Association Analysis Between Intratumoral and Peritumoral MRI Radiomics Features and Overall Survival of Neoadjuvant Therapy in Rectal Cancer.

BACKGROUND: The use of peritumoral features to determine the survival time of patients with rectal cancer (RC) is still imprecise. PURPOSE: To explore the correlation between intratumoral, peritumoral and combined features, and overall survival (OS). STUDY TYPE: Retrospective. POPULATION: One hundred sixty-six RC patients (53 women, 113 men; average age: 55 ± 12 years) who underwent radical resection after neoadjuvant therapy. FIELD STRENGTH/SEQUENCE: 3 T; T2WI sagittal, T1WI axial, T2WI axial with fat suppression, and high-resolution T2WI axial sequences, enhanced T1WI axial and sagittal sequences with fat suppression. ASSESSMENT: Radiologist A segmented 166 patients, and radiologist B randomly segmented 30 patients. Intratumoral and peritumoral features were extracted, and features with good stability (ICC ≥0.75) were retained through intra-observer analysis. Seven classifiers, including Logistic Regression (LR), Support Vector Machine (SVM), K-Nearest Neighbors (KNN), Random Forest (RF), Extremely randomized trees (ET), eXtreme Gradient Boosting (XGBoost), and LightGBM (LGBM), were applied to select the classifier with the best performance. Next, the Rad-score of best classifier and the clinical features were selected to establish the models, thus, nomogram was built to identify the association with 1-, 3-, and 5-year OS. STATISTICAL TESTS: LASSO, regression analysis, ROC, DeLong method, Kaplan-Meier curve. P < 0.05 indicated a significant difference. RESULTS: Only Node (irregular tumor nodules in the surrounding mesentery) and ExtraMRF (lymph nodes outside the perirectal mesentery) were significantly different in 20 clinical features. Twelve intratumoral, 3 peritumoral, and 14 combined features related to OS were selected. LR, SVM, and RF classier showed the best efficacy in the intratumoral, peritumoral, and combined model, respectively. The combined model (AUC = 0.954 and 0.821) had better survival association than the intratumoral model (AUC = 0.833 and 0.813) and the peritumoral model (AUC = 0.824 and 0.687). DATA CONCLUSION: The proposed peritumoral model with radiomics features may serve as a tool to improve estimated survival time. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 4.

Association of elevated circulating monocyte-platelet aggregates with hypercoagulability in patients with nephrotic syndrome.

BACKGROUND: Hypercoagulability emerges as a central pathological feature and clinical complication in nephrotic syndrome. Increased platelet activation and aggregability are closely related to hypercoagulability in nephrotic syndrome. Monocyte-platelet aggregates (MPAs) have been proposed to represent a robust biomarker of platelet activation. The aim of this study was to investigate levels of the circulating MPAs and MPAs with the different monocyte subsets to evaluate the association of MPAs with hypercoagulability in nephrotic syndrome. METHODS: Thirty-two patients with nephrotic syndrome were enrolled. In addition, thirty-two healthy age and sex matched adult volunteers served as healthy controls. MPAs were identified by CD14 monocytes positive for CD41a platelets. The classical (CD14 + + CD16-, CM), the intermediate (CD14 + + CD16+, IM) and the non-classical (CD14 + CD16++, NCM) monocytes, as well as subset specific MPAs, were measured by flow cytometry. RESULTS: Patients with nephrotic syndrome showed a higher percentage of circulating MPAs as compared with healthy controls (p < 0.001). The percentages of MPAs with CM, IM, and NCM were higher than those of healthy controls (p = 0.012, p < 0.001 and p < 0.001, respectively). Circulating MPAs showed correlations with hypoalbuminemia (r=-0.85; p < 0.001), hypercholesterolemia (r = 0.54; p < 0.001), fibrinogen (r = 0.70; p < 0.001) and D-dimer (r = 0.37; p = 0.003), but not with hypertriglyceridemia in nephrotic syndrome. The AUC for the prediction of hypercoagulability in nephrotic syndrome using MPAs was 0.79 (95% CI 0.68-0.90, p < 0.001). The sensitivity of MPAs in predicting hypercoagulability was 0.71, and the specificity was 0.78. CONCLUSION: Increased MPAs were correlated with hypercoagulability in nephrotic syndrome. MPAs may serve as a potential biomarker for thrombophilic or hypercoagulable state and provide novel insight into the mechanisms of anticoagulation in nephrotic syndrome.

