Transcription Factors AhR and ARNT Regulate the Expression of CYP6SX1 and CYP3828A1 Involved in Insecticide Detoxification in Bradysia odoriphaga.

Aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mediate the responses of adaptive metabolism to various xenobiotics. Here, we found that BoAhR and BoARNT are highly expressed in the midgut of Bradysia odoriphaga larvae. The expression of BoAhR and BoARNT was significantly increased after exposure to imidacloprid and phoxim. The knockdown of BoAhR and BoARNT significantly decreased the expression of CYP6SX1 and CYP3828A1 as well as P450 enzyme activity and caused a significant increase in the sensitivity of larvae to imidacloprid and phoxim. Exposure to ß-naphthoflavone (BNF) significantly increased the expression of BoAhR, BoARNT, CYP6SX1, and CYP3828A1 as well as P450 activity and decreased larval sensitivity to imidacloprid and phoxim. Furthermore, CYP6SX1 and CYP3828A1 were significantly induced by imidacloprid and phoxim, and the silencing of these two genes significantly reduced larval tolerance to imidacloprid and phoxim. Taken together, the BoAhR/BoARNT pathway plays key roles in larval tolerance to imidacloprid and phoxim by regulating the expression of CYP6SX1 and CYP3828A1.

Functional Characterization of CYP6QE1 and CYP6FV21 in Resistance to λ-Cyhalothrin and Imidacloprid in Bradysia odoriphaga.

Cytochrome P450 monooxygenases (P450s) belong to a family of metabolic enzymes that are involved in the detoxification of insecticides. In this study, our bioassay results showed that a field-collected population of Bradysia odoriphaga displayed a moderate resistance to λ-cyhalothrin and imidacloprid. Compared to susceptible population, CYP6QE1 and CYP6FV21 were significantly overexpressed in the field population. The expression of CYP6QE1 and CYP6FV21 was more abundant in the third and fourth larval stages, and CYP6QE1 and CYP6FV21 were most highly expressed in the midgut and Malpighian tubules. Exposure to λ-cyhalothrin and imidacloprid significantly increased the expression levels of CYP6QE1 and CYP6FV21. Furthermore, the silencing of CYP6QE1 and CYP6FV21 significantly increased the susceptibility of B. odoriphaga larvae to λ-cyhalothrin and imidacloprid. The overexpression of CYP6QE1 and CYP6FV21 significantly enhanced the tolerance of transgenic Drosophila melanogaster lines to λ-cyhalothrin and imidacloprid. In addition, molecular docking revealed that these two P450 proteins have strong binding affinity toward λ-cyhalothrin and imidacloprid insecticides. Taken together, these results indicate that the overexpression of CYP6QE1 and CYP6FV21 is responsible for resistance to λ-cyhalothrin and imidacloprid in B. odoriphaga.

Urinary-derived extracellular vesicle microRNAs as non-invasive diagnostic biomarkers for early-stage renal cell carcinoma.

BACKGROUND AND AIMS: The potential of urinary-derived extracellular vesicle (uEV) microRNAs (miRNAs) as noninvasive molecular biomarkers for identifying early-stage renal cell carcinoma (RCC) patients is rarely explored. The present study aims to explore the possibility of uEV miRNAs as novel molecular biomarkers for distinguishing early-stage RCC. MATERIALS AND METHODS: uEVs were extracted by ExoQuick-TC™ kit and miRNA concentrations were measured by RT-qPCR. ROC curves and bioinformatics analysis were employed to predict the diagnostic efficacy and regulatory mechanisms of dysregulated miRNAs. RESULTS: Through a multiphase case-control study on uEV miRNAs screening, training, and validation in RCC cells (ACHN, Caki-1) and control cells (HK-2) and in uEVs of 125 RCC patients and 128 age- and sex-matched controls, we successfully identified four uEVs miRNAs (miR-135b-5p, miR-196b-5p, miR-200c-3p, and miR-203a-3p) were significantly and stably upregulated in RCC in vitro and in vivo. When adjusted with estimated glomerular filtration rate (eGFR), the AUC of the three-uEV miRNA panel (miR-135b-5p, miR-200c-3p, and miR-203a-3p) was 0.785 (95 % CI = 0.729-0.842, P < 0.0001) for discriminating RCC patients from controls. Notably, this panel exhibited similar performance in distinguishing early-stage (stage Ⅰ) RCC patients, with an AUC of 0.786 (95 %CI = 0.727-0.844, P < 0.0001). Bioinformatics analysis predicted that candidate miRNAs were involved in cancer progressing. CONCLUSION: Our study identified a four uEV miRNAs panel (miR-135b-5p, miR-196b-5p, miR-200c-3p, and miR-203a-3p) may serve as an auxiliary noninvasive indication of early-stage RCC.

