Evaluation of combined detection of multigene mutation and SDC2/SFRP2 methylation in stool specimens for colorectal cancer early diagnosis.

PURPOSE: Molecular diagnostics of colorectal cancer (CRC) can be used as an auxiliary approach for patients recommended for colonoscopy, providing more CRC supplemental diagnosis options. This study investigated whether combined detection of KRAS/BRAF/APC mutation and SDC2/SFRP2 methylation can serve as auxiliary diagnostics in clinical management. METHODS: KRAS/BRAF/APC mutation and SDC2/SFRP2 methylation in stool samples from healthy donors, patients with CRC, advanced adenoma (AA), non-advanced adenoma (NAA), or other gastroenterological diseases were evaluated using quantitative PCR (qPCR) or methylation-specific quantitative PCR (MSP). Test accuracy was determined by evaluating the tests' sensitivity, specificity, positive/negative predictive value (PPV/NPV), or positive/negative likelihood ratio (PLR/NLR). RESULTS: The combined fecal KRAS/BRAF/APC mutation and SFRP2/SDC2 methylation detection test achieved a sensitivity of 88.57% with a PPV of 93.64% and a PLR of 7.10 for CRC patients. In comparison, the corresponding parameters for multigene mutation were 46.67%, 92.59%, and 36.26 and 83.81%, 93.94%, and 7.47, for DNA methylation, separately. The sensitivity of the combined test, gene mutation test, and DNA methylation test approach was 75%, 28.26%, and 72.83%. Furthermore, the specificity of this approach in the NAA group was 79.49%. Meanwhile, the overall diagnostic specificity for the combined test in NAA, healthy control, and interference groups was 88.42%. In addition, the sensitivity of the combined detection method increased with the disease stage in CRC patients and elevated along with the lesion size (≥ 1 cm) in AA patients. CONCLUSION: Combined detection of fecal KRAS/BRAF/APC mutation and SFRP2/SDC2 methylation has potential application value for the auxiliary diagnosis of CRC and AA.

Evaluation of Combined use of Fecal Multigene Mutation Test and Fecal Immunochemical Test for Colorectal Cancer Screening.

BACKGROUND: The aim was to investigate the value of concomitant use of fecal KRAS-APC-p53-BRAF mutation test and a fecal immunochemical test (FIT) for colorectal cancer (CRC) screening. METHODS: Stool samples of 279 subjects were collected from the Fujian provincial hospital and divided into five groups: CRC (n = 82); advanced adenoma (AA, n = 76); non-advanced adenoma (NAA, n = 24); healthy control (n = 85); and interference group (n = 12). All stool samples were tested using a fecal multigene mutation (KRAS-APC-p53-BRAF) Kit and FIT. RESULTS: The sensitivity of combined use of fecal multigene mutation test and FIT for detecting CRC [84.15% (69/ 82)] was significantly higher than that of fecal multigene mutation test [47.56% (39/82), p < 0.001] or FIT [71.95% (59/82), p < 0.001] alone. The sensitivity of combined use for detection of AA [48.68% (37/76)] was also significantly higher than that of multigene mutation test [26.32% (20/76), p < 0.001] or FIT [28.95% (22/76), p < 0.001] alone. The specificity of combined use for detection of NAA and healthy control was 87.16%. CONCLUSIONS: The combination of fecal multigene (KRAS-APC-p53-BRAF) mutation test and FIT has greater sensitivity than alone and may be a useful noninvasive method for CRC screening.

Ultrapulse carbon dioxide laser treatment for bilateral facial nevus comedonicus: A case report.

Nevus comedonicus (NC), a rare skin ailment with an aggregation of dilated follicular orifices filled with keratinous material, is difficult to treat. Several drugs have been assessed for the treatment of NC, but with limited success. Surgery requires much experience and the recurrence rate is high. Various types of laser have been tried, with promising outcomes. A 54-year-old male patient with bilateral facial NC was admitted on July 8, 2014. A coin-sized area was first treated successfully with ultrapulse CO2 laser. The remaining lesions were treated during three subsequent sessions at 2-week intervals. There were no complications. There was no recurrence after 2 years. This case suggests that ultrapulse CO2 laser could efficiently alleviate NC. Ultrapulse CO2 laser treatment should be further studied for its application in the treatment of NC.

