KAT8/SIRT7-mediated Fascin-K41 acetylation/deacetylation regulates tumor metastasis in esophageal squamous cell carcinoma.

Fascin actin-bundling protein 1 (Fascin) is highly expressed in a variety of cancers, including esophageal squamous cell carcinoma (ESCC), working as an important oncogenic protein and promoting the migration and invasion of cancer cells by bundling F-actin to facilitate the formation of filopodia and invadopodia. However, it is not clear how exactly the function of Fascin is regulated by acetylation in cancer cells. Here, in ESCC cells, the histone acetyltransferase KAT8 catalyzed Fascin lysine 41 (K41) acetylation, to inhibit Fascin-mediated F-actin bundling and the formation of filopodia and invadopodia. Furthermore, NAD-dependent protein deacetylase sirtuin (SIRT) 7-mediated deacetylation of Fascin-K41 enhances the formation of filopodia and invadopodia, which promotes the migration and invasion of ESCC cells. Clinically, the analysis of cancer and adjacent tissue samples from patients with ESCC showed that Fascin-K41 acetylation was lower in the cancer tissue of patients with lymph node metastasis than in that of patients without lymph node metastasis, and low levels of Fascin-K41 acetylation were associated with a poorer prognosis in patients with ESCC. Importantly, K41 acetylation significantly blocked NP-G2-044, one of the Fascin inhibitors currently being clinically evaluated, suggesting that NP-G2-044 may be more suitable for patients with low levels of Fascin-K41 acetylation, but not suitable for patients with high levels of Fascin-K41 acetylation. © 2024 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinoma.

Anillin (ANLN) is a mitosis-related protein that promotes contractile ring formation and cytokinesis, but its cell cycle-dependent degradation mechanisms in cancer cells remain unclear. Here, we show that high expression of ANLN promotes cytokinesis and proliferation in esophageal squamous cell carcinoma (ESCC) cells and is associated with poor prognosis in ESCC patients. Furthermore, the findings of the study showed that the deubiquitinating enzyme USP10 interacts with ANLN and positively regulates ANLN protein levels. USP10 removes the K11- and K63-linked ubiquitin chains of ANLN through its deubiquitinase activity and prevents ANLN ubiquitin-mediated degradation. Importantly, USP10 promotes contractile ring assembly at the cytokinetic furrow as well as cytokinesis by stabilizing ANLN. Interestingly, USP10 and the E3 ubiquitin ligase APC/C co-activator Cdh1 formed a functional complex with ANLN in a non-competitive manner to balance ANLN protein levels. In addition, the macrolide compound FW-04-806 (F806), a natural compound with potential for treating ESCC, inhibited the mitosis of ESCC cells by targeting USP10 and promoting ANLN degradation. F806 selectively targeted USP10 and inhibited its catalytic activity but did not affect the binding of Cdh1 to ANLN and alters the balance of the USP10-Cdh1-ANLN complex. Additionally, USP10 expression was positively correlated with ANLN level and poor prognosis of ESCC patients. Overall, targeting the USP10-ANLN axis can effectively inhibit ESCC cell-cycle progression.

Complete Genome Sequence of Bacillus amyloliquefaciens EA19, an Endophytic Bacterium with Biocontrol Potential Isolated from Erigeron annuus.

Bacillus amyloliquefaciens strain EA19 is an endophyte isolated from Erigeron annuus with antifungal activity against Blumeria graminis f. sp. tritici, Magnaporthe oryzae, and Fusarium graminearum. The genome sequence of this strain is 3.96 Mb and contains 3,421 coding sequences, which will facilitate an understanding of the mechanisms of biocontrol.

P300/CBP-associated factor (PCAF)-mediated acetylation of Fascin at lysine 471 inhibits its actin-bundling activity and tumor metastasis in esophageal cancer.

BACKGROUND: Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post-translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. METHODS: Immunoprecipitation and glutathione-S-transferase pull-down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP-associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF-mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live-cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. RESULTS: Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti-AcK471-Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin-K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin-K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin-K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471-acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin-bundling activity. Clinically, high levels of AcK471-Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease-free survival of ESCC patients. CONCLUSIONS: Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin-K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin-bundling activity.

Preoperative identification of microvascular invasion in hepatocellular carcinoma by XGBoost and deep learning.