The Orco gene involved in recognition of host plant volatiles and sex pheromone in the chive maggot Bradysia odoriphaga.

The insect olfactory recognition system plays a crucial role in the feeding and reproductive behaviors of insects. The odorant receptor co-receptor (Orco), as an obligatory chaperone, is critical for odorant recognition by way of forming heteromeric complexes with conventional odorant receptors (ORs). To investigate the biological functions of Orco in perceiving host plant volatiles and sex pheromone, the Orco gene was identified from the chive maggot Bradysia odoriphaga transcriptome data. Multiple sequence alignment reveals that BodoOrco exhibits an extremely high sequence identity with Orcos from other dipteran insects. The expression of BodoOrco is significantly higher in adults than in larvae and pupae, and the BodoOrco gene is primarily expressed in the antennae of both sexes. Furthermore, the Y-tube assay indicated that knockdown of BodoOrco leads to significant reductions in B. odoriphaga adults' response to all tested host plant volatiles. The dsOrco-treated unmated male adults show less attraction to unmated females and responded slowly compared with dsGFP control group. These results indicated that BodoOrco is involved in recognition of sex pheromone and host plant volatiles in B. odoriphaga and has the potential to be used as a target for the design of novel active compounds for developing ecofriendly pest control strategies.

Transcription factor CncC potentially regulates cytochrome P450 CYP321A1-mediated flavone tolerance in Helicoverpa armigera.

Insect P450s play crucial roles in metabolizing insecticides and toxic plant allelochemicals. In this study, our results demonstrate that Helicoverpa armigera can adapt to a lower concentration of flavone (a flavonoid phytochemical), and P450 activities and CYP321A1 transcript levels significantly increase after exposure to flavone. RNAi-mediated knockdown of CYP321A1 significantly reduced the tolerance of H. armigera larvae to flavone. In addition, the regulatory mechanisms driving CYP321A1 induction following exposure to flavone were investigated. Flavone exposure significantly increased H2O2 generation in the larval midgut. The mRNA levels of HaCncC and HaMaf-s significantly increased in the midgut of H. armigera after exposure to flavone. Knockdown of HaCncC significantly inhibited expression of flavone-induced CYP321A1 and resulted in a decrease in flavone induction of CYP321A1. HaCncC knockdown significantly reduced the tolerance of H. armigera larvae to flavone. Taken together, these results indicate that HaCncC regulates expression of the CYP321A1 gene responsible for flavone tolerance in H. armigera.

Activation of the CncC pathway is involved in the regulation of P450 genes responsible for clothianidin resistance in Bradysia odoriphaga.

