CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.

Causal Effect of Selenium Levels on Osteoporosis: A Mendelian Randomization Study.

Prior research has demonstrated equivocal associations between selenium (Se) concentrations and osteoporosis (OP), yielding inconclusive findings. The purpose of the current study was to examine the potential correlation between Se levels and the risk of OP by using the Mendelian randomization (MR) study design. The genetic variants related to Se levels were obtained from a meta-analysis of a Genome-Wide Association Study (GWAS) conducted on toenail Se levels (n = 4162) and blood Se levels (n = 5477). The data summary for OP and bone mineral density (BMD) was obtained by utilizing the GWAS database. To examine the association between Se levels and BMD and OP, we employed three statistical methods: inverse variance weighted, weighted median, and MR-Egger. The reliability of the analysis was verified by sensitivity testing. All three methods of MR analysis revealed that Se levels had no effect on OP risk. In addition, the sensitivity analysis revealed no heterogeneity or pleiotropy, and the significance of the overall effect remained unaffected by single-nucleotide polymorphisms (SNPs), as determined by the leave-one-out analysis, indicating that our findings are relatively reliable. The results of our study indicate that there is no causal association between Se levels and the risk of OP. However, additional investigation is necessary to ascertain whether there is a potential association between these variables.

Enhanced resistance to Bacillus thuringiensis Cry1Ac toxin mediated by the activation of prophenoloxidase in a cosmopolitan pest.

Plutella xylostella has evolved resistance to Bacillus thuringiensis Cry1Ac toxin over a long evolutionary period. Enhanced immune response is an important factor in insect resistance to a variety of insecticides, and whether phenoloxidase (PO), an immune protein, is involved in resistance to Cry1Ac toxin in P. xylostella remains unclear. Here, spatial and temporal expression patterns showed that prophenoloxidase (PxPPO1 and PxPPO2) in the Cry1S1000-resistant strain was more highly expressed in eggs, 4th instar, head, and hemolymph than those in G88-susceptible strain. The results of PO activity analysis showed that after treatment with Cry1Ac toxin PO activity was about 3 times higher than that before treatment. Furthermore, knockout of PxPPO1 and PxPPO2 significantly increased the susceptibility to Cry1Ac toxin. These findings were further supported by the knockdown of Clip-SPH2, a negative regulator of PO, which resulted in increased PxPPO1 and PxPPO2 expression and Cry1Ac susceptibility in the Cry1S1000-resistant strain. Finally, the synergistic effect of quercetin showed that larval survival decreased from 100 % to <20 % compared to the control group. This study will provide a theoretical basis for the analysis of immune-related genes (PO) genes involved in the resistance mechanism and pest control of P. xylostella.

Analysis of the Effect of Plutella xylostella Polycalin and ABCC2 Transporter on Cry1Ac Susceptibility by CRISPR/Cas9-Mediated Knockout.

Many insects, including the Plutella xylostella (L.), have developed varying degrees of resistance to many insecticides, including Bacillus thuringiensis (Bt) toxins, the bioinsecticides derived from Bt. The polycalin protein is one of the potential receptors for Bt toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of P. xylostella, but whether polycalin is associated with the resistance of Bt toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the Pxpolycalin gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of Pxpolycalin showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the Pxpolycalin gene and its transcript level were not linked to Cry1Ac resistance, whereas both the PxABCC2 gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the Pxpolycalin gene in a short term. Furthermore, the knockout of polycalin and ATP-binding cassette transporter subfamily C2 (ABCC2) genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to Bt toxins.

Dynamical analysis of a modified Leslie-Gower Holling-type II predator-prey stochastic model in polluted environments with interspecific competition and impulsive toxicant input.

In this paper, we use a mean-reverting Ornstein-Uhlenbeck process to simulate the stochastic perturbations in the environment, and then a modified Leslie-Gower Holling-type II predator-prey stochastic model in a polluted environment with interspecific competition and pulse toxicant input is proposed. Through constructing V-function and applying Ito^'s formula, the sharp sufficient conditions including strongly persistent in the mean, persistent in the mean and extinction are established. In addition, the theoretical results are verified by numerical simulation.

Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth.

The CRISPR/Cas9 system is an efficient tool for reverse genetics validation, and the application of this system in the cell lines provides a new perspective on target gene analysis for the development of biotechnology tools. However, in the cell lines of diamondback moth, Plutella xylostella, the integrity of the CRISPR/Cas9 system and the utilization of this cell lines still need to be improved to ensure the application of the system. Here, we stabilize the transfection efficiency of the P. xylostella cell lines at different passages at about 60% by trying different transfection reagents and adjusting the transfection method. For Cas9 expression in the CRIPSPR/Cas9 system, we identified a strong endogenous promoter: the 217-2 promoter. The dual-luciferase and EGFP reporter assay demonstrated that it has a driving efficiency close to that of the IE1 promoter. We constructed pB-Cas9-Neo plasmid and pU6-sgRNA plasmid for CRISPR/Cas9 system and subsequent cell screening. The feasibility of the CRISPR/Cas9 system in P. xylostella cell lines was verified by knocking out endogenous and exogenous genes. Finally, we generated a transgenic Cas9 cell line of P. xylostella that would benefit future exploitation, such as knock-in and multi-threaded editing. Our works provides the validity of the CRISPR/Cas9 system in the P. xylostella cell lines and lays the foundation for further genetic and molecular studies on insects, particularly favoring gene function analysis.

Mapping and Ablation of Isolated Frequent Symptomatic Premature Atrial Contractions in Patients With Structurally Normal Heart.

Background: The present study investigated the safety and efficacy of mapping and ablating isolated premature atrial contractions (PACs) in patients with a structurally normal heart, as well as whether the elimination of PACs by radiofrequency catheter ablation (RFCA) improved symptoms and the quality of life. Methods: Forty-three consecutive patients with frequent, symptomatic, and drug-refractory PACs, but without atrial tachyarrhythmias (≥5 beats), were enrolled. In all patients, we performed physical, laboratory, and imaging examinations to exclude structural heart disease. The quality of life questionnaire SF-36 before and 3 months after RFCA was performed in each patient. Results: Twenty-three men and 20 women with an average age of 52.6 ± 17.6 years were finally enrolled. The mean number of PACs was 21,685 ± 9,596 per 24 h, and the mean PACs' burden was 28.9 ± 13.7%. Short runs of tachycardia (<5 atrial beats) were observed in 32 patients (74.4%). All patients underwent successful RFCA without complications. The activation time at the successful ablation sites preceded the onset of the P-wave by 36 ± 7.6 ms. During 15 ± 8 months of follow-up, the recurrence of PACs was observed in 2 patients. The 24-h PAC burden was significantly reduced 3 months after RFCA (mean 0.5%, p < 0.05). The quality of life scores were significantly increased 3 months after RFCA (all p < 0.05). Conclusions: RFCA was feasible, safe, and effective to eliminate isolated frequent, symptomatic, and drug-refractory PACs in patients with a structurally normal heart. The elimination of PACs by RFCA significantly improved symptoms and the quality of life.

Persistence and extinction of a modified Leslie-Gower Holling-type â ¡ predator-prey stochastic model in polluted environments with impulsive toxicant input.

In this paper, a modified Leslie-Gower Holling-type Ⅱ two-predator one-prey stochastic model in polluted environments with impulsive toxicant input is proposed where we use an Ornstein-Uhlenbeck process to improve the stochasticity of the environment. The sharp sufficient conditions for persistence in the mean and extinction are established. The results reveal that the persistence and extinction of the species have close relationships with the toxicant and environmental stochasticity. In addition, the theoretical results are verified by numerical simulation.

Chocolate Consumption and Risk of Heart Failure: A Meta-Analysis of Prospective Studies.

