Development of leafhopper cell culture to trace the early infection process of a nucleorhabdovirus, rice yellow stunt virus, in insect vector cells.

BACKGROUND: In China, the rice pathogen Rice yellow stunt virus (RYSV), a member of the genus Nucleorhabdovirus in the family Rhabdoviridae, was a severe threat to rice production during the1960s and1970s. Fundamental aspects of the biology of this virus such as protein localization and formation of the RYSV viroplasm during infection of insect vector cells are largely unexplored. The specific role(s) of the structural proteins nucleoprotein (N) and phosphoprotein (P) in the assembly of the viroplasm during RYSV infection in insect vector is also unclear. METHODS: In present study, we used continuous leafhopper cell culture, immunocytochemical techniques, and transmission electron microscopy to investigate the subcellular distributions of N and P during RYSV infection. Both GST pull-down assay and yeast two-hybrid assay were used to assess the in vitro interaction of N and P. The dsRNA interference assay was performed to study the functional roles of N and P in the assembly of RYSV viroplasm. RESULTS: Here we demonstrated that N and P colocalized in the nucleus of RYSV-infected Nephotettix cincticeps cell and formed viroplasm-like structures (VpLSs). The transiently expressed N and P are sufficient to form VpLSs in the Sf9 cells. In addition, the interactions of N/P, N/N and P/P were confirmed in vitro. More interestingly, the accumulation of RYSV was significantly reduced when the transcription of N gene or P gene was knocked down by dsRNA treatment. CONCLUSIONS: In summary, our results suggest that N and P are the main viral factors responsible for the formation of viroplasm in RYSV-infected insect cells. Early during RYSV infection in the insect vector, N and P interacted with each other in the nucleus to form viroplasm-like structures, which are essential for the infection of RYSV.

Decreased IL-27 Negatively Correlated with Th17 Cells in Non-Small-Cell Lung Cancer Patients.

The presence of Th17 cells and IL-27 is observed in a variety of inflammatory associated cancers. However, there are some data on the role of Th17 cells and IL-27 in the regulation of immune reactions in non-small-cell lung cancer (NSCLC). The aim of this study is to assess the variation of Th17 cells and IL-27 in the peripheral blood (PB) of patients with NSCLC. The proportion of Th17 cells in peripheral blood mononuclear cells (PBMCs) was evaluated by flow cytometry. The serum concentrations of IL-27 and IL-17 were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of RORγt and IL-27 in the peripheral blood was examined by real-time quantitative polymerase chain reaction (QPCR). Expression of IL-27 was lower in NSCLC patients compared with normal controls. The frequency of Th17 cells was increased in NSCLC patients, accompanied by the upregulation of IL-17 and RORγt. IL-27 negatively correlated with the number of Th17 cells and the RORγt mRNA. Our results indicate that IL-27 might inhibit Th17 differentiation in NSCLC patients and better understanding of the regulatory effects of IL-27 on Th17 cells may shed light on potential new targets in cancer prevention and therapy.

Cut&tag: a powerful epigenetic tool for chromatin profiling.

Analysis of transcription factors and chromatin modifications at the genome-wide level provides insights into gene regulatory processes, such as transcription, cell differentiation and cellular response. Chromatin immunoprecipitation is the most popular and powerful approach for mapping chromatin, and other enzyme-tethering techniques have recently become available for living cells. Among these, Cleavage Under Targets and Tagmentation (CUT&Tag) is a relatively novel chromatin profiling method that has rapidly gained popularity in the field of epigenetics since 2019. It has also been widely adapted to map chromatin modifications and TFs in different species, illustrating the association of these chromatin epitopes with various physiological and pathological processes. Scalable single-cell CUT&Tag can be combined with distinct platforms to distinguish cellular identity, epigenetic features and even spatial chromatin profiling. In addition, CUT&Tag has been developed as a strategy for joint profiling of the epigenome, transcriptome or proteome on the same sample. In this review, we will mainly consolidate the applications of CUT&Tag and its derivatives on different platforms, give a detailed explanation of the pros and cons of this technique as well as the potential development trends and applications in the future.