BACKGROUND: Insect cytochrome P450 monooxygenases (P450s) play a key role in the detoxification metabolism of insecticides and their overexpression is often associated with insecticide resistance. Our previous research showed that the overexpression of four P450 genes is responsible for clothianidin resistance in B. odoriphaga. In this study, we characterized another P450 gene, CYP6FV21, associated with clothianidin resistance. However, the molecular basis for the overexpression of P450 genes in clothianidin-resistant strain remains obscure in B. odoriphaga. RESULTS: In this study, the CYP6FV21 gene was significantly overexpressed in the clothianidin-resistant (CL-R) strain. Clothianidin exposure significantly increased the expression level of CYP6FV21. Knockdown of CYP6FV21 significantly increased the susceptibility of B. odoriphaga larvae to clothianidin. The transcription factor Cap 'n' Collar isoform-C (CncC) was highly expressed in the midgut of larvae in B. odoriphaga. The expression level of CncC was higher in the CL-R strain compared with the susceptible (SS) strain. Clothianidin exposure caused reactive oxygen species (ROS) accumulation and significantly increased the expression level of CncC. Knockdown of CncC caused a significant decrease in the expression of CYP3828A1 and CYP6FV21, and P450 enzyme activity, and led to a significant increase in mortality after exposure to lethal concentration at 30% (LC30 ) of clothianidin. After treatment with CncC agonist curcumin, the P450 activity and the expression levels of CYP3828A1 and CYP6FV21 significantly increased, and larval sensitivity to clothianidin decreased. The ROS scavenger N-acetylcysteine (NAC) treatment significantly inhibited the expression levels of CncC, CYP3828A1 and CYP6FV21 in response to clothianidin exposure and increased larval sensitivity to clothianidin. CONCLUSION: Taken together, these results indicate that activation of the CncC pathway by the ROS burst plays a critical role in clothianidin resistance by regulating the expression of CYP3828A1 and CYP6FV21 genes in B. odoriphaga. This study provides more insight into the mechanisms underlying B. odoriphaga larval resistance to clothianidin. © 2023 Society of Chemical Industry.

Increased serum piwi-interacting RNAs as a novel potential diagnostic tool for brucellosis.

Background: Piwi-interacting RNAs (piRNAs) have emerged as potential novel indicators for various diseases; however, their diagnostic value for brucellosis remains unclear. This study aimed to evaluate the diagnostic potential of altered serum piRNAs in patients with brucellosis. Methods: Illumina sequencing via synthesis (SBS) technology was used to screen the serum piRNA profile in brucellosis patients, and markedly dysregulated piRNAs were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) assay in two sets from a cohort of 73 brucellosis patients and 65 controls. Results: Illumina SBS technology results showed that seven piRNAs were markedly elevated in brucellosis patients compared to normal controls. The seven upregulated piRNAs were further validated individually by qRT-PCR, of which three piRNAs (piR-000753, piR-001312, and piR-016742) were confirmed to be significantly and steadily increased in the patients (> 2-fold, P < 0.01). The area under the receiver operating characteristic (ROC) curve (AUCs) for the three piRNAs ranged from 0.698 to 0.783. The AUC for the three piRNAs combination was 0.772, with a specificity of 86% and a positive predictive value of 90%, respectively. Conclusions: The three-piRNA panel identified in this study has potential as a novel blood-based auxiliary tool for brucellosis detection.

Overexpression of Multiple Cytochrome P450 Genes Conferring Clothianidin Resistance in Bradysia odoriphaga.

Cytochrome P450 monooxygenases (P450s) play important roles in the detoxification metabolism of xenobiotics and are involved in the resistance of insects to many insecticides. In this study, piperonyl butoxide (PBO), an inhibitor of P450 enzyme activity, significantly increased the toxicity of clothianidin in the clothianidin-resistant (CL-R) population of Bradysia odoriphaga. The enzyme activity of P450 in the CL-R population was significantly higher than that in the SS population. Furthermore, four P450 genes were found to be significantly overexpressed in the CL-R population. Tissue-specific expression analysis indicates that CYP9J57, CYP3828A1, CYP6SX1, and CYP6QE1 were most highly expressed in the midgut and/or Malpighian tubules. After exposure to LC30 of clothianidin, the expression levels of the four P450 genes were significantly upregulated. The RNAi-mediated knockdown of CYP9J57, CYP3828A1, and CYP6QE1 significantly increased the susceptibility of B. odoriphaga to clothianidin. These results suggest that P450 genes are involved in clothianidin resistance in B. odoriphaga. This provides a better understanding of P450-mediated clothianidin resistance in B. odoriphaga and will contribute to the management of insect resistance to insecticides.

Astrocytic phagocytosis contributes to demyelination after focal cortical ischemia in mice.