Greatly isolated heterogeneous circulating tumor cells using hybrid engineered cell membrane-camouflaged magnetic nanoparticles.

BACKGROUND: Circulating tumor cells (CTCs) are considered as a useful biomarker for early cancer diagnosis, which play a crucial role in metastatic process. Unfortunately, the tumor heterogeneity and extremely rare occurrence rate of CTCs among billions of interfering leukocytes seriously hamper the sensitivity and purity of CTCs isolation. METHODS: To address these, we firstly used microfluidic chips to detect the broad-spectrum of triple target combination biomarkers in CTCs of 10 types of cancer patients, including EpCAM, EGFR and Her2. Then, we constructed hybrid engineered cell membrane-camouflaged magnetic nanoparticles (HE-CM-MNs) for efficient capture of heterogeneous CTCs with high-purity, which was enabled by inheriting the recognition ability of HE-CM for various CTCs and reducing homologous cell interaction with leukocytes. Compared with single E-CM-MNs, HE-CM-MNs showed a significant improvement in the capture efficiency for a cell mixture, with an efficiency of 90%. And the capture efficiency of HE-CM-MNs toward 12 subpopulations of tumor cells was ranged from 70 to 85%. Furthermore, by using HE-CM-MNs, we successfully isolated heterogeneous CTCs with high purity from clinical blood samples. Finally, the captured CTCs by HE-CM-MNs could be used for gene mutation analysis. CONCLUSIONS: This study demonstrated the promising potential of HE-CM-MNs for heterogeneous CTCs detection and downstream analysis.

Mechanistic study of transcription factor Sox18 during heart development.

Heart development is a delicate and complex process regulated by coordination of various signaling pathways. In this study, we investigated the role of sox18 in heart development by modulating Wnt/ß-Catenin signaling pathways. Our spatiotemporal expression analysis revealed that sox18 is mainly expressed in the heart, branchial arch, pharyngeal arch, spinal cord, and intersegmental vessels at the tailbud stage of Xenopus tropicalis embryo. Overexpression of sox18 in the X. tropicalis embryos causes heart edema, while loss-of-function of sox18 can change the signal of developmental heart marker gata4 at different stages, suggesting that sox18 plays an essential role in the development of the heart. Knockdown of SOX18 in human umbilical vein endothelial cells suggests a link between Sox18 and ß-CATENIN, a key regulator of the Wnt signaling pathway. Sox18 negatively regulates islet1 and tbx3, the downstream factors of Wnt/ß-Catenin signaling, during the linear heart tube formation and the heart looping stage. Taken together, our findings highlight the crucial role of Sox18 in the development of the heart via inhibiting Wnt/ß-Catenin signaling.

Decidual cell FKBP51-progesterone receptor binding mediates maternal stress-induced preterm birth.

Depression and posttraumatic stress disorder increase the risk of idiopathic preterm birth (iPTB); however, the exact molecular mechanism is unknown. Depression and stress-related disorders are linked to increased FK506-binding protein 51 (FKBP51) expression levels in the brain and/or FKBP5 gene polymorphisms. Fkbp5-deficient (Fkbp5-/-) mice resist stress-induced depressive and anxiety-like behaviors. FKBP51 binding to progesterone (P4) receptors (PRs) inhibits PR function. Moreover, reduced PR activity and/or expression stimulates human labor. We report enhanced in situ FKBP51 expression and increased nuclear FKBP51-PR binding in decidual cells of women with iPTB versus gestational age-matched controls. In Fkbp5+/+ mice, maternal restraint stress did not accelerate systemic P4 withdrawal but increased Fkbp5, decreased PR, and elevated AKR1C18 expression in uteri at E17.25 followed by reduced P4 levels and increased oxytocin receptor (Oxtr) expression at 18.25 in uteri resulting in PTB. These changes correlate with inhibition of uterine PR function by maternal stress-induced FKBP51. In contrast, Fkbp5-/- mice exhibit prolonged gestation and are completely resistant to maternal stress-induced PTB and labor-inducing uterine changes detected in stressed Fkbp5+/+ mice. Collectively, these results uncover a functional P4 withdrawal mechanism mediated by maternal stress-induced enhanced uterine FKBP51 expression and FKPB51-PR binding, resulting in iPTB.