Epidemiological studies have shown inconsistent findings on the association between chocolate consumption and risk of heart failure (HF). We, therefore, performed a meta-analysis of prospective studies to determine the role of chocolate intake in the prevention of HF. We searched databases of PubMed, Web of Science, and Scopus through December 2016 and scrutinized the reference lists of relevant literatures to identify eligible studies. Study-specific hazard ratios (HRs) and 95% confidence intervals (CIs) were aggregated using random effect models. The dose-response relationship between chocolate consumption and incident HF was also assessed. This meta-analysis is registered with PROSPERO, number CRD42017054230. Five prospective studies with 106,109 participants were finally included. Compared to no consumption of chocolate, the pooled HRs (95% CIs) of HF were 0.86 (0.82-0.91) for low-to-moderate consumption (<7 servings/week) and 0.94 (0.80-1.09) for high consumption (≥7 servings/week). In dose-response meta-analysis, we detected a curve linear relationship between chocolate consumption and risk of HF (p for nonlinearity = 0.005). Compared with non-consumption, the HRs (95% CIs) of HF across chocolate consumption levels were 0.92 (0.88-0.97), 0.86 (0.78-0.94), 0.93 (0.85-1.03), and 1.07 (0.92-1.23) for 1, 3, 7, and 10 servings/week, respectively. In conclusion, chocolate consumption in moderation may be associated with a decreased risk of HF.

A Single-Cell Roadmap of Lineage Bifurcation in Human ESC Models of Embryonic Brain Development.

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/ß-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders.

CORTECON: a temporal transcriptome analysis of in vitro human cerebral cortex development from human embryonic stem cells.

Many neurological and psychiatric disorders affect the cerebral cortex, and a clearer understanding of the molecular processes underlying human corticogenesis will provide greater insight into such pathologies. To date, knowledge of gene expression changes accompanying corticogenesis is largely based on murine data. Here we present a searchable, comprehensive, temporal gene expression data set encompassing cerebral cortical development from human embryonic stem cells (hESCs). Using a modified differentiation protocol that yields neurons suggestive of prefrontal cortex, we identified sets of genes and long noncoding RNAs that significantly change during corticogenesis and those enriched for disease-associations. Numerous alternatively spliced genes with varying temporal patterns of expression are revealed, including TGIF1, involved in holoprosencephaly, and MARK1, involved in autism. We have created a database (http://cortecon.neuralsci.org/) that provides online, query-based access to changes in RNA expression and alternatively spliced transcripts during human cortical development.

miRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure.

microRNAs (miRNAs) are crucial for cellular development and homeostasis. In order to better understand regulation of miRNA biosynthesis, we studied cleavage of primary miRNAs by Drosha. While Drosha knockdown triggers an expected decrease of many mature miRNAs in human embryonic stem cells (hESC), a subset of miRNAs are not reduced. Statistical analysis of miRNA secondary structure and fold change of expression in response to Drosha knockdown showed that absence of mismatches in the central region of the hairpin, 5 and 9-12 nt from the Drosha cutting site conferred decreased sensitivity to Drosha knockdown. This suggests that, when limiting, Drosha processes miRNAs without mismatches more efficiently than mismatched miRNAs. This is important because Drosha expression changes over cellular development and the fold change of expression for miRNAs with mismatches in the central region correlates with Drosha levels. To examine the biochemical relationship directly, we overexpressed structural variants of miRNA-145, miRNA-137, miRNA-9, and miRNA-200b in HeLa cells with and without Drosha knockdown; for these miRNAs, elimination of mismatches in the central region increased, and addition of mismatches decreased their expression in an in vitro assay and in cells with low Drosha expression. Change in Drosha expression can be a biologically relevant mechanism by which eukaryotic cells control miRNA profiles. This phenomenon may explain the impact of point mutations outside the seed region of certain miRNAs.

Transjugular approach for radiofrequency ablation of premature ventricular contractions originating from the superior tricuspid annulus.