PURPOSE: Microvascular invasion (MVI) is a valuable predictor of survival in hepatocellular carcinoma (HCC) patients. This study developed predictive models using eXtreme Gradient Boosting (XGBoost) and deep learning based on CT images to predict MVI preoperatively. METHODS: In total, 405 patients were included. A total of 7302 radiomic features and 17 radiological features were extracted by a radiomics feature extraction package and radiologists, respectively. We developed a XGBoost model based on radiomics features, radiological features and clinical variables and a three-dimensional convolutional neural network (3D-CNN) to predict MVI status. Next, we compared the efficacy of the two models. RESULTS: Of the 405 patients, 220 (54.3%) were MVI positive, and 185 (45.7%) were MVI negative. The areas under the receiver operating characteristic curves (AUROCs) of the Radiomics-Radiological-Clinical (RRC) Model and 3D-CNN Model in the training set were 0.952 (95% confidence interval (CI) 0.923-0.973) and 0.980 (95% CI 0.959-0.993), respectively (p = 0.14). The AUROCs of the RRC Model and 3D-CNN Model in the validation set were 0.887 (95% CI 0.797-0.947) and 0.906 (95% CI 0.821-0.960), respectively (p = 0.83). Based on the MVI status predicted by the RRC and 3D-CNN Models, the mean recurrence-free survival (RFS) was significantly better in the predicted MVI-negative group than that in the predicted MVI-positive group (RRC Model: 69.95 vs. 24.80 months, p < 0.001; 3D-CNN Model: 64.06 vs. 31.05 months, p = 0.027). CONCLUSION: The RRC Model and 3D-CNN models showed considerable efficacy in identifying MVI preoperatively. These machine learning models may facilitate decision-making in HCC treatment but requires further validation.

Multiphase convolutional dense network for the classification of focal liver lesions on dynamic contrast-enhanced computed tomography.

BACKGROUND: The accurate classification of focal liver lesions (FLLs) is essential to properly guide treatment options and predict prognosis. Dynamic contrast-enhanced computed tomography (DCE-CT) is still the cornerstone in the exact classification of FLLs due to its noninvasive nature, high scanning speed, and high-density resolution. Since their recent development, convolutional neural network-based deep learning techniques has been recognized to have high potential for image recognition tasks. AIM: To develop and evaluate an automated multiphase convolutional dense network (MP-CDN) to classify FLLs on multiphase CT. METHODS: A total of 517 FLLs scanned on a 320-detector CT scanner using a four-phase DCE-CT imaging protocol (including precontrast phase, arterial phase, portal venous phase, and delayed phase) from 2012 to 2017 were retrospectively enrolled. FLLs were classified into four categories: Category A, hepatocellular carcinoma (HCC); category B, liver metastases; category C, benign non-inflammatory FLLs including hemangiomas, focal nodular hyperplasias and adenomas; and category D, hepatic abscesses. Each category was split into a training set and test set in an approximate 8:2 ratio. An MP-CDN classifier with a sequential input of the four-phase CT images was developed to automatically classify FLLs. The classification performance of the model was evaluated on the test set; the accuracy and specificity were calculated from the confusion matrix, and the area under the receiver operating characteristic curve (AUC) was calculated from the SoftMax probability outputted from the last layer of the MP-CDN. RESULTS: A total of 410 FLLs were used for training and 107 FLLs were used for testing. The mean classification accuracy of the test set was 81.3% (87/107). The accuracy/specificity of distinguishing each category from the others were 0.916/0.964, 0.925/0.905, 0.860/0.918, and 0.925/0.963 for HCC, metastases, benign non-inflammatory FLLs, and abscesses on the test set, respectively. The AUC (95% confidence interval) for differentiating each category from the others was 0.92 (0.837-0.992), 0.99 (0.967-1.00), 0.88 (0.795-0.955) and 0.96 (0.914-0.996) for HCC, metastases, benign non-inflammatory FLLs, and abscesses on the test set, respectively. CONCLUSION: MP-CDN accurately classified FLLs detected on four-phase CT as HCC, metastases, benign non-inflammatory FLLs and hepatic abscesses and may assist radiologists in identifying the different types of FLLs.

Resistance to pyrethroid and organophosphate insecticides, and the geographical distribution and polymorphisms of target-site mutations in voltage-gated sodium channel and acetylcholinesterase 1 genes in Anopheles sinensis populations in Shanghai, China.