DeepHPV: a deep learning model to predict human papillomavirus integration sites.

Human papillomavirus (HPV) integrating into human genome is the main cause of cervical carcinogenesis. HPV integration selection preference shows strong dependence on local genomic environment. Due to this theory, it is possible to predict HPV integration sites. However, a published bioinformatic tool is not available to date. Thus, we developed an attention-based deep learning model DeepHPV to predict HPV integration sites by learning environment features automatically. In total, 3608 known HPV integration sites were applied to train the model, and 584 reviewed HPV integration sites were used as the testing dataset. DeepHPV showed an area under the receiver-operating characteristic (AUROC) of 0.6336 and an area under the precision recall (AUPR) of 0.5670. Adding RepeatMasker and TCGA Pan Cancer peaks improved the model performance to 0.8464 and 0.8501 in AUROC and 0.7985 and 0.8106 in AUPR, respectively. Next, we tested these trained models on independent database VISDB and found the model adding TCGA Pan Cancer performed better (AUROC: 0.7175, AUPR: 0.6284) than the model adding RepeatMasker peaks (AUROC: 0.6102, AUPR: 0.5577). Moreover, we introduced attention mechanism in DeepHPV and enriched the transcription factor binding sites including BHLHA15, CHR, COUP-TFII, DMRTA2, E2A, HIC1, INR, NPAS, Nr5a2, RARa, SCL, Snail1, Sox10, Sox3, Sox4, Sox6, STAT6, Tbet, Tbx5, TEAD, Tgif2, ZNF189, ZNF416 near attention intensive sites. Together, DeepHPV is a robust and explainable deep learning model, providing new insights into HPV integration preference and mechanism. Availability: DeepHPV is available as an open-source software and can be downloaded from https://github.com/JiuxingLiang/DeepHPV.git, Contact: huzheng1998@163.com, liangjiuxing@m.scnu.edu.cn, lizheyzy@163.com.

Acute muscle wasting rate assessment and long-term mortality in critically ill trauma.

BACKGROUND AND OBJECTIVES: To evaluate the relationship between acute muscle wasting rate and long-term mortality in critically ill trauma. METHODS AND STUDY DESIGN: A single-center, retrospective study was conducted in critically ill trauma. Patients with Computed Tomography scans including the L3 vertebra within 24 hours and at 1 week after trauma were recruited. Acute muscle wasting rate was defined as the mean percent variation per day of skeletal muscle index in the first week after trauma. Multivariate logistic regression analysis and receiver operating characteristic curve analysis were performed to determine whether acute muscle wasting rate could help predict hospital malnutrition and 1-year mortality. RESULTS: Skeletal muscle index was 49.3±10.7 cm2/m2 at baseline and decreased to 45.1±9.6 cm2/m2 (p<0.001) at 1 week and 39.8±10.8cm2/m2 (p<0.001) at 1 month after trauma. A sustained decrease of skeletal muscle index was observed from baseline up to 6 months (33.7±8.4cm2/m2, p<0.001) post trauma, and lasted for 1 year (37.7±5.6cm2/m2, p=0.004). Logistic regression analysis showed that acute muscle wasting rate was an independent risk factor for hospital malnutrition and 1-year mortality. Every 1% absolute increase of acute muscle wasting rate was associated with 1.82-fold higher odds of 1-year mortality in critically ill trauma. The area under curve of acute muscle wasting rate was 0.813 for hospital malnutrition prediction and 0.715 for 1-year mortality prediction. CONCLUSIONS: Acute muscle wasting rate was independently associated with higher 1-year mortality and hospital malnutrition in critically ill trauma.

Leptin-Mediated Induction of IL-6 Expression in Hofbauer Cells Contributes to Preeclampsia Pathogenesis.