Dynamic Modeling for Chatter Analysis in Micro-Milling by Integrating Effects of Centrifugal Force, Gyroscopic Moment, and Tool Runout.

During micro-milling, regenerative chatter will decrease the machining accuracy, destabilize the micro-milling process, shorten the life of the micro-mill, and increase machining failures. Establishing a mathematical model of chatter vibration is essential to suppressing the adverse impact of chatter. The mathematical model must include the dynamic motions of the cutting system with the spindle-holder-tool assembly and tool runout. In this study, an integrated model was developed by considering the centrifugal force induced by rotational speeds, the gyroscopic effect introduced by high speeds, and the tool runout caused by uncertain factors. The tool-tip frequency-response functions (FRFs) obtained by theoretical calculations and the results predicted by simulation experiments were compared to verify the developed model. And stability lobe diagrams (SLDs) and time-domain responses are depicted and analyzed. Furthermore, experiments on tool-tip FRFs and micro-milling were conducted. The results validate the effectiveness of the integrated model, which can calculate the tool-tip FRFs, SLDs, and time responses to analyze chatter stability by considering the centrifugal force, gyroscopic effect, and tool runout.

Nutritional and volatile profiles of pulp and flavedo from four local pummelo cultivars grown in Fujian province of China.

The nutritional and volatile profiles of pulp and flavedo samples from four distinct local pummelo landraces ("Siji," "Pingshan," "Wendan," and "Guanxi") cultivated in Fujian province of China were investigated. "Guanxi" pummelo exhibited relatively high contents of vitamin C (42.01 mg/100 mL) and phenols (360.61 mg/L) and displayed a robust antioxidant capacity (41.15 mg/100 mL). Conversely, the red pulp from "Pingshan" demonstrated relatively high values of carotenoids (55.96 µg/g) and flavonoids (79.79 mg/L). Considerable differences were observed in volatile compositions between the two fruit tissues and among the four genotypes. A total of 166 and 255 volatile compounds were detected in the pulp and flavedo samples, respectively. Notably, limonene and ß-myrcene were identified as the principal volatile compounds in flavedo, whereas hexanal was highly abundant in the pulp of "Siji," "Pingshan," and "Guanxi." "Wendan" displayed distinct separation from the other three pummelo cultivars in principal component analysis based on the pulp volatile compositions. This distinction was attributed to the higher number and content of volatile compounds in "Wendan" pulp, particularly the remarkable enrichment of ß-myrcene. The newly characterized pummelo landraces and genotype/tissue-dependent variations in volatiles provide essential information for the genetic improvement of pummelo aroma, as well as for fruit processing and utilization.

Parametric Neural Network-Based Model Free Adaptive Tracking Control Method and Its Application to AFS/DYC System.

This paper deals with adaptive nonlinear identification and trajectory tracking problem for model free nonlinear systems via parametric neural network (PNN). Firstly, a more effective PNN identifier is developed to obtain the unknown system dynamics, where a parameter error driven updating law is synthesized to ensure good identification performance in terms of accuracy and rapidity. Then, an adaptive tracking controller consisting of a feedback control term to compensate the identified nonlinearity and a sliding model control term to deal with the modeling error is established. The Lyapunov approach is synthesized to ensure the convergence characteristics of the overall closed-loop system composed of the PNN identifier and the adaptive tracking controller. Simulation results for an AFS/DYC system are presented to confirm the validity of the proposed approach.

Fruit quality and volatile constituents of a new very early-ripening pummelo (Citrus maxima) cultivar 'Liuyuezao'.

