene expression of EGFR in activated HSC in vitro. The Egr-1 binding site located in the egfr promoter is found to be cis-activating element in regulating the promoter activity of the gene. EGCG inhibits the trans-activation activity of Egr-1 in activated HSC by suppressing gene expression of the transcription factor. The interruption of the ERK signaling pathway by EGCG reduces the trans-activation activity of Egr-1 and the promoter activity of EGFR gene in HSC. Taken together, our results demonstrate that EGCG suppresses gene expression of EGFR in rat activated HSC in vitro mediated by reducing the trans-activation activity of Egr-1.

Designing DNA interstrand lock for locus-specific methylation detection in a nanopore.

DNA methylation is an important epigenetic regulation of gene transcription. Locus-specific DNA methylation can be used as biomarkers in various diseases including cancer. Many methods have been developed for genome-wide methylation analysis, but molecular diagnotics needs simple tools to determine methylation states at individual CpG sites in a gene fragment. In this report, we utilized the nanopore single-molecule sensor to investigate a base-pair specific metal ion/nucleic acids interaction, and explored its potential application in locus-specific DNA methylation analysis. We identified that divalent Mercury ion (Hg²âº) can selectively bind a uracil-thymine mismatch (U-T) in a dsDNA. The Hg²âº binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in a 5-methylcytosine-thymine mismatch (mC-T). By nanopore detection of dsDNA stability, single bases of uracil and 5-methylcytosine can be distinguished. Since uracil is converted from cytosine by bisulfite treatment, cytosine and 5'-methylcytosine can be discriminated. We have demonstrated the methylation analysis of multiple CpGs in a p16 gene CpG island. This single-molecule assay may have potential in detection of epigenetic cancer biomarkers in biofluids, with an ultimate goal for early diagnosis of cancer.