BACKGROUND: In the final phase of China's national programme to eliminate malaria by 2020, it is vitally important to monitor the resistance of malaria vectors for developing effective vector control strategies. In 2017 Shanghai declared that it had eliminated malaria; however, the insecticide resistance status of the primary malaria vector Anopheles sinensis remains unknown. METHODS: We examined the pyrethroid and organophosphate resistance of An. sinensis via a bioassay of two populations from the Chongming District of Shanghai. The voltage-gated sodium channel (VGSC) and acetylcholinesterase 1 (ace-1) genes were partially sequenced to examine the association between resistance phenotype and target site genotype. In addition, the geographical distribution, polymorphism and genotype frequencies of insecticide resistance genes were examined using samples collected during routine mosquito surveillance in 2016 and 2017 from Chongming, Songjiang, Jiading and Qingpu Districts. RESULTS: In Chongming District, the An. sinensis population near Dongtan National Nature Reserve showed resistance to pyrethroids, sensitivity to organophosphate, no knockdown resistance (kdr) mutations in the VGSC gene, and a low frequency (1.71%) of the ace-1 119S allele. An An. sinensis population near the Chongming central area (CM-Xinhe population) showed high resistance to pyrethroids and organophosphates and high frequencies of kdr 1014F and 1014C (80.73%) and ace-1 119S (85.98%) alleles. A significant association was detected between the homozygous kdr mutation 1014F/1014F and pyrethroid resistance in the CM-Xinhe population, indicating that the kdr mutation is probably recessive. Eight kdr genotypes with 1014F and 1014C substitutions were detected in the four surveyed districts of Shanghai. TTT and GGC/AGC were the dominant kdr allele and ace-1 genotype, respectively, and were prevalent in most Shanghai An. sinensis populations. CONCLUSIONS: On the basis of our assessment of insecticide resistance gene mutations in Shanghai, we identified a kdr mutation-free population in Chongming Dongtan. However, high frequencies of target-site mutations of insecticide resistance genes were observed in most areas of Shanghai. Bioassays of An. sinensis populations in the central Chongming District indicated the high insecticide resistance status of An. sinensis populations in Shanghai. We accordingly recommend a restriction on insecticide usage and development of effective integrated pest/vector management interventions to support disease control efforts.

The Association of Serum Vitamin D Deficiency and Metabolic Risk Factors in Chinese Adults with Prediabetes: A Cross-Sectional Study.

The association of serum vitamin D deficiency and metabolic risk factors in Chinese adults with prediabetes (PreDM) has not been investigated. The present study aimed to investigate the association of serum vitamin D deficiency and metabolic risk factors in Chinese adults with PreDM. In this cross-sectional study, we stratified 412 PreDM patients into vitamin D sufficient, vitamin D insufficient and vitamin D deficient subgroups. The physical examination data was collected. Serum 25-hydroxyvitamin D3 [25(OH)D3] were measured by high performance liquid chromatography. The prevalence of vitamin D deficiency and insufficiency in PreDM patients were 30.58% and 26.70%, respectively. Compared with the vitamin D deficient group, the prevalence of metabolic syndrome, central obesity, hyperglycemia and hypertension were higher than those in the vitamin D insufficient or sufficient group (p<0.05). Moreover, the prevalence of dyslipidemia in the vitamin D deficient group was higher than those in the vitamin D sufficient group (p<0.05). We observed an inverse relationship between 25(OH)D3 levels and waist circumference, triglyceride, and serum uric acid (ß=-0.315; ß=-0.134; ß=-0.239), a positive relationship between 25(OH)D3 levels and high-density lipoprotein cholesterol (ß=0.197) after adjusting for age, sex and body mass index. Vitamin D deficiency is very common among PreDM patients in China and this deficiency is related to metabolic risk factors.

New strains of Japanese encephalitis virus circulating in Shanghai, China after a ten-year hiatus in local mosquito surveillance.