'Liuyuezao' (LYZ) pummelo (Citrus maxima) originated from a spontaneous bud sport on a 'Guanxi' (GXB) pummelo tree and was released as a new very early-season cultivar. The objective of this study was to present the sensory and nutritional profiles of LYZ fruits, and compare it with other major commercialized pummelo cultivars including GXB, 'Sanhong' (SH) and 'Hongrou' (HR). LYZ had higher contents of organic acids (12.01 mg/g), phenols (669.01 mg/L), vitamin C (75.73 mg/100 mL) and stronger antioxidant capacity (77.65 mg/100 mL) but lower levels of soluble sugars (62.85 mg/g), carotenoids (0.25 mg/L) and flavonoids (46.3 mg/L) when compared to the other pummelos. Moreover, a smaller number (49) and much less content (7.63) of fruit volatiles were detected in LYZ than them in GXB, SH and HR. The relatively high levels of fructose (20.6 mg/g) and organic acids and low levels of volatile compounds in LYZ mainly contributed to its sweet and mildly sour taste and moderate aroma of pummelo note. LYZ is presented as an alternative pummelo cultivar with the potential for commercialization.

Metabolite signatures of diverse Camellia sinensis tea populations.

The tea plant (Camellia sinensis) presents an excellent system to study evolution and diversification of the numerous classes, types and variable contents of specialized metabolites. Here, we investigate the relationship among C. sinensis phylogenetic groups and specialized metabolites using transcriptomic and metabolomic data on the fresh leaves collected from 136 representative tea accessions in China. We obtain 925,854 high-quality single-nucleotide polymorphisms (SNPs) enabling the refined grouping of the sampled tea accessions into five major clades. Untargeted metabolomic analyses detect 129 and 199 annotated metabolites that are differentially accumulated in different tea groups in positive and negative ionization modes, respectively. Each phylogenetic group contains signature metabolites. In particular, CSA tea accessions are featured with high accumulation of diverse classes of flavonoid compounds, such as flavanols, flavonol mono-/di-glycosides, proanthocyanidin dimers, and phenolic acids. Our results provide insights into the genetic and metabolite diversity and are useful for accelerated tea plant breeding.

[Expression of FAP and alpha-SMA during the incised wound healing in mice skin].

UNLABELLED: OBJECTIVE To investigate the time-dependent expression of fibroblast activation protein (FAP) and alpha-smooth muscle actin(alpha-SMA) during the incised wound healing of the skin in mice. METHODS: The expression of FAP and alpha-SMA in incised wound of mice skin was detected by immunohistochemistry and Western blot. RESULTS: By immunohistochemistry, the expression of FAP and alpha-SMA in the normal skin and the skin 1 h after injury maintained at a very low level, but the positive cells expressing FAP and alpha-SMA started to elevate 6 h after injury and reached its peak on 5 d for FAP and on 3 d for alpha-SMA, then gradually decreased to the normal level on 14 d. The expression of FAP and alpha-SMA was observed throughout the wound healing stages 1 d after injuries by Western blot as well with a peak expression occurring on 5 d for FAP and on 3 d for alpha-SMA after injury. CONCLUSION: FAP may be a potentially useful marker for wound age determination and alpha-SMA may be used as an effective indicator for the mid- and late stage incised wound of mice skin. The combination use of FAP and alpha-SMA may be potentially effective indicators for wound age determination.

Nutrient-starved incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an unconventional frontline tuberculosis drug characterized by high in vivo sterilizing activity, but poor in vitro activity. The study on the mechanism of action of PZA has attracted significant attention because of the peculiarity of PZA and its ability to shorten the tuberculosis chemotherapy. In this study, we examined the effect of nutrient-starved conditions on PZA activity in vitroat acid pH. We also examined the effect of fatty acids, benzoic acid and salicylic acid on PZA activity. The results showed that nutrient-starved conditions lowered the membrane potential of Mycobacterium tuberculosis and enhanced the activity of PZA, with 5- and 10-day starvation conditions resulting in greater enhancement than 3-day starvation. Fatty acids, benzoic acid and salicylic acid enhanced PZA activity in both normal and starved bacilli, especially in starved bacilli. These findings provide further support for the recent model of PZA action and may have implications for developing new drugs that shorten therapy.

[Nutrient starved incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis].