Leptin plays a crucial role in regulating energy homoeostasis, neuroendocrine function, metabolism, and immune and inflammatory responses. The adipose tissue is a main source of leptin, but during pregnancy, leptin is also secreted primarily by the placenta. Circulating leptin levels peak during the second trimester of human pregnancy and fall after labor. Several studies indicated a strong association between elevated placental leptin levels and preeclampsia (PE) pathogenesis and elevated serum interleukin-6 (IL-6) levels in PE patients. Therefore, we hypothesized that a local increase in placental leptin production induces IL-6 production in Hofbauer cells (HBCs) to contribute to PE-associated inflammation. We first investigated HBCs-specific IL-6 and leptin receptor (LEPR) expression and compared their immunoreactivity in PE vs. gestational age-matched control placentas. Subsequently, we examined the in vitro regulation of IL-6 as well as the phosphorylation levels of intracellular signaling proteins STAT3, STAT5, NF-κB, and ERK1/2 by increasing recombinant human leptin concentrations (10 to 1000 ng/mL) in primary cultured HBCs. Lastly, HBC cultures were incubated with leptin ± specific inhibitors of STAT3 or STAT5, or p65 NF-κB or ERK1/2 MAPK signaling cascades to determine relevant cascade(s) involved in leptin-mediated IL-6 regulation. Immunohistochemistry revealed ~three- and ~five-fold increases in IL-6 and LEPR expression, respectively, in HBCs from PE placentas. In vitro analysis indicated that leptin treatment in HBCs stimulate IL-6 in a concentration-dependent manner both at the transcriptional and secretory levels (p < 0.05). Moreover, leptin-treated HBC cultures displayed significantly increased phosphorylation levels of STAT5, p65 NF-κB, and ERK1/2 MAPK and pre-incubation of HBCs with a specific ERK1/2 MAPK inhibitor blocked leptin-induced IL-6 expression. Our in situ results show that HBCs contribute to the pathogenesis of PE by elevating IL-6 expression, and in vitro results indicate that induction of IL-6 expression in HBCs is primarily leptin-mediated. While HBCs display an anti-inflammatory phenotype in normal placentas, elevated levels of leptin may transform HBCs into a pro-inflammatory phenotype by activating ERK1/2 MAPK to augment IL-6 expression.

DeepEBV: a deep learning model to predict Epstein-Barr virus (EBV) integration sites.

MOTIVATION: Epstein-Barr virus (EBV) is one of the most prevalent DNA oncogenic viruses. The integration of EBV into the host genome has been reported to play an important role in cancer development. The preference of EBV integration showed strong dependence on the local genomic environment, which enables the prediction of EBV integration sites. RESULTS: An attention-based deep learning model, DeepEBV, was developed to predict EBV integration sites by learning local genomic features automatically. First, DeepEBV was trained and tested using the data from the dsVIS database. The results showed that DeepEBV with EBV integration sequences plus Repeat peaks and 2-fold data augmentation performed the best on the training dataset. Furthermore, the performance of the model was validated in an independent dataset. In addition, the motifs of DNA-binding proteins could influence the selection preference of viral insertional mutagenesis. Furthermore, the results showed that DeepEBV can predict EBV integration hotspot genes accurately. In summary, DeepEBV is a robust, accurate and explainable deep learning model, providing novel insights into EBV integration preferences and mechanisms. AVAILABILITYAND IMPLEMENTATION: DeepEBV is available as open-source software and can be downloaded from https://github.com/JiuxingLiang/DeepEBV.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Evolutionary analysis of a newly isolated Banna virus strain from Yunnan, China.

Banna virus (BAV) is a typical arbovirus whose infection is associated with fever and viral encephalitis. The whole genome of BAV is composed of 12 RNA segments. BAVs, which have been divided into three genotypes (A, B, and C) based on phylogenetic analysis of segment 12 or segment 9, are currently undergoing rapid evolution. Recent studies have shown that BAV variation can exceed intraspecific limits and generate novel viruses. In the current study, a new BAV strain, named 113c5, was isolated from Culex tritaeniorhynchus and found to be a member of genotype A2 based on phylogenetic analysis of segments 9 and 12. The complete genome sequence of the new BAV strain described in the current study might contribute to the surveillance of evolutionary characteristics of BAVs.