BACKGROUND: Continuous vector pathogen surveillance is essential for preventing outbreaks of mosquito-borne diseases. Several mosquito species acting as vectors of Japanese encephalitis virus (JEV), dengue virus, Zika virus, malaria parasites and other pathogens are primary mosquito species in Shanghai, China. However, few surveys of human pathogenic arboviruses in mosquitoes in Shanghai have been reported in the last ten years. Therefore, in this study, we evaluated mosquito activity in Shanghai, China during 2016 and tested for the presence of alphaviruses, flaviviruses, orthobunyaviruses and several parasitic pathogens. RESULTS: Five pooled samples were JEV-positive [4/255 pools of Culex tritaeniorhynchus and 1/256 pools of Cx. pipiens (s.l.)] based on analysis of the NS5 gene. Alphaviruses, orthobunyaviruses, Plasmodium and filariasis were not found in this study. Phylogenetic and molecular analyses revealed that the JEV strains belonged to genotype I. Moreover, newly detected Shanghai JEV strains were genetically close to previously isolated Shandong strains responsible for transmission during the 2013 Japanese encephalitis (JE) outbreak in Shandong Province, China but were more distantly related to other Shanghai strains detected in the early 2000s. The E proteins of the newly detected Shanghai JEV strains differed from that in the live attenuated vaccine SA14-14-2-derived strain at six amino residues: E130 (Ile→Val), E222 (Ala→Ser), E327 (Ser→Thr), E366 (Arg→Ser/Pro), E393 (Asn→Ser) and E433 (Val→Ile). However, no differences were observed in key amino acid sites related to antigenicity. Minimum JEV infection rates were 1.01 and 0.65 per 1000 Cx. tritaeniorhynchus and Cx. pipiens (s.l.), respectively. CONCLUSIONS: Five new Shanghai JEV genotype I strains, detected after a ten-year hiatus in local mosquito surveillance, were genetically close to strains involved in the 2013 Shandong JE outbreak. Because JEV is still circulating, vaccination in children should be extensively and continuously promoted. Moreover, JEV mosquito surveillance programmes should document the genotype variation, intensity and distribution of circulating viruses for use in the development and implementation of disease prevention and control strategies.

Higher serum carotenoids associated with improvement of non-alcoholic fatty liver disease in adults: a prospective study.

PURPOSE: Previous studies have suggested that serum carotenoids might be inversely associated with non-alcoholic fatty liver disease (NAFLD), but little data came from longitudinal studies. We prospectively examined the associations between serum-carotenoid levels and NAFLD severity and the intermediary effects of retinol-binding protein 4 (RBP4), HOMA insulin-resistance index (HOMA-IR), body mass index (BMI), and serum triglycerides in middle-aged and elderly Chinese adults. METHODS: This prospective study included 3336 Chinese adults (40-75 years). We assessed serum concentrations of carotenoids at baseline and determined serum RBP4, triglycerides, and HOMA-IR levels at year 3. Abdominal ultrasonography was conducted to assess the presence and degree of NAFLD at years 3 and 6. RESULTS: The 2687 subjects who completed both NAFLD tests were classified into stable, improved and progressed groups according to changes in the degree of NAFLD between two visits. Analyses of covariance showed that ln-transformed serum concentrations of α-carotene, ß-cryptoxanthin, ß-carotene, lycopene, lutein/zeaxanthin, and total carotenoids were positively associated with NAFLD improvement (all p-trend < 0.05). After multivariable adjustment, mean differences in serum carotenoids were higher by 29.6% (ß-carotene), 18.2% (α-carotene), 15.6% (ß-cryptoxanthin), 11.5% (lycopene), 8.9% (lutein/zeaxanthin), and 16.6% (total carotenoids) in the improved vs. progressed subjects. Path analyses indicated the carotenoid-NAFLD association was mediated by lowering serum RBP4, triglycerides, HOMA-IR, and BMI, which were positively associated with the prevalence and progression of NAFLD. CONCLUSIONS: In middle-aged and elderly adults, higher serum-carotenoid concentrations were favorably associated with NAFLD improvement, mediated by reducing serum RBP4, triglycerides, HOMA-IR, and BMI. TRIAL REGISTRATIONS: This study has been registered at http://www.clinicaltrials.gov as NCT03179657.

Monitoring of malaria vectors at the China-Myanmar border while approaching malaria elimination.

BACKGROUND: Tengchong County was one of the counties located at the China-Myanmar border with high malaria incidence in the previous decades. As the pilot county for malaria elimination at the border area, Tengchong County is aiming to be the first county to achieve malaria elimination goal. A cross-sectional entomological survey was carried out to evaluate the feasibility of elimination approach and assess the receptivity of malaria reintroduction. METHODS: Light traps associated with live baits were used to investigate the abundance of adult mosquitoes in nine villages in Tengchong County. Light traps were set to collect adult mosquitoes in both human houses and cowsheds from dusk till dawn in each site. RESULTS: A total of 4948 adult Anopheles mosquitoes were collected from May to December in two villages. Of the mosquitoes were captured, 24.2% were in human houses and 75.8% in cowsheds. The peak of abundance occurred in July for An. sinensis and in September-October for An. minimus (s.l.) Ten Anopheles species were collected, the most prevalent being An. sinensis (50.3%), An. peditaeniatus (31.6%) and An. minimus (s.l.) (15.8%), contributing to 97.6% of the sample. Potential breeding sites were also investigated and a total of 407 larvae were collected, with An. sinensis (50.1%) and An. minimus (s.l.) (46.2%) as predominant species. Ponds and rice fields were the two preferred breeding sites for Anopheles mosquitoes; however, the difference between the number of adults and larvae captured suggest other breeding sites might exist. Both An. sinensis and An. minimus (s.l.) were found zoophilic with human blood index as 0.21 and 0.26, respectively. No Plasmodium positive Anopheles specimens were found by PCR among 4,000 trapped mosquitoes. CONCLUSIONS: Although no indigenous malaria cases have been reported in Tengchong County since 2013, there is still a risk from the presence of vectors in the context of human population movements from neighboring malaria endemic areas. The presence of An. sinensis, associated to rice fields, is particularly worrying. Sustained entomological surveillance is strongly suggested even after malaria elimination certification.