Ischemic stroke can cause secondary myelin damage in the white matter distal to the primary injury site. The contribution of astrocytes during secondary demyelination and the underlying mechanisms are unclear. Here, using a mouse of distal middle cerebral artery occlusion, we show that lipocalin-2 (LCN2), enriched in reactive astrocytes, expression increases in nonischemic areas of the corpus callosum upon injury. LCN2-expressing astrocytes acquire a phagocytic phenotype and are able to uptake myelin. Myelin removal is impaired in Lcn2-/- astrocytes. Inducing re-expression of truncated LCN2(Δ2-20) in astrocytes restores phagocytosis and leads to progressive demyelination in Lcn2-/- mice. Co-immunoprecipitation experiments show that LCN2 binds to low-density lipoprotein receptor-related protein 1 (LRP1) in astrocytes. Knockdown of Lrp1 reduces LCN2-induced myelin engulfment by astrocytes and reduces demyelination. Altogether, our findings suggest that LCN2/LRP1 regulates astrocyte-mediated myelin phagocytosis in a mouse model of ischemic stroke.

Altered serum human cytomegalovirus microRNA levels are common and closely associated with the inflammatory status in patients with fever.

Background: Fever has a complicated etiology, and diagnosing its causative factor is clinically challenging. Human cytomegalovirus (HCMV) infection causes various diseases. However, the clinical relevance, prevalence, and significance of HCMV microRNAs (miRNA) in association with fever remain unclear. In the present study, we analyzed the HCMV miRNA expression pattern in the serum of patients with fever and evaluate its clinical associations with occult HCMV infection status in immune disorders. Methods: We included serum samples from 138 patients with fever and 151 age-gender-matched controls in this study. First, the serum levels of 24 HCMV miRNAs were determined using a hydrolysis probe-based stem-loop quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay in the training set. The markedly altered miRNAs were verified in the validation and testing sets. The serum HCMV IgG/IgM and DNA titers in the testing cohort were also assessed using enzyme-linked immunosorbent assay (ELISA) and RT-qPCR, respectively. Results: The majority of HCMV miRNAs were markedly upregulated in the serum of fever patients. We selected the five most significantly altered HCMV miRNAs: hcmv-miR-US4-3p, hcmv-miR-US29-3p, hcmv-miR-US5-2-3p, hcmv-miR-UL112-3p, and hcmv-miR-US33-3p for validation. These miRNAs were also significantly elevated in the serum of fever patients in the validation and testing sets compared with the controls. Logistic regression analysis revealed that the five miRNAs were novel potential risk factors for fever. Notably, the serum levels of four of the five confirmed HCMV miRNAs were significantly associated with blood C-reaction protein concentrations. Moreover, the five HCMV miRNA levels were closely correlated with the HCMV DNA titers in the testing cohort. Conclusion: HCMV infection and activation are common in fever patients and could be novel risk factors for fever. These differentially expressed HCMV miRNAs could enable HCMV activation status monitoring in immune disorders.

Involvement of Chemosensory Protein BodoCSP1 in Perception of Host Plant Volatiles in Bradysia odoriphaga.

Chemosensory proteins (CSPs) can bind and transport odorant molecules and play important roles in insect chemoreception. In this study, we focused on the roles of a chemosensory protein (BodoCSP1) in perception of host plant volatiles in Bradysia odoriphaga. The expression of BodoCSP1 was significantly higher in adults than in larvae and pupae, without a significant difference between male and female adults. Recombinant protein BodoCSP1 exhibited relatively high binding affinities to 9 out of 10 tested ligands (Ki < 10 µM). Behavioral assays revealed that adults of B. odoriphaga showed a significant preference for five compounds. The predicted three-dimensional (3D) structure of BodoCSP1 has the typical six α-helices that form the hydrophobic ligand-binding pocket. Molecular docking and site-directed mutagenesis combined with ligand-binding assays indicated that Val48 and Thr66 may be the key binding site in BodoCSP1 for host plant volatiles. RNAi results indicated that dsBodoCSP1-treated adults showed significant reductions in response to diallyl disulfide, dipropyl disulfide, and allyl methyl disulfide. These results indicated that BodoCSP1 plays essential functions in the perception of host plant volatiles in B. odoriphaga.