OBJECTIVE: To investigate the mechanism of the action and the target of pyrazinamide (PZA), by examining the effect of nutrient-starved conditions on PZA activity. METHODS: M. tuberculosis starved in PBS for 3, 5 or 10 days were treated with PZA, and with benzoic acid, salicylic acid or fatty acid in combination with PZA. The activity of PZA against M.tuberculosis was determined by the number of colony-forming unit (CFU). The membrane potential of M.tuberculosis was determined by flow-cytometry with Rhodamine to investigate the mechanism of PZA activity. RESULTS: The CFU of M. tuberculosis treated with PZA for 0, 3, 5, 10 days nutrient starvation compared with their respective controls, decreased by 23.08%, 37.75%, 82.32%, 81.03%, respectively. The membrane potential of M. tuberculosis in nutrient-starved cultures declined rapidly over the first 5 days' starvation. The starved bacilli treated with PZA had significantly lower level of membrane potential compared with the control. The effect of PZA on lowering the membrane potential was antagonized by addition of glucose, which provided energy for the bacilli. Fatty acids, benzoic acid and salicylic acid-enhanced PZA activity in both normal and starved bacilli, especially in starved bacilli. CONCLUSION: Nutrient starved incubation conditions and weak acids could enhance pyrazinamide activity against Mycobacterium tuberculosis. PZA can impair Mycobacterium tuberculosis cell viability by disrupting the energy supply system and reducing membrane potential of the cells.

Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism.

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates.

Nonlinear systems identification and control via dynamic multitime scales neural networks.

This paper deals with the adaptive nonlinear identification and trajectory tracking via dynamic multilayer neural network (NN) with different timescales. Two NN identifiers are proposed for nonlinear systems identification via dynamic NNs with different timescales including both fast and slow phenomenon. The first NN identifier uses the output signals from the actual system for the system identification. In the second NN identifier, all the output signals from nonlinear system are replaced with the state variables of the NNs. The online identification algorithms for both NN identifier parameters are proposed using Lyapunov function and singularly perturbed techniques. With the identified NN models, two indirect adaptive NN controllers for the nonlinear systems containing slow and fast dynamic processes are developed. For both developed adaptive NN controllers, the trajectory errors are analyzed and the stability of the systems is proved. Simulation results show that the controller based on the second identifier has better performance than that of the first identifier.

Expression of HMGB1 and RAGE in rat and human brains after traumatic brain injury.

BACKGROUND: Increasing evidence suggests that an inflammatory reaction contributes to the secondary brain injury that plays a critical role in the clinical outcome of patients with traumatic brain injury (TBI). Recently, high-mobility group box 1 (HMGB1) has been identified as a key cytokine in the inflammatory reaction and may represent a new target for the treatment of TBI. However, the expression of HMGB1 during this injury process has not yet been studied. METHODS: In this study, the levels of both HMGB1 and receptor for advanced glycation end products (RAGE) in the rat brain were analyzed by Western blot at different time points after TBI. Immunohistochemistry was also performed to examine the expression pattern of HMGB1 and RAGE in both the rat and the human brain after TBI. RESULTS: In the rat brain, HMGB1 levels significantly declined below the basal level at 6 hours after TBI and then gradually returned to the basal level 2 days later. RAGE expression increased 6 hours after TBI and reached its peak after 1 day; this level then slowly decreased but remained higher than the sham-injury group until 6 days after TBI. In both rat and human brains, HMGB1 either disappeared or was translocated from the nucleus to the cytoplasm at early stages after TBI and then was localized to the cytoplasm of phagocytic microglia at later stages. RAGE expression increased in the region surrounding the contused area after TBI in both rat and human brains. At later stages, RAGE was mainly expressed in microglia. CONCLUSION: HMGB1 is involved in both early and later stages after TBI. Targeting HMGB1 signaling may be a promising therapeutic approach for the treatment of TBI.

Multiplex detection of mutations in clinical isolates of rifampin-resistant Mycobacterium tuberculosis by short oligonucleotide ligation assay on DNA chips.

A new approach, short-oligonucleotide-ligation assay on DNA chip (SOLAC), is developed to detect mutations in rifampin-resistant Mycobacterium tuberculosis. The method needs only four common probes to detect 15 mutational variants of the rpoB gene within 12 h. Fifty-five rifampin-resistant M. tuberculosis isolates were analyzed, resulting in 87.3% accuracy and 83.6% concordance relative to DNA sequencing.