A Nanoscale Mutation-Sensitive On/Off Switch Based Assays for the Detection of Hepatitis B Virus Lamivudine-Resistant Mutations.

Long-term usage of lamivudine in the treatment of chronic hepatitis B virus (HBV) infection induces the emergence of drug resistance. Sensitive and specific methods aimed at detecting the mutants are clinically useful and required. The purpose of this study was to develop methods for detecting the mutations of YMDD, rtL180M, and rtV173L by nanoscale mutation-sensitive switch consisting of high fidelity polymerase and phosphorothioate-modified allele specific primers. Four assays for these hotspot mutations have been developed with the sensitivity of 100 copies and specificity of at least three log scales for matched templates over mismatched templates. In the condition of multiplex PCR, the sensitivities of these assays are approximately 1000 copies and specificities with two log scales in discrimination of mutant alleles over wild type sequences. These newly developed assays are rapid, accurate, and cost-efficient in detection of lamivudine-related HBV mutants.

NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages.

Idiopathic pulmonary fibrosis (IPF) is a progressive and highly lethal inflammatory interstitial lung disease characterized by aberrant extracellular matrix deposition. Macrophage activation by cytokines released from repetitively injured alveolar epithelial cells regulates the inflammatory response, tissue remodeling, and fibrosis throughout various phases of IPF. Our previous studies demonstrate that nuclear factor of activated T cells cytoplasmic member 3 (NFATc3) regulates a wide array of macrophage genes during acute lung injury pathogenesis. However, the role of NFATc3 in IPF pathophysiology has not been previously reported. In the current study, we demonstrate that expression of NFATc3 is elevated in lung tissues and pulmonary macrophages in mice subjected to bleomycin (BLM)-induced pulmonary fibrosis and IPF patients. Remarkably, NFATc3 deficiency (NFATc3+/-) was protective in bleomycin (BLM)-induced lung injury and fibrosis. Adoptive transfer of NFATc3+/+ macrophages to NFATc3+/- mice restored susceptibility to BLM-induced pulmonary fibrosis. Furthermore, in vitro treatment with IL-33 or conditioned medium from BLM-treated epithelial cells increased production of CCL2 and CXCL2 in macrophages from NFATc3+/+ but not NFATc3+/- mice. CXCL2 promoter-pGL3 Luciferase reporter vector showed accentuated reporter activity when co-transfected with the NFATc3 expression vector. More importantly, exogenous administration of recombinant CXCL2 into NFATc3+/- mice increased fibrotic markers and exacerbated IPF phenotype in BLM treated mice. Collectively, our data demonstrate, for the first time, that NFATc3 regulates pulmonary fibrosis by regulating CCL2 and CXCL2 gene expression in macrophages.

NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophage.

Idiopathic pulmonary fibrosis (IPF) is a progressive and highly lethal inflammatory interstitial lung disease characterized by aberrant extracellular matrix deposition. Macrophage activation by cytokines released from repetitively injured alveolar epithelial cells regulates the inflammatory response, tissue remodeling, and fibrosis throughout various phases of IPF. Our previous studies demonstrate that nuclear factor of activated T cells cytoplasmic member 3 (NFATc3) regulates a wide array of macrophage genes during acute lung injury pathogenesis. However, the role of NFATc3 in IPF pathophysiology has not been previously reported. In the current study, we demonstrate that expression of NFATc3 is elevated in lung tissues and pulmonary macrophages in mice subjected to bleomycin (BLM)-induced pulmonary fibrosis and IPF patients. Remarkably, NFATc3 deficiency (NFATc3+/-) was protective in bleomycin (BLM)-induced lung injury and fibrosis. Adoptive transfer of NFATc3+/+ macrophages to NFATc3+/- mice restored susceptibility to BLM-induced pulmonary fibrosis. Furthermore, in vitro treatment with IL-33 or conditioned medium from BLM-treated epithelial cells increased production of CCL2 and CXCL2 in macrophages from NFATc3+/+ but not NFATc3+/- mice. CXCL2 promoter-pGL3 Luciferase reporter vector showed accentuated reporter activity when co-transfected with the NFATc3 expression vector. More importantly, exogenous administration of recombinant CXCL2 into NFATc3+/- mice increased fibrotic markers and exacerbated IPF phenotype in BLM treated mice. Collectively, our data demonstrate, for the first time, that NFATc3 regulates pulmonary fibrosis by regulating CCL2 and CXCL2 gene expression in macrophages.