Zika Virus-Infected Decidual Cells Elicit a Gestational Age-Dependent Innate Immune Response and Exaggerate Trophoblast Zika Permissiveness: Implication for Vertical Transmission.

Vertical transmission of the Zika virus (ZIKV) causes severe fetal defects, but the exact pathogenic mechanism is unclear. We identified up to a 10,480-fold higher expression of viral attachment factors AXL, GAS6, and PROS1 and a 3880-fold increase in ZIKV infectiousness/propagation in human term decidual stromal cells versus trophoblasts. Moreover, levels of viral attachment factors and ZIKV are significantly increased, whereas expression of innate immune response genes are significantly decreased, in human first trimester versus term decidual cells. ZIKV-infected decidual cell supernatants increased cytotrophoblasts infection up to 252-fold compared with directly infected cytotrophoblasts. Tizoxanide treatment efficiently inhibited Zika infection in both maternal and fetal cells. We conclude that ZIKV permissiveness, as well as innate immune responsiveness of human decidual cells, are gestational age dependent, and decidual cells augment ZIKV infection of primary human cytotrophoblast cultures, which are otherwise ZIKV resistant. Human decidual cells may act as reservoirs for trimester-dependent placental transmission of ZIKV, accounting for the higher Zika infection susceptibility and more severe fetal sequelae observed in early versus late pregnancy. Moreover, tizoxanide is a promising agent in preventing perinatal Zika transmission as well as other RNA viruses such as coronavirus.

A Resource and Task Scheduling Based Multi-Objective Optimization Model and Algorithms in Elastic Optical Networks.

The elastic optical network (EON) adopting virtual network function (VNF) is a new type of network, in which the routing, spectrum, and data center allocation are key and challenging problems, and solving these three problems simultaneously can not only improve the network efficiency for network providers, but also let users obtain better service. However, few existing works handle these three problems simultaneously. To tackle the three problems simultaneously, given a set of network function chains (i.e., a set of tasks), we set up a new multi-objective optimization model in which the total length of paths for all tasks is minimized, the totally occupied spectrums are minimized, and the loads on all data centers are most balanced, simultaneously. To solve the model, we design two new evolutionary algorithms. The experiments are conducted on 16 cases of 4 widely used types of networks, and the results indicate that the proposed model and algorithms are effective.

Dual Inhibition of EGFR and IGF-1R Signaling Leads to Enhanced Antitumor Efficacy against Esophageal Squamous Cancer.

Both the epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) have been implicated in the development of cancers, and the increased expression of both receptors has been observed in esophageal cancer. However, the tyrosine kinase inhibitors of both receptors have thus far failed to provide clinical benefits for esophageal cancer patients. Studies have confirmed the complicated crosstalks that exist between the EGFR and IGF-1R pathways. The EGFR and IGF-1R signals act as mutual compensation pathways, thereby conveying resistance to EGFR or IGF-1R inhibitors when used alone. This study evaluated the antitumor efficacy of the EGFR/HER2 inhibitors, gefitinib and lapatinib, in combination with the IGF-1R inhibitor, linsitinib, on the esophageal squamous cell carcinoma (ESCC). Gefitinib or lapatinib, in combination with linsitinib, synergistically inhibited the proliferation, migration, and invasion of ESCC cells, caused significant cell cycle arrest, and induced marked cell apoptosis. Their combination demonstrated stronger inhibition on the activation of EGFR, HER2, and IGF-1R as well as the downstream signaling molecules. In vivo, the addition of linsitinib to gefitinib or lapatinib also potentiated the inhibition effects on the growth of xenografts. Our results suggest the next clinical exploration of the combination of gefitinib or lapatinib with linsitinib in the treatment of ESCC patients.

Thrombin-Induced Decidual Colony-Stimulating Factor-2 Promotes Abruption-Related Preterm Birth by Weakening Fetal Membranes.