Co-circulation of Aedes flavivirus, Culex flavivirus, and Quang Binh virus in Shanghai, China.

BACKGROUND: With increases in global travel and trade, the spread of arboviruses is undoubtedly alarming. Pathogen detection in field-caught mosquitoes can provide the earliest possible warning of transmission. Insect-specific flavivirus (ISFV) has been first detected in 1991 and documented worldwide in the latest ten years. Although infection with ISFVs is apparently limited to insects, an increase in the infection rate of mosquito-borne flaviviruses may be able to induce cytopathic effects in vertebrate cells during co-infection with other human pathogens. However, little is known whether ISFVs persist in most regions of China. METHODS: During the mosquito activity season in 2016, a surveillance program was carried out to detect ISFVs in mosquitoes in metropolitan Shanghai, China. The presence of ISFVs was randomly tested in different species of mosquitoes using RT-PCR-based and hemi-nested PCR assays, following by the sequencing of PCR products. Sequences from positive pooled samples were compared with those deposited in GenBank. Thereafter, sequences of representative insect flaviviruses were used for further phylogenetic and molecular evolutionary analyses. RESULTS: Our investigations showed: (1) the presence of Aedes flavivirus (AEFV) in 11/161 pooled samples (nine pools in Songjiang District, one pool in Huangpu District, and one pool in Qingpu District) of Aedes albopictus, (2) the presence of Quang Binh virus (QBV) in 10/195 pooled samples (all in Chongming District) of Culex tritaeniorhynchus; and (3) the presence of Culex flavivirus (CxFV) in 9/228 pooled samples (six pools in Pudong New Area, two pools in Huangpu District, and one pool in Chongming District) of Cx. pipiens. Furthermore, phylogenetic analyses of the gene sequences of envelope proteins indicated that Shanghai CxFV strains belonged to the Asia/USA genotype. The overall maximum likelihood estimation values (and 95% confidence interval) for CxFV, QBV, and AEFV in mosquitoes collected in Shanghai in 2016 were 1.34 (0.66-2.45), 1.65 (0.87-2.85), and 1.51 (0.77-2.70) per 1000, respectively. CONCLUSIONS: This study reveals the presence and the geographical distribution of ISFVs, and determines the genetic variation and the infection rate of ISFVs in Shanghai, China. At least, three insect flaviviruses including ISFVs, AEFV, CxFV, and QBV, co-circulate in this area. To our knowledge, this is the first report of AEFV in China.

Molecular phylogeny of Anopheles hyrcanus group members based on ITS2 rDNA.

BACKGROUND: The Anopheles hyrcanus group includes 25 species, and is widely distributed in the Oriental and Palaearctic regions. Several species within this group are vectors of malaria, lymphatic filariasis and Japanese encephalitis. It is difficult or impossible to identify cryptic species based on their morphological characteristics, with some closely related species of the Hyrcanus Group have similar adult morphological characteristics. Thus, their molecular identification has been an important complementary method to traditional morphological taxonomy. METHODS: We used 461 ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) sequences relating to 19 species to reconstruct the molecular phylogeny of the Hyrcanus Group across its range. In addition, we compared the performance of rDNA ITS2 to that of mitochondrial DNA (mtDNA) cytochrome c oxidase subunit 1 gene (cox1) to assess the genetic divergence of Hyrcanus Group sibling species. RESULTS: Based on Kimura's 2-parameter (K2P) distance model, the average conspecific ITS2 divergence was 0.003, whereas sequence divergence between species averaged 0.480. Average ITS2 sequence divergences were almost 160 times higher among the Hyrcanus Group members than within each species. Two sets of sibling species, An. lesteri Baisas & Hu, 1936 and An. paraliae Sandosham, 1959; and An. sinensis Wiedemann, 1828, An. belenrae Rueda, 2005, and An. kleini Rueda, 2005, were resolved by ITS2. Each of these species was represented as an independent lineage in the phylogenetic tree. Results suggest that An. pseudopictus Grassi, 1899 and An. hyrcanus (Pallas, 1771) are most likely a single species. We uncovered two new ITS2 lineages that require further study before resolving their true taxonomic status, and designed a diagnostic polymerase chain reaction (PCR) assay to distinguish five morphologically similar species. CONCLUSIONS: Nuclear and mitochondrial genes generally provided consistent results for subgroup division. Compared to cox1, ITS2 is a more reliable tool for studying phylogenetic relationships among closely related mosquito taxa. Based on species-specific differences in ITS2 sequences, the multiplex PCR assay developed here can be used to improve the efficiency of vector identification. Thus, this research will promote the progress of malaria vector surveillance in both epidemic and non-epidemic areas of South and East Asia.