NRF-1 directly regulates TFE3 and promotes the proliferation of renal cancer cells.

The role of transcription factor binding to IGHM enhancer 3 (TFE3) in renal cell carcinoma (RCC) is not well understood. Nuclear respiratory factor 1 (NRF-1) may be the positive upstream regulatory gene of TFE3. The aim of the present study was to determine whether NRF-1 could directly regulate the expression of TFE3 and regulate tumorigenesis and progression of RCC through TFE3. Short hairpin RNA (shRNA) was used to silence the expression of NRF-1 in the 786-O human kidney adenocarcinoma cell line and the 293T human embryonic kidney cell line. Luciferase reporter assays were used to determine the relationship between NRF-1 and TFE3. The CHIP experiment was used to verify the actual binding of NRF-1 and TFE3 promoter regions. MitoTimer staining was used to measure mitochondrial biosynthesis. Flow cytometry was used to detect cell cycle and apoptosis. The 786-O and 293T cells were used to examine the underlying mechanism of action. The results demonstrated that NRF-1 could bind to the promoter region of the TFE3 gene and directly regulate the expression of TFE3. Following NRF-1 knockdown, the protein levels of phosphorylated (p)-AKT and p-S6 of mTOR pathway was inhibited, cell cycle progression was blocked, the levels of apoptosis increased, and mitochondrial generation was reduced. Following overexpression of TFE3, the levels of mTOR-associated markers were restored in NRF-1 knockdown cells. These findings suggest that NRF-1 may regulate the mTOR pathway through TFE3 and regulate the energy metabolism, proliferation and growth of cancer cells by directly regulating the expression of TFE3.

Genome-Wide Association Mapping Unravels the Genetic Control of Seed Vigor under Low-Temperature Conditions in Rapeseed (Brassica napus L.).

Low temperature inhibits rapid germination and successful seedling establishment of rapeseed (Brassica napus L.), leading to significant productivity losses. Little is known about the genetic diversity for seed vigor under low-temperature conditions in rapeseed, which motivated our investigation of 13 seed germination- and emergence-related traits under normal and low-temperature conditions for 442 diverse rapeseed accessions. The stress tolerance index was calculated for each trait based on performance under non-stress and low-temperature stress conditions. Principal component analysis of the low-temperature stress tolerance indices identified five principal components that captured 100% of the seedling response to low temperature. A genome-wide association study using ~8 million SNP (single-nucleotide polymorphism) markers identified from genome resequencing was undertaken to uncover the genetic basis of seed vigor related traits in rapeseed. We detected 22 quantitative trait loci (QTLs) significantly associated with stress tolerance indices regarding seed vigor under low-temperature stress. Scrutiny of the genes in these QTL regions identified 62 candidate genes related to specific stress tolerance indices of seed vigor, and the majority were involved in DNA repair, RNA translation, mitochondrial activation and energy generation, ubiquitination and degradation of protein reserve, antioxidant system, and plant hormone and signal transduction. The high effect variation and haplotype-based effect of these candidate genes were evaluated, and high priority could be given to the candidate genes BnaA03g40290D, BnaA06g07530D, BnaA09g06240D, BnaA09g06250D, and BnaC02g10720D in further study. These findings should be useful for marker-assisted breeding and genomic selection of rapeseed to increase seed vigor under low-temperature stress.