Preterm premature rupture of membranes (PPROM) and thrombin generation by decidual cell-expressed tissue factor often accompany abruptions. Underlying mechanisms remain unclear. We hypothesized that thrombin-induced colony-stimulating factor-2 (CSF-2) in decidual cells triggers paracrine signaling via its receptor (CSF2R) in trophoblasts, promoting fetal membrane weakening and abruption-associated PPROM. Decidua basalis sections from term (n = 10), idiopathic preterm birth (PTB; n = 8), and abruption-complicated pregnancies (n = 8) were immunostained for CSF-2. Real-time quantitative PCR measured CSF2 and CSF2R mRNA levels. Term decidual cell (TDC) monolayers were treated with 10-8 mol/L estradiol ± 10-7 mol/L medroxyprogesterone acetate (MPA) ± 1 IU/mL thrombin pretreatment for 4 hours, washed, and then incubated in control medium with estradiol ± MPA. TDC-derived conditioned media supernatant effects on fetal membrane weakening were analyzed. Immunostaining localized CSF-2 primarily to decidual cell cytoplasm and cytotrophoblast cell membranes. CSF-2 immunoreactivity was higher in abruption-complicated or idiopathic PTB specimens versus normal term specimens (P < 0.001). CSF2 mRNA was higher in TDCs versus cytotrophoblasts (P < 0.05), whereas CSF2R mRNA was 1.3 × 104-fold higher in cytotrophoblasts versus TDCs (P < 0.001). Thrombin enhanced CSF-2 secretion in TDC cultures fourfold (P < 0.05); MPA reduced this effect. Thrombin-pretreated TDC-derived conditioned media supernatant weakened fetal membranes (P < 0.05), which MPA inhibited. TDC-derived CSF-2, acting via trophoblast-expressed CSFR2, contributes to thrombin-induced fetal membrane weakening, eliciting abruption-related PPROM and PTB.

Prevalence and genotype distribution of group A rotavirus circulating in Shanxi Province, China during 2015-2019.

BACKGROUND: Group A rotavirus (RVA), despite being a leading cause of gastroenteritis in infants and young children, is less studied in Shanxi Province, China. The current study was conducted to determine the prevalence and genetic characterization of RVA in hospitalized children younger than 10 years of age with the diagnosis of acute gastroenteritis in Shanxi Province, China. METHODS: A hospital-based active surveillance of rotavirus gastroenteritis was conducted at Children's Hospital of Shanxi from Jan 1, 2015, through Dec 31, 2019. Rotavirus was detected in stool samples by real-time quantitative reverse transcription PCR (qRT-PCR). G- and P-genotypes were determined by reverse transcription PCR (RT-PCR) and nucleotide sequencing. RESULTS: A total of 961 children younger than 10 years of age was enrolled over the study period, of whom 183 (19.0%) were positive for RVA. The highest RVA-infection frequency (23.7%) was found among children aged 12-23 months, and the seasonal peak was in December. G9P[8] was most prevalent (76.0%), followed by G3P[8] (7.1%), G2P[4] (3.3%), G1P[8] (0.5%) and G9P[4] (0.5%). CONCLUSIONS: These results report for the first time that RVA was one of the main causes of severe infectious gastroenteritis in children, and a high proportion of G9P[8] strains circulating in most areas of Shanxi Province. While the protective efficacy of the rotavirus vaccines has been demonstrated against G9P[8] strains, our results highlight that the dominant strains have not been effectively controlled in China.

The mitohormetic response as part of the cytoprotection mechanism of berberine : Berberine induces mitohormesis and mechanisms.

It was well-known that Berberine, a major bioactive compound extracted from natural plants Coptis chinensis, has anti-diabetic effects for decades in china. Other types of pharmacological activities, such as anti-inflammatory, antimicrobial, hypolipidemic, and anti-cancer effects, have also been examined. At cellular level, these pharmacological activities were mostly an inhibitory effect. However, the cytoprotective effect of berberine was also observed in various types of cells, such as neurons, endothelial cells, fibroblasts, and ß-cells. The paradoxical result may be closely associated with characteristics and distribution of berberine within cells, and they can be explained mechanically by mitohormesis, one particular form of hormesis. Here, we reviewed the mitohormetic response and assessed the berberine-induced effects and the possible signaling pathway involved. These findings may contribute to better clinical applications of berberine and indicate that some mitochondria-targeted conventional drugs should be considered carefully in clinical application.