Transcriptome Analyses Shed New Insights into Primary Metabolism and Regulation of Blumeria graminis f. sp. tritici during Conidiation.

Conidia of the obligate biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt) play a vital role in its survival and rapid dispersal. However, little is known about the genetic basis for its asexual reproduction. To uncover the primary metabolic and regulatory events during conidiation, we sequenced the transcriptome of Bgt epiphytic structures at 3 (vegetative hyphae growth), 4 (foot cells initiation), and 5 (conidiophore erection) days post-inoculation (dpi). RNA-seq analyses identified 556 and 404 (combined 685) differentially expressed genes (DEGs) at 4 and 5 dpi compared with their expression levels at 3 dpi, respectively. We found that several genes involved in the conversion from a variety of sugars to glucose, glycolysis, the tricarboxylic acid cycle (TAC), the electron transport chain (ETC), and unsaturated fatty acid oxidation were activated during conidiation, suggesting that more energy supply is required during this process. Moreover, we found that glucose was converted into glycogen, which was accumulated in developing conidiophores, indicating that it could be the primary energy storage molecule in Bgt conidia. Clustering for the expression profiles of 91 regulatory genes showed that calcium (Ca2+), H2O2, and phosphoinositide (PIP) signaling were involved in Bgt conidiation. Furthermore, a strong accumulation of H2O2 in developing conidiophores was detected. Application of EGTA, a Ca2+ chelator, and trifluoperazine dihydrochloride (TFP), a calmodulin (CaM) antagonist, markedly suppressed the generation of H2O2, affected foot cell and conidiophore development and reduced conidia production significantly. These results suggest that Ca2+ and H2O2 signaling play important roles in conidiogenesis and a crosslink between them is present. In addition to some conidiation-related orthologs known in other fungi, such as the velvet complex components, we identified several other novel B. graminis-specific genes that have not been previously found to be implicated in fungal conidiation, reflecting a unique molecular mechanism underlying asexual development of cereal powdery mildews.

Molecular phylogeny of Anopheles hyrcanus group (Diptera: Culicidae) based on mtDNA COI.

BACKGROUND: The Anopheles hyrcanus group, which includes at least 25 species, is widely distributed in the Oriental and Palearctic regions. Some group members have been incriminated as vectors of malaria and other mosquito-borne diseases. It is difficult to identify Hyrcanus Group members by morphological features. Thus, molecular phylogeny has been proposed as an important complementary method to traditional morphological taxonomy. METHODS: Based on the GenBank database and our original study data, we used 466 mitochondrial DNA COI sequences belonging to 18 species to reconstruct the molecular phylogeny of the Hyrcanus Group across its worldwide geographic range. RESULTS: The results are as follows. 1) The average conspecific K2P divergence was 0.008 (range 0.002-0.017), whereas sequence divergence between congroup species averaged 0.064 (range 0.026-0.108). 2) The topology of COI tree of the Hyrcanus Group was generally consistent with classical morphological taxonomy in terms of species classification, but disagreed in subgroup division. In the COI tree, the group was divided into at least three main clusters. The first cluster contained An. nimpe; the second was composed of the Nigerrimus Subgroup and An. argyropus; and the third cluster was comprised of the Lesteri Subgroup and other unassociated species. 3) Phylogenetic analysis of COI indicated that ancient hybridizations probably occurred among the three closely related species, An. sinensis, An. belenrae, and An. kleini. 4) The results supported An. paraliae as a probable synonym of An. lesteri, and it was possible that An. pseudopictus and An. hyrcanus were the same species, as evident from their extremely low interspecific genetic divergence (0.020 and 0.007, respectively) and their phylogenetic positions. CONCLUSIONS: In summary, we reconstructed the molecular phylogeny and analysed genetic divergence of the Hyrcanus Group using mitochondrial COI sequences. Our results suggest that in the future of malaria surveillance, we should not only pay much attention to those known vectors of malaria, but also their closely related species.