Discovery and validation of extracellular vesicle-associated miRNAs as noninvasive detection biomarkers for early-stage non-small-cell lung cancer.

miRNAs in circulating extracellular vesicles (EVs) are promising biomarkers for cancer. However, their diagnostic ability for early-stage non-small-cell lung cancer (NSCLC) is not well known. In this study, the circulating EV miRNAs profiling was initially performed in 36 untreated NSCLC patients and 36 healthy controls by TaqMan Low Density Array (TLDA). Subsequently, we performed quantitative reverse-transcription PCR assay (RT-qPCR) validation in several independent cohorts that included 159 NSCLC patients, 120 age/sex-matched healthy controls and 31 benign nodule patients enrolled from three different clinical centres. In addition, 38 cases of NSCLC were analysed before and after surgery. We demonstrated that miR-520c-3p and miR-1274b were significantly and steadily increased in NSCLC patients in comparison with healthy controls and benign nodule patients (P < 0.001) and decreased markedly after tumour resection (P < 0.001). The areas under the curve (AUCs) of the ROC curve of the two-miRNA panel were 0.857 (95% CI, 0813-0.901; P < 0.0001) and 0.845 (95% CI, 0.793-0.896; P < 0.0001) for NSCLC and NSCLC stage I, respectively. Furthermore, the panel was able to differentiate NSCLC from benign nodules with an AUC of 0.823 (95% CI, 0.730-0.915; P < 0.0001). Furthermore, logistic regression analysis revealed the two-miRNA panel as an independent risk factor for NSCLC (OR = 16.128, P < 0.0001). In conclusion, miR-520c-3p and miR-1274b have biomarker potential for early diagnosis of NSCLC in multiple centres.

SIDT1-dependent absorption in the stomach mediates host uptake of dietary and orally administered microRNAs.

Dietary microRNAs have been shown to be absorbed by mammals and regulate host gene expression, but the absorption mechanism remains unknown. Here, we show that SIDT1 expressed on gastric pit cells in the stomach is required for the absorption of dietary microRNAs. SIDT1-deficient mice show reduced basal levels and impaired dynamic absorption of dietary microRNAs. Notably, we identified the stomach as the primary site for dietary microRNA absorption, which is dramatically attenuated in the stomachs of SIDT1-deficient mice. Mechanistic analyses revealed that the uptake of exogenous microRNAs by gastric pit cells is SIDT1 and low-pH dependent. Furthermore, oral administration of plant-derived miR2911 retards liver fibrosis, and this protective effect was abolished in SIDT1-deficient mice. Our findings reveal a major mechanism underlying the absorption of dietary microRNAs, uncover an unexpected role of the stomach and shed light on developing small RNA therapeutics by oral delivery.

[The miR-184 level in the seminal plasma exosome of male infertility patients and its clinical significance].

OBJECTIVE: To study the miR-184 level in the seminal plasma exosome of male infertility patients and its clinical significance. METHODS: Between 2015 and 2019, we collected 285 seminal plasma samples from 97 azoospermia (AS) and 96 asthenospermia (AZS) patients and 92 age-matched normal fertile controls in Jiangsu Provincial Hospital of Traditional Chinese Medicine, General Hospital of Eastern Theater Command and the First Hospital Affiliated to Wenzhou Medical University, identified the isolated seminal plasma exosomes by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot, and detected the miR-184 level in the seminal plasma exosomes by quantitative real-time PCR (qRT-PCR). We determined the clinical value of the miR-184 level and its correlation with semen parameters by multiple statistics, predicted the target genes and involved pathways of miR-184 by bioinformatic algorithms, and analyzed their relationship with male infertility. RESULTS: NTA, TEM and Western blot exhibited plenty of exosomes in the seminal plasma of the patients. The results of qRT-PCR showed that the miR-184 level in the seminal plasma exosome was dramatically decreased in the AS patients compared with that in the normal fertile controls (0.227 ［0.092, 0.790］ vs 0.650 ［0.408, 1.061］, P < 0.01), but increased in AZS males in comparison with that in the control group (1.176 ［0.661, 1.946］ vs 0.650 ［0.408, 1.061］, P < 0.01). The areas under the ROC curve (AUC) for differentiating the AS and AZS patients from the controls were 0.866 (95% CI: 0.815－0.916) and 0.724 (95% CI: 0.653－0.795), respectively, and that for differentiating the AS from the AZS group was 0.964 (95% CI: 0.943－0.985). The miR-184 level in the seminal plasma exosome of the AZS patients was correlated positively with the sperm count (r = 0.243, P = 0.017) but negatively with the percentage of progressively motile sperm (r = －0.407, P = 0.006). Bioinformatics analysis indicated that the downstream target genes of miR-184 were significantly enriched in the protein regulatory pathways closely related to male reproduction and spermatogenesis. CONCLUSIONS: The miR-184 level in the seminal plasma exosome of infertility patients is significantly different from that of normal fertile males, which may serve as a potential auxiliary marker for the diagnosis of and participate in the development and progression of male infertility.