Screening and analysis of immune-related genes of Aedes aegypti infected with DENV2.

Dengue virus type Ⅱ (DENV2) is a primary serotype responsible for the dengue fever epidemic, and Aedes aegypti is the main DENV2 vector. Understanding the Aedes aegypti immune mechanism against DENV2 is the basis for research on immune blockade in mosquitoes. Some preliminary studies lack validation in the literature, so this study was performed to further study and validate the potential target genes to provide a further basis for screening key target genes. We screened 51 genes possibly related to Aedes aegypti infection and immunity from the literature for further verification. First, bioinformatic methods such as GO, KEGG and PPI analysis were used, and then RT-qPCR was used to detect the changes in mRNA expression in the midguts and salivary glands of Aedes aegypti infected with DENV2.Bioinformatic analysis showed that mostly genes of the glucose metabolism pathway and myoprotein were influenced. In salivary glands, the Gst (xa) and Toll (xb) expression levels were significantly correlated with DENV2 load (y, lg[DENV2 RNA copies]), y = -3436xa+0.2287xb+3.8194 (adjusted R2 = 0.5563, F = 9.148, PF = 0.0045). In midguts, DENV2 load was significantly correlated with the relative Fba(R2 = 0.4381, t = 2.497, p < 0.05, df = 8), UcCr(R2 = 0.4072, t = 2.344, p < 0.05, df = 8) and Gbps1(R2 = 0.4678, t = 2.652, p < 0.05, df = 8) expression levels, but multiple regression did not yield significant results. This study shows that genes related to glucose metabolism and muscle proteins contribute to the interaction between Aedes aegypti and dengue virus. It was confirmed that SAAG-4, histone H4, endoplasmin, catalase and other genes are involved in the regulation of DENV2 infection in Aedes aegypti. It was revealed that GST and Toll in salivary glands may have antagonistic effects on the regulation of DENV2 load. Fba, UcCr and Gbps1 in the midgut may increase DENV2 load. These study results further condensed the potential target gene range of the Aedes aegypti immune mechanism against DENV2 infection and provided basic information for research on the Aedes aegypti in vivo blockade strategy against DENV2.

Analysis of KRAS mutations in circulating tumor DNA and colorectal cancer tissue.

The mutation status of KRAS is important for anti-EGFR therapy in colorectal cancer (CRC) patients; however, detection of KRAS mutations in circulating tumor DNA (ctDNA) is problematic. We investigated tissue and plasma assays for KRAS mutations in CRC patients. The KRAS status of 407 CRC patients was evaluated using integration of amplification refractory mutation system polymerase chain reaction (PCR), melting curves and wild type DNA blocking (IAMB) in tissue and plasma samples. Disparate cases were re-evaluated by Sanger sequencing of tissue samples. General characteristics and tumor biomarkers including CEA, CA19-9 and CA125 were characterized. The prevalence of KRAS mutations was 40.8% in plasma and 49.1% in tissue. The overall percent agreement, positive percent agreement and negative percent agreement were 82.3, 76.3 and 90.8%, respectively. Older patients and higher TNM stage exhibited increased sensitivity for detecting KRAS mutations in plasma. We found 54.1% of patients with KRAS mutations using parallel analysis of tissue and plasma; only 36.4% of patients were detected by series analysis. We found that plasma based KRAS detection with IAMB technology is an alternative to tissue based KRAS testing. KRAS mutations can be identified more easily when both assays are used together.