[Discovery of Culex inatomii (Diptera : Culicidae) in Chongming, Shanghai].

Objective: To record the discovery of Culex inatomii in Chongming, Shanghai. Methods: Larvae and adult mosquitos of Cx. inatomii were collected in Dongtan of Chongming Island from May to November in 2015 and 2016, and their morphological characteristics were observed. The genomic DNA was extracted from adult mosquitos, PCR was performed to amplify the cytochrome c oxidaseⅠ(COⅠ) gene. Multiple alignment of COⅠ sequence was conducted with ClustalW2. Pairwise distances within and between species were calculated using MEGA v5.10 based on COⅠ sequences of Cx. inatomii, Cx. modestus, Cx. pipiens pallens, and Cx. tritaeniorhynchus. The phylogenetic tree of the above four species was constructed using neighbor joining, maximum likelihood, and Bayesian methods. Results: One hundred and fifty-six adult female mosquitos and 36 larvae of Cx. inatomii were collected. Larvae were reared to adult stage in the laboratory (17 female, 19 male). Morphologically, the subapical lobe of the sidepiece in male genitalia was divided into two parts, the anterior part having 2 bladed setae, and the posterior part having 1 bladed setae and 1 lanceolar strong setae. This strucutre can be used to distinguish Cx. inatomii from Cx. modestus. PCR of COⅠ resulted in products of approximately 650 bp. They were sequenced and the sequencing result was submitted to GenBank (accession number, KX555565-KX555570). Multiple sequence alignment revealed a 96% sequence similarity of COⅠ between Cx. inatomii and Cx. modestus. The genetic distance between Cx. inatomii and Cx. modestus was 0.047, and that within them each was 0.003 and 0.011, respectively. The phylogenetic tree showed that the four species clustered as a monophyletic clade, and each formed an individual lineage. Cx. inatomii had a closer relationship with Cx. modestus, while distant from the other two species. Conclusion: We recorded the discovery of Cx. inatomii in Chongming, Shanghai.

Noninvasive evaluation of radiation-enhanced glioma cells invasiveness by ultra-high-field (1)H-MRS in vitro.

INTRODUCTION: Glioma is the most common type of the primary CNS tumor. Radiotherapy is an important treatment measure after surgery. However, its highly invasive character is the main reason of postoperative recurrence. The aim of the study was to probe the correlation between the invasion ability and the metabolite characteristics of glioma cells at the cellular level after irradiation by using 14.7T high-resolution nuclear proton magnetic resonance spectroscopy ((1)H-MRS). METHODS: To determine the matrix metalloproteinase-2 (MMP-2) activity and metabolite ratios of glioma cells after irradiation with different doses of X-rays, U87 and C6 glioma cells were exposed to X-ray irradiation of 0, 1, 5, 10, and 15Gy. After 20h, the perchloric acid (PCA) extraction method was used to evaluate water-soluble metabolites [choline (Cho), creatine (Cr), and N-acetylaspartate (NAA)], and (1)H-MRS patterns and changes in metabolite ratios were observed in vitro by 14.7T high resolution (1)H-MRS. Matrigel invasion assays and gelatin zymography were performed to test the invasion ability of U87 and C6 glioma cells. RESULTS: Good MR spectra were obtained from PCA method extracts of U87 and C6 glioma cells. Both radiation-induced MMP-2 activity and the Cho/Cr and Cho/NAA ratios increased after irradiation, and their increase occurred in a dose-dependent manner. The MMP-2 activity and the Cho/Cr and Cho/NAA ratios of glioma cells increased after irradiation up to 10Gy and decreased thereafter. In particular, the Cho/NAA ratio of U87 cells increased from 3.55±0.06 (0Gy) to 9.13±0.30 (10Gy) and then declined to 5.94±0.15 (15Gy). Furthermore, the invasion ability of glioma cells had a strong positive correlation with the Cho/Cr and Cho/NAA ratios. Both the Cho/Cr ratio and the Cho/NAA ratio of U87 glioma cells were highly positively correlated with the number of invading cells in the Matrigel invasion assay. The Pearson's correlation coefficient (r) values of U87 cells were 0.89 (Cho/Cr ratio versus invasion ability) and 0.91 (Cho/NAA ratio versus invasion ability) (P<0.01). C6 cells exhibited similar changes to those of U87 cells. CONCLUSIONS: In vitro high-resolution (1)H-MRS is useful for detecting glioma invasiveness at the cellular level.