Different expression pattern of human cytomegalovirus-encoded microRNAs in circulation from virus latency to reactivation.

BACKGROUND: Human cytomegalovirus (HCMV) is a beta-hersvirinae that has a high latent infection rate worldwide and can cause serious consequences in immunocompromised patients when reactivation; however, the mechanism of how HCMV convert from latent to reactivation has rarely been investigated. In the present study, we aimed to perform a comprehensive analysis of the HCMV-encoded microRNA (miRNA) profile in serum of patients upon HCMV reactivation from latency and to further evaluate its clinical significance for the disease monitoring and preventing usefulness. METHODS: Serum samples from 59 viremia patients and 60 age-gender matched controls were enrolled in this study for screening and validation of different expression of HCMV miRNAs. Serum concentrations of 22 known HCMV miRNAs were determined by a hydrolysis probe-based stem-loop quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. HCMV DNA was measured by quantitative real-time PCR (qPCR) with the whole blood sample. Serum HCMV IgG and IgM were assessed using enzyme linked immunosorbent assay (ELISA). Another 47 samples from 5 patients at different time points were collected to evaluate the monitoring effectiveness and disease prediction ability of differential expression HCMV-miRNAs during the antiviral treatment. RESULTS: The RT-qPCR analysis revealed that the serum levels of 16 of the 22 examined HCMV miRNAs were elevated in HCMV viremia patients compared with controls, and a profile of 8 HCMV miRNAs including hcmv-miR-US25-2-3p, hcmv-miR-US4-5p, hcmv-miR-US25-2-5p, hcmv-miR-US25-1-3p, hcmv-miR-US25-1, hcmv-miR-UL36, hcmv-miR-UL148D, hcmv-miR-US29-3p were markedly elevated (fold change > 2, P < 0.01). Receiver operating characteristic curve (ROC) analysis were performed on the selected HCMV-miRNAs in all of the patients and controls that enrolled in this study, and which ranged from 0.72 to 0.80 in the autoimmune patients. In addition, hcmv-miR-US25-1-3p levels were significantly correlated with HCMV DNA load (r = 0.349, P = 0.007), and were obviously higher in the reactivation set than the latency set in the autoimmune patients, which could be a predictor for the monitoring of the antiviral treatment. CONCLUSIONS: HCMV miRNAs profile showed markedly shift-switch from latency to reactivation in circulation from HCMV infected patients and hcmv-miR-US25-1-3p may be served as a predictor for the switch upon reactivation from latency in patients suffered with autoimmune diseases.

A novel HER2-targeting antibody 5G9 identified by large-scale trastuzumab-based screening exhibits potent synergistic antitumor activity.

BACKGROUND: Pertuzumab is currently used in combination with trastuzumab as the first-line treatment for HER2-positive metastatic breast cancer. However, pertuzumab was originally developed independently from trastuzumab and was later incidentally found to have synergistic efficacy when combined with trastuzumab, it remains to be seen whether a more potent synergistic efficacy partner exists for trastuzumab. METHODS: A trastuzumab-based functional assay was used to screen anti-HER2 antibodies harboring trastuzumab-synergistic antitumor activity. The lead candidate 5G9, in combination with trastuzumab, was further characterized for its bioactivities in cell proliferation, cell apoptosis, antigen-antibody endocytosis and HER2-mediated cell signaling pathway blocking. Finally, animal models were used to evaluate the in vivo synergistic antitumor efficacy of 5G9 in combination with trastuzumab. FINDINGS: Compared to pertuzumab, 5G9 demonstrated more potent synergistic cell growth inhibitory activity when combined with trastuzumab (85% vs. 55%, P<0.001). In addition, 5G9 exhibited a higher internalization rate than pertuzumab (20% vs. 9%, P<0.05), and was able to further synergize with trastuzumab to promote antigen-antibody endocytosis. The internalization rate of the combination of 5G9 and trastuzumab was higher than that of pertuzumab and trastuzumab (35% vs. 14%, P<0.001). In vivo animal studies demonstrated that 5G9 in combination with trastuzumab showed more potent synergistic antitumor efficacy than the combination of pertuzumab and trastuzumab. INTERPRETATION: 5G9, together with trastuzumab, may provide a potential opportunity for more efficacious treatment of HER2-positive cancers. FUNDING: National Natural Science Foundation of China; State Key Laboratory of Analytical Chemistry for Life Science.