We report a 33-year-old man with idiopathic frequent premature ventricular contractions (PVCs) originating from the superior tricuspid annulus. The PVCs were successfully abolished via a transjugular approach because of poor contact of the ablation catheter via a femoral vein approach.

Perimembranous ventricular septal defect with aneurysm: two options for transcatheter closure.

We present our experience with 2 options for device closure of perimembranous ventricular septal defect with aneurysm. Thirty-four patients with perimembranous ventricular septal defect with aneurysm, aged from 14 to 42 years, underwent transcatheter closure with modified double-disk occluders. A sheath was used to deliver the occluder after establishment of a stable "arteriovenous loop" under fluoroscopy. Electrocardiography and transthoracic echocardiography were used for follow-up. All but 1 patient experienced successful transcatheter closure of perimembranous ventricular septal defect with aneurysm, when occluders were used in 2 different positions. There were 19 patients whose perimembranous ventricular septal defects were closed at the inlet of the aneurysm and 15 patients whose defects were closed at the outlet. Eight patients had a residual shunt immediately after the procedure, which disappeared during follow-up. One patient developed minor aortic regurgitation. Four patients who manifested different types of conductive block were all in the group that underwent closure at the inlet of the aneurysm. No other complications were observed during follow-up.We infer that perimembranous ventricular septal defect with aneurysm can be successfully closed with modified double-disk occluders. Each of the 2 options that we have presented for transcatheter closure of perimembranous ventricular septal defect with aneurysm has its advantages and disadvantages. Ultimately, the configuration of the lesion should decide the type and position of the device.

Targeted gene addition to human mesenchymal stromal cells as a cell-based plasma-soluble protein delivery platform.

BACKGROUND AIMS: Gene-modified mesenchymal stromal cells (MSC) provide a promising tool for cell and gene therapy-based applications by potentially acting as a cellular vehicle for protein-replacement therapy. However, to avoid the risk of insertional mutagenesis, targeted integration of a transgene into a 'safe harbor' locus is of great interest. METHODS: We sought to determine whether zinc finger nuclease (ZFN)-mediated targeted addition of the erythropoietin (Epo) gene into the chemokine [C-C motif] receptor 5 (CCR5) gene locus, a putative safe harbor locus, in MSC would result in stable transgene expression in vivo. RESULTS: Whether derived from bone marrow (BM), umbilical cord blood (UCB) or adipose tissue (AT), 30-40% of human MSC underwent ZFN-driven targeted gene addition, as determined by a combination of fluorescence-activated cell sorting (FACS)- and polymerase chain reaction (PCR)-based analyzes. An enzyme-linked immunosorbent assay (ELISA)-based analysis of gene-targeted MSC expressing Epo from the CCR5 locus showed that these modified MSC were found to secrete a significant level of Epo (c. 2 IU/10(6)cells/24 h). NOD/SCID/gammaC mice injected with ZFN-modified MSC expressing Epo exhibited significantly higher hematocrit and Epo plasma levels for several weeks post-injection, compared with mice receiving control MSC. CONCLUSIONS: These data demonstrate that MSC modified by ZFN-driven targeted gene addition may represent a cellular vehicle for delivery of plasma-soluble therapeutic factors.

Generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human TF lentivirus set.

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.(1) The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.(2) Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.(3) iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells. Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

The use of SC1 (Pluripotin) to support mESC self-renewal in the absence of LIF.

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.(1) However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.(2) The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.(3) Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

Inflammation in lone atrial fibrillation.