Association of dietary and serum vitamin E with bone mineral density in middle-aged and elderly Chinese adults: a cross-sectional study.

Previous studies have suggested that vitamin E (VE) may affect bone health, but the findings have been inconclusive. We examined the relationship between VE status (in both diet and serum) and bone mineral density (BMD) among Chinese adults. This community-based study included 3203 adults (2178 women and 1025 men) aged 40-75 years from Guangzhou, People's Republic of China. General and dietary intake information were collected using structured questionnaire interviews. The serum α-tocopherol (TF) level was quantified by reversed-phase HPLC. The BMD of the whole body, the lumbar spine and left hip sites (total, neck, trochanter, intertrochanter and Ward's triangle) were measured using dual-energy X-ray absorptiometry. In women, the dietary intake of VE was significantly and positively associated with BMD at the lumbar spine, total hip, intertrochanter and femur neck sites after adjusting for covariates (P(trend): 0·001-0·017). Women in quartile 3 of VE intake typically had the highest BMD; the covariate-adjusted mean BMD were 2·5, 3·06, 3·41 and 3·54% higher, respectively, in quartile 3 (v. 1) at the four above-mentioned sites. Similar positive associations were observed between cholesterol-adjusted serum α-TF levels and BMD at each of the studied bone sites (P(trend): 0·001-0·022). The covariate-adjusted mean BMD were 1·24-4·83% greater in quartile 4 (v. 1) in women. However, no significant associations were seen between the VE levels (dietary or serum) and the BMD at any site in men. In conclusion, greater consumption and higher serum levels of VE are associated with greater BMD in Chinese women but not in Chinese men.

[Research Progress in Classification of Anopheles hyrcanus Group (Diptera : Culicidae)].

Objective: The Anopheles hyrcanus group is widely distributed in the Palearctic and Oriental regions. Among the reported members, 25 species have definitive scientific names. Some are determined as vectors of malaria, lymphatic filariasis, and Japanese encephalitis. However, it is extremely difficult to morphologically discriminate the group members. With the development of techniques like crossing experiment, karyotype analysis, and molecular phylogeny, the classification of the Hyrcanus Group has been changed. In this review we update the list of the Hyrcanus Group, including addition of newly discovered species An. belenrae, An. kleini, An. xui, and An. hyrcanus spIR, and merges of An. junlianensis, An. yatsushiroensis, and An. kunmingensis as synonyms. We also discuss the relationships between An. hyrcanus and An. pseudopictus, An. lesteri and An. paraliae, as well as An. kleini and An. engarensis. This review will help to clearly define the relationships among the species.

Association of dietary consumption and serum levels of vitamin A and ß-carotene with bone mineral density in Chinese adults.

BACKGROUND: Former studies suggested an adverse effect of hypervitaminosis A on bone health, while the effects of retinol and its precursor (ß-carotene) remain uncertain in populations consuming vitamin A (VA) mainly from plant sources. OBJECTIVE: We investigated the association of serum, dietary retinol, and ß-carotene with bone mineral density (BMD) in Chinese adults. METHODS: We recruited 2101 women and 1053 men (aged 40-75 years) in Guangzhou, China. Dietary intake was assessed through face-to-face interviews with food-frequency questionnaires at baseline and 3 years later. Serum levels of retinol and ß-carotene were determined by HPLC using a baseline specimen, and the BMD for the whole body (WB), lumbar spine (LS), total hip (TH), and femur neck (FN) were measured using dual energy X-ray absorptiometry at follow-up. RESULTS: In general, greater levels of serum retinol, ß-carotene, and the ß-carotene-to-retinol ratio were associated with a higher BMD after adjustment for potential covariates in the total sample. BMD values in the top (vs. bottom) quartile were increased by 2.06% (TH) for retinol; 2.87% (WB), 2.51% (LS), 3.10% (FN) for ß-carotene; 2.21% (WB) and 2.05% (FN) for the ß-carotene-to-retinol ratio in the total sample (all p<0.05). A significant positive association with BMD was observed for dietary intake of ß-carotene and total VA in retinol equivalents at the hip sites in the total sample. CONCLUSION: Higher circulating and dietary levels of VA and ß-carotene and higher serum ß-carotene-to-retinol ratios were positively associated with BMD in Chinese adults consuming relatively low levels of VA, mainly from plant foods.