Gender difference analysis of Xp11.2 translocation renal cell carcinomas's attack rate: a meta-analysis and systematic review.

BACKGROUND: Xp11.2 translocation renal cell carcinoma (tRCC) is recently recognized. As Xp11.2 tRCC involved gene translocation and fusion in X chromosome and the number of X chromosomes in female is twice of male, we wondered whether the gender difference of attack rate is consistent with the proportion of the X chromosome. METHODS: In the present paper, meta-analysis was performed to find out the difference of morbidity between male and female. RESULTS: Nine studies with 209 cases calculated. Odds ratios (ORs) and ORs with 95% confidence intervals (CIs) were calculated for attack rate of Xp11.2 RCC with different gender. The result showed that the attack rate of female was higher than that of male with pooled OR of 2.84 (95% CI = 1.48-5.45), while the rate rises even further in adult (OR = 3.37, 95% CI =2.19-5.18). In other types of common kidney cancer, the OR value is less than 1, which means that the incidence of female is lower than that of male. CONCLUSIONS: The result showed that the incidence rate of female patients is much higher than that of male patients with Xp11.2 tRCC, it was reasonable to indicate that this particular incidence rate is related to the X chromosome.

Characterization of clinical enterococci isolates, focusing on the vancomycin-resistant enterococci in a tertiary hospital in China: based on the data from 2013 to 2018.

BACKGROUND: Vancomycin-resistant Enterococcus spp. (VRE) have spread all over the world. The present study aims to investigate the species distribution, specimen type and susceptibilities of Enterococcal species collected from Nanjing Drum Tower Hospital from 2013 to 2018. Additionally, distribution of VRE and prevalence of van gene among VRE isolates were also analyzed. METHODS: The susceptibilities of 3913 Enterococcus isolates were retrospectively investigated. Among these strains, 60 VRE strains were further anazlyed in this study. The minimum inhibitory concentrations (MICs) of the VRE strains towards vancomycin, teicoplanin and linezolid were determined by E-test. Polymerase chain reaction (PCR) and DNA sequencing were used to investigate the prevalence of van genes among VRE. Furthermore, the sequence types (STs) of VRE strains were explored by multi-locus sequence typing (MLST). RESULTS: Among the 3913 enterococci isolates, Enterococcus faecalis (n = 1870, 47.8%) and Enterococcus faecium (1738, 44.4%) were the main isolates. These Enterococcus strains were mainly isolated from urine (n = 1673, 42.8%), followed by secretions (n = 583, 14.9%) and ascites (n = 554, 14.2%). VRE displayed a decreasing trend year by year. Molecular analysis revealed that 49 out of 60 VRE isolates carried vanA gene, 10 carried vanM, and 1 carried both vanA and vanM genes. Sixteen distinct STs were identified among the 58 VREM, with ST78 (n = 16), ST192 (n = 8) and ST570 (n = 7) being the most dominant ones. CONCLUSIONS: E. faecalis and E. faecium were the major enterococci strains which are the main pathogens of urinary traction infections; vanA and vanM were the main determinants conferring resistance to vancomycin; ST78, ST192 and ST570 were the leading STs of VREM which displayed a decreasing trend of prevalence year by year.