Vector Competence and Vertical Transmission of Zika Virus in Aedes albopictus (Diptera: Culicidae).

Zika virus (ZIKV) is an emerging mosquito-borne pathogen belonging to the genus Flavivirus of the family Flaviviridae. Aedes albopictus is widely distributed in China. However, little is known about the vector competence of Ae. albopictus in China. The present study presents the oral susceptibility and vector competence of Ae. albopictus Guangzhou strain to ZIKV. Additionally, vertical transmission of ZIKV is described. The results demonstrated the susceptibility of local Ae. albopictus mosquitoes to ZIKV with an extrinsic incubation period of 6 days. Disseminated infection was observed in Ae. albopictus starting on day 2 postinfection (PI). Starting on day 6 PI, the saliva of Ae. albopictus exhibited ZIKV infection, and the transmission rate was 36.4%. Vertical transmission was observed during the first gonotrophic cycle. The minimum infection rate was observed in third-to-fourth instar larvae.

Susceptibility of Armigeres subalbatus Coquillett (Diptera: Culicidae) to Zika virus through oral and urine infection.

BACKGROUND: Zika virus (ZIKV) disease outbreaks have been occurring in South America since 2015, and has spread to North America. Because birth defects and cases of Guillain Barré have been associated with infection with ZIKV, this has drawn global attention. ZIKV is generally considered an Aedes-transmitted pathogen. The transmission of ZIKV through blood by Aedes mosquito bites has been recognized as the major transmission route. However, it is not clear whether there are other transmission routes that can cause viral infection in mosquitos. The aim of the present study is to describe the susceptibility of Armigeres subalbatus, which often develop in human waste lagoons, to ZIKV, through oral infection in adult mosquitoes and urine infection in larvae. METHODOLOGY/PRINCIPAL FINDINGS: Five-day-old female Ar. subalbatus ingested infectious blood meals containing ZIKV. After 4, 7, and 10 days of ingesting infectious blood meals, ZIKV could be detected in the midguts, salivary glands, ovaries, and collected saliva of mosquitoes. The ZIKV infection rate (IR) on day 10 reached 40% in salivary glands and 13% in saliva, indicating that these mosquitoes were able to transmit ZIKV. In addition, ZIKV infection was also discovered in mosquito ovaries, suggesting the possibility of vertical transmission of virus. Moreover, Ar. subalbatus transmitted ZIKV to infant mice bitten by infectious mosquitoes. In a second experiment, 1st-instar larvae of Ar. subalbatus were reared in water containing ZIKV and human urine. After pupation, pupae were placed in clean water and transferred to a mosquito cage for emergence. Although ZIKV RNA was detected in all of the larvae tested, ZIKV was not detected in the saliva of any adult Ar. subalbatus. Considering that there are more uncontrollable factors in nature than in the laboratory environment, the possibility that the virus is transmitted to adult mosquitoes via larvae is very small period. CONCLUSIONS/SIGNIFICANCE: Adult Ar. subalbatus could be infected with ZIKV and transmit ZIKV through mosquito bites. Therefore, in many rural areas in China and in undeveloped areas of other Asian countries, the management of human waste lagoons in the prevention and control of Zika disease should be considered. Corresponding adjustments and modifications should also be made in prevention and control strategies against ZIKV.

ZeoR: A Candidate for Assays Testing Enzymatic Activities and Off-Target Effects of Gene-Editing Enzymes.

The target lengths of gene-editing enzymes vary from approximately 20 to 80 nucleotides. The ideal reporter genes should be able to tolerate the insertion of target sequences to differentiate the phenotypic changes between in-frame and frame shift mutations for evaluating the enzymatic activities and off-target effects of gene-editing enzymes such as zinc finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats-associated system. This study demonstrated that the zeocin resistance gene (ZeoR) tolerated the insertion of long targets for a variety of tests using a strategy of inserting tandem repeats of NNT. Four genes such as CCR5, AR, HPV 16E7 early gene, and EGFR applications of ZeoR in quantitatively analyzing the off-targets of CRISPR were successful. The data indicated that evaluating the enzymatic activities and off-target effects with assays employing ZeoR had great potential in facilitating the application of gene editing to human gene therapy.