A simple microsphere-based mold to rapidly fabricate microwell arrays for multisize 3D tumor culture.

Three-dimensional (3D) tumor has been considered as the best in vitro model for cancer research. In recent years, various methods have been developed to controllable prepare multisize 3D tumors. Nonetheless, reported technologies are still problematic and difficult to produce 3D tumors with highly uniform size and cell content. Here, a novel and simple microsphere-based mold approach is proposed to rapidly fabricate spherical microwell arrays for multisize 3D tumors formation, culture, and recovery. Larger amounts of HepG2 3D tumors with excellent quality and uniformity can be efficiently generated using this method. In addition, the tumor size can also be simply controlled by adjusting the diameter of the microwell arrays. All experimental results indicated that the proposed method offers a promising platform to generate and recover highly controlled multisize 3D tumors for various cell-based biomedical research.

Complete genome sequence of a novel bovine hepacivirus from Yunnan, China.

We detected a novel bovine hepacivirus N (HNV) subtype, IME_BovHep_01, in the serum of cattle in Tengchong, Yunnan, China, by high-throughput sequencing. The complete genome of IME_BovHep_01, was sequenced using an Illumina MiSeq sequencer and found to be 8850 nt in length, encoding one hypothetical protein. BLASTn analysis showed that the genome sequence shared similarity with the bovine hepacivirus isolate BovHepV_209/Ger/2014, with 88% query coverage and 70.8% identity. However, the highest similarity was to bovine hepacivirus N strain BRBovHep_RS963, for which only a partial genome sequence is available, with 68% query coverage and 81.5% identity. Sequence comparisons and phylogenetic analysis suggested that IME_BovHep_01 is a novel HNV subtype. Importantly, IME_BovHep_01 is the first member of this new genotype for which the complete genome sequence was determined.

CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system has emerged as a powerful tool for knock-in of DNA fragments via donor plasmid and homology-independent DNA repair mechanism; however, conventional integration includes unnecessary plasmid backbone and may result in the unfaithful expression of the modified endogenous genes. Here, we report an efficient and precise CRISPR/Cas9-mediated integration strategy using a donor plasmid that harbors 2 of the same cleavage sites that flank the cassette at both sides. After the delivery of donor plasmid, together with Cas9 mRNA and guide RNA, into cells or fertilized eggs, concurrent cleavages at both sides of the exogenous cassette and the desired chromosomal site result in precise targeted integration without plasmid backbone. We successfully used this approach to precisely integrate the EGFP reporter gene into the myh6 locus or the GAPDH locus in Xenopus tropicalis or human cells, respectively. Furthermore, we demonstrate that replacing conventional terminators with the endogenous 3UTR of target genes in the cassette greatly improves the expression of reporter gene after integration. Our efficient and precise method will be useful for a variety of targeted genome modifications, not only in X. tropicalis, but also in mammalian cells, and can be readily adapted to many other organisms.-Mao, C.-Z., Zheng, L., Zhou, Y.-M., Wu, H.-Y., Xia, J.-B., Liang, C.-Q., Guo, X.-F., Peng, W.-T., Zhao, H., Cai, W.-B., Kim, S.-K., Park, K.-S., Cai, D.-Q., Qi, X.-F. CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis.

Complete genome sequence of a novel negevirus isolated from Culex tritaeniorhynchus in China.

A novel negevirus, tentatively named Manglie virus (MaV), was isolated from Culex tritaeniorhynchus from the village of Manglie, Yunnan, China, in August 2011. It was identified by high-throughput sequencing of cell culture supernatants, and the complete genome was sequenced using an Illumina MiSeq sequencer. The complete MaV genome comprised 9,218 nt encoding three hypothetical proteins and had a poly(A) tail. BLASTn analysis showed that the genome had the greatest similarity to Ngewotan virus strain Nepal22, with query coverage of 100% and 79% identity. Genomic and phylogenetic analyses demonstrated that MaV should be considered a novel negevirus.