OBJECTIVES: This study was designed to evaluate the correlation between lone atrial fibrillation and inflammation. METHODS: A total of 411 subjects were enrolled in this study, including 333 patients with lone atrial fibrillation, and 78 controls. C-reactive protein (CRP) and echocardiography were evaluated, and the electrocardiograph was monitored to identify cardiac rhythm at the time of blood sampling. According to the rhythm, paroxysmal atrial fibrillation was divided into presence and absence of atrial fibrillation. RESULTS: Subjects with lone atrial fibrillation had higher CRP levels than controls (media, 1.00 mg/L; IQR, 1.00-2.54 versus media, 1.00 mg/L; IQR, 1.00-1.55; p = 0.016) and subjects with persistent atrial fibrillation had higher CRP levels than those with paroxysmal atrial fibrillation (media, 1.62 mg/L; IQR, 1.00-3.98 versus media, 1.00 mg/L, IQR, 1.00-2.10; p = 0.022), and so did presence of atrial fibrillation rather than absence of atrial fibrillation (media, 2.11 mg/L; IQR, 1.00-3.60 versus media, 1.00 mg/L; IQR, 1.00-1.76; p = 0.000) in paroxysmal atrial fibrillation. However, there was no significant difference in CRP levels between persistent atrial fibrillation and presence of atrial fibrillation in paroxysmal atrial fibrillation (p = 0.992). Neither was there any difference between absence of atrial fibrillation in paroxysmal atrial fibrillation and controls (p = 0.483). In patients with lone atrial fibrillation, atrial fibrillation rhythm (B = 4.85, 95%CI: 2.61-8.99) was the only independent predictor of elevated CRP levels after adjusted covariants. CONCLUSIONS: Patients with lone atrial fibrillation had elevated CRP levels only when they were in atrial fibrillation rhythm and an elevated CRP level was not related to duration of time or history of atrial fibrillation.

The morphology changes in limb leads after ablation of verapamil-sensitive idiopathic left ventricular tachycardia and their correlation with recurrence.

OBJECTIVES: This study was designed to explore the morphology changes in limb leads of ECGs after successful ablation of verapamil sensitive idiopathic left ventricular tachycardia (ILVT) and their correlation with tachycardia recurrence. METHODS: Between January 2001 and December 2006, 116 patients who underwent successful ablation of ILVT were included in the study. Twelve-lead surface ECG recordings during sinus rhythm were obtained in all patients before and after ablation to compare morphology changes in limb leads. RESULTS: The ECG morphology changes after ablation were divided into two categories: one with new or deepening Q wave in inferior leads and/or disappearance of Q wave in leads I and aVL, and the other without change. The changes in any Lead II, III, or aVF after ablation occurred significantly more in patients without recurrence of ventricular tachycardia (VT) (P < 0.0001, 0.002, and 0.0001, respectively). The patients with recurrence of VT tended to have no ECG changes, compared with those without recurrence of VT (P = 0.009). The sensitivity of leads II, III, and aVF changes in predicting nonrecurrence VT were 66.7%, 78.7%, and 79.6%, specificity were 100%, 75%, and 87.5%, and nonrecurrence predictive value of 100%, 97.7%, and 98.9%, respectively. When inferior leads changes were combined, they could predict all nonrecurrence patients with 100% specificity. CONCLUSIONS: Successful radiofrequency ablation of ILVT could result in morphology changes in limb leads of ECG, especially in inferior leads. The combined changes in inferior leads can be used as an effective endpoint in ablation of this ILVT.

Self-renewal of embryonic stem cells by a small molecule.

A cell-based screen of chemical libraries was carried out to identify small molecules that control the self-renewal of ES cells. A previously uncharacterized heterocycle, SC1, was discovered that allows one to propagate murine ES cells in an undifferentiated, pluripotent state under chemically defined conditions in the absence of feeder cells, serum, and leukemia inhibitory factor. Long-term SC1-expanded murine ES cells can be differentiated into cells of the three primary germ layers in vitro and also can generate chimeric mice and contribute to the germ line in vivo. Biochemical and cellular experiments suggest that SC1 works through dual inhibition of RasGAP and ERK1. Molecules of this kind may not only facilitate practical applications of stem cells in research and therapy, but also provide previously undescribed insights into the complex biology of stem cells.