The genetic diversity and population structure of domestic Aedes aegypti (Diptera: Culicidae) in Yunnan Province, southwestern China.

BACKGROUND: There was no record of Aedes aegypti in Yunnan Province, China, until 2002, but this species is now continuously found in nine cities (or counties). Until now, little was known about the genetic diversity and population structure of this invasive species. Thus, a detailed understanding of the invasion strategies, colonisation and dispersal of this mosquito from a population genetics perspective is urgently needed for controlling and eliminating this disease vector. METHODS: The genetic diversity and population structure of Ae. aegypti communities were analysed by screening nine microsatellite loci from 833 Ae. aegypti mosquitoes sampled from 28 locations in Yunnan Province. RESULTS: In total, 114 alleles were obtained, and the average polymorphic information content (PIC) value was 0.672. The value of the alleles per locus ranged from 2.90 to 5.18, with an average of 4.04. The value of He ranged from 0.353 to 0.681, and the value of Ho within populations ranged from 0.401 to 0.689. Of the 28 locations, two showed significant departures from the Hardy-Weinberg equilibrium (HWE) with P-values less than 0.05, and a bottleneck effect was detected among locations from Ruili and the border areas with the degree of 60% and 50%, respectively. Combined with the F-statistics (FIT = 0.222; FCT = 0.145), the analysis of molecular variance (AMOVA) revealed that there was substantial molecular variation among individuals, accounting for 77.76% of the sample, with a significant P-value (<0.0001). The results suggest that genetic differences in Ae. aegypti originated primarily among individuals rather than among populations. Furthermore, the STRUCTURE and UPGMA cluster analyses showed that Ae. aegypti from the border areas were genetically isolated compared to those from the cities Ruili and Jinghong, consistent with the results of the Mantel test (R 2 = 0.245, P < 0.0001). CONCLUSIONS: Continuous invasion contributes to the maintenance of Ae. aegypti populations' genetic diversity and different invasion accidents result in the genetic difference among Ae. aegypti populations of Yunnan Province.

Vector competence and transovarial transmission of two Aedes aegypti strains to Zika virus.

Zika virus (ZIKV) has become a serious threat to global health since the outbreak in Brazil in 2015. Additional Chinese cases have continuously been reported since the first case of laboratory-confirmed ZIKV infection in China on 6 February 2016. Aedes aegypti is the most important vector for ZIKV. This study shows that two strains from China exhibit high levels of midgut infection and highly disseminated infection of salivary glands and ovaries. Both strains can transmit ZIKV to infant mice bitten by infectious mosquitoes. Moreover, the results provide the evidence of transovarial transmission of ZIKV in mosquitoes. The study indicates that the two Ae. aegypti strains are not only effective transmission vectors but also persistent survival hosts for ZIKV during unfavorable inter-epidemic periods. This function as a reservoir of infection has epidemiological implications that further enhance the risk of potential future outbreaks.

Identification of Differentially Expressed Genes In Deltamethrin-Resistant Culex pipiens quinquefasciatus.

Culex quinquefasciatus is one of China's major house-dwelling mosquito species and an important vector of filariasis and encephalitis. Chemical treatments represent one of the most successful approaches for comprehensive mosquito prevention and control. However, the widespread use of chemical pesticides has led to the occurrence and development of insecticide resistance. Therefore, in-depth studies of resistance to insecticides are of vital importance. In this study, we performed a gene expression analysis to investigate genes from Cx. quinquefasciatus that may confer pyrethroid resistance. We aimed to understand the mechanisms of Cx. quinquefasciatus resistance to pyrethroid insecticides and provide insights into insect resistance management. Using a resistance bioassay, we determined the deltamethrin LC50 values (lethal concentration required to kill 50% of the population) for Cx. quinquefasciatus larvae in the F21, F23, F24, F26, F27, and F30 generations. The 7 tested strains exhibited pesticide resistance that was 25.25 to 87.83 times higher than that of the SanYa strain. Moreover, the expression of the OBPjj7a (odorant-binding protein OBPjj7a), OBP28 (odorant-binding protein OBP28), and E2 (ubiquitin-conjugating enzyme) genes was positively correlated with deltamethrin resistance ( R2 = 0.836, P = 0.011; R2 = 0.788, P = 0.018; and R2 = 0.850, P = 0.009, respectively) in Cx. quinquefasciatus. The expression of 4 additional genes, H/ACA, S19, SAR2, and PGRP, was not correlated with deltamethrin resistance. In summary, this study identified 3 Cx. quinquefasciatus genes with potential involvement in deltamethrin resistance, and these results may provide a theoretical basis for the control of mosquito resistance and insights into resistance detection.

Culex pipiens quinquefasciatus: a potential vector to transmit Zika virus.

Zika virus (ZIKV) has become a threat to global health since the outbreak in Brazil in 2015. Although ZIKV is generally considered an Aedes-transmitted pathogen, new evidence has shown that parts of the virus closely resemble Culex-transmitted viruses. Therefore, it is important to evaluate the competence of Culex species for ZIKV to understand their potential as vectors. In this study, female Culex pipiens quinquefasciatus were orally exposed to ZIKV. Mosquito midguts, salivary glands and ovaries were tested for ZIKV to measure infection and dissemination at 2, 4, 6, 8, 12, 16 and 18 days post exposure (pe). In addition, saliva was collected from mosquitoes after infection and infant mice were bitten by infected mosquitoes to measure the transmission ability of Cx. p. quinquefasciatus. The results showed that the peak time of virus appearance in the salivary glands was day 8 pe, with 90% infection rate and an estimated virus titer of 3.92±0.49 lg RNA copies/mL. Eight of the nine infant mice had positive brains after being bitten by infected mosquitoes, which meant that Cx. p. quinquefasciatus could be infected with and transmit ZIKV following oral infection. These laboratory results clearly demonstrate the potential role of Cx. p. quinquefasciatus as a vector of ZIKV in China. Because there are quite different vector management strategies required to control Aedes (Stegomyia) species and Cx. p. quinquefasciatus, an integrated approach may be required should a Zika epidemic occur.

Epigallocatechin-3-gallate regulates the expression of Kruppel-like factor 4 through myocyte enhancer factor 2A.

Epigallocatechin-3-gallate (EGCG), a powerful antioxidant and free ion scavenger found in green tea, exhibits inhibitory effects on different stages of tumorigenesis. Within gastric cancer cells, the transcription factor Kruppel-like factor 4 (KLF4) is downregulated, and it is possible that EGCG exerts its anti-tumorigenic function through modulation of KLF4 expression. In order to examine the effects of EGCG on KLF4 in a gastric tumor model, we treated the gastric cancer cell line NCI-N87 with EGCG. We found that EGCG treatment results in increased expression of KLF4 and alters expression of the KLF4 target genes p21, CDK4, and cyclin D1. EGCG inhibits the growth of NCI-N87 cells in a time- and dose-dependent manner through arresting the cell cycle in the G0/G1 phase. Furthermore, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and 4',6-diamidino-2-phenylindole staining revealed that EGCG is able to promote apoptosis of NCI-N87 cells. The suppressive effects of EGCG on cell growth and cell cycle protein expression are eliminated by decreasing KLF4 mRNA using siRNA and are magnified by overexpressing KLF4. Using KLF4 reporter constructs, we verified that the elevated expression induced by EGCG was mediated by increasing levels of activated MEF2A, which bound to the promoter region of KLF4. Taken together, this is the first time that EGCG is reported to increase the expression of KLF4, suggesting a novel mechanisms in gastric cancer treatment.