Development and application of a colloidal-gold immunochromatographic strip for detecting Getah virus antibodies.

Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: • We established a rapid antibody detection method that can monitor GETV infection • We developed colloidal gold test strips with high sensitivity and specificity • The development of colloidal gold test strips will aid in the field serologic detection of GETV.

Quasi-Classical Trajectory Calculation of Rate Constants Using an Ab Initio Trained Machine Learning Model (aML-MD) with Multifidelity Data.

Machine learning (ML) provides a great opportunity for the construction of models with improved accuracy in classical molecular dynamics (MD). However, the accuracy of a ML trained model is limited by the quality and quantity of the training data. Generating large sets of accurate ab initio training data can require significant computational resources. Furthermore, inconsistent or incompatible data with different accuracies obtained using different methods may lead to biased or unreliable ML models that do not accurately represent the underlying physics. Recently, transfer learning showed its potential for avoiding these problems as well as for improving the accuracy, efficiency, and generalization of ML models using multifidelity data. In this work, ab initio trained ML-based MD (aML-MD) models are developed through transfer learning using DFT and multireference data from multiple sources with varying accuracy within the Deep Potential MD framework. The accuracy of the force field is demonstrated by calculating rate constants for the H + HO2 → H2 + 3O2 reaction using quasi-classical trajectories. We show that the aML-MD model with transfer learning can accurately predict the rate constants while reducing the computational cost by more than five times compared to the use of more expensive quantum chemistry training data sets. Hence, the aML-MD model with transfer learning shows great potential in using multifidelity data to reduce the computational cost involved in generating the training set for these potentials.

PDZK1 improves ventricular remodeling in hypertensive rats by regulating the stability of the Mas receptor.

Ventricular remodeling is one of the main causes of mortality from heart failure due to hypertension. Exploring its mechanism and finding therapeutic targets have become urgent scientific problems to be solved. A number of studies have shown that Mas, as an Ang-(1-7) specific receptor, was significantly reduced in myocardial tissue of rats undergoing hypertensive ventricular remodeling. It has been reported that Mas receptor levels are significantly downregulated in myocardium undergoing ventricular remodeling, but studies focused on intracellular and post-translational modifications of Mas are lacking. The results of this research are as follows: (1) PDZK1 interacts with the carboxyl terminus of Mas through its PDZ1 domain; (2) the expression of PDZK1 and Mas is decreased in rats undergoing hypertensive ventricular remodeling, and PDZK1 upregulation can ameliorate hypertensive myocardial fibrosis and myocardial hypertrophy; (3) PDZK1 enhances the stability of Mas protein through the proteasome pathway, and the proteasome inhibitor MG132 promotes hypertensive ventricular remodeling. PDZK1 improves ventricular remodeling in hypertensive rats by regulating Mas receptor stability. This study provides a scientific basis for the prevention and treatment of ventricular remodeling.

Identify nitrogen transport paths and sources contribution in karst valley depression area using isotopic approach.

Karst groundwater provides drinking water for a quarter of Earth's population. However, in intensive agricultural regions worldwide, karst water is commonly polluted by nitrate (NO3-), particularly in the valley depression areas with well hydrological connectivity. The valley depression aquifers are particularly vulnerable to anthropogenic pollution because their pipes and sinkholes respond quickly to rainfall events and anthropogenic inputs. Identifying nitrate sources and transport paths in the valley depression areas is key to understanding the nitrogen cycle and effectively preventing and controlling NO3- pollution. This study collected high-resolution samples at four sites including one surface stream-SS, two sinkholes-SH and reservoir-Re, during the wet season in the headwater sub-catchment. The chemical component concentrations and stable isotopes (δ15N-NO3- and δ18O-NO3-) were analyzed. The stable isotope analysis model in R language (SIAR) was used to quantitatively analyze the contribution rate of NO3- sources. The results showed that the down section site (Re) has the highest [NO3--N], followed by SH and the lowest SS. The sources calculation of SIAR demonstrated that, during the non-rainfall period, soil organic nitrogen was the primary source of the down section site, followed by fertilizer and the upper reaches sinkholes. During the rainfall period, fertilizer was the primary source of the down section site, followed by soil organic nitrogen and from upper reaches sinkholes. Rainfall events accelerated fertilizer-leaching into the groundwater. Slight denitrification may have occurred at the sampling sites but the assimilation of Re and SH could not occur. In conclusion, agricultural activities were still the primary influencing factor of [NO3--N] in the study area. Therefore, the focus of NO3- prevention and control in the valley depression areas should consider the methods and timing of fertilization and the spatial distribution of sinkholes. To reduce nitrogen flux in the valley depression area, effective management policy should consider, e.g., prolongation of water residence time by wetland, and blocking nitrogen loss paths by sinkholes.

Analysis of Spatiotemporal Changes of Ecological Environment Quality and Its Coupling Coordination with Urbanization in the Yangtze River Delta Urban Agglomeration, China.

It is inevitable that urban agglomeration will have a coercive impact on the regional Ecological Environment Quality (EEQ) as a consequence of high-speed urbanization. Balancing the EEQ and urbanization development has become a problem worthy of attention. In order to objectively evaluate the EEQ of the Yangtze River Delta Urban Agglomeration (YRDUA) and explore the impact of the urbanization process on it, this paper is based on the Modified Remote Sensing Ecological Index (MRSEI) and the Comprehensive Night Light Index (CNLI), respectively, and conducts a quantitative assessment of the YRDUA in China from 2000 to 2020. The results show that: (1) From 2000 to 2020, the MRSEI of the YRDUA first decreased and then increased, and the ecological environment quality degraded first and then improved; however, there were significant differences between regions. The ecological environment quality in the south is obviously better than that in the north, and the ecological environment quality in the north changes more drastically, and the low value area of MRSEI will gradually move downstream as time changes; (2) During the study period, the YRDUA formed a hierarchical and progressive urbanization pattern. The inland urbanization process expanded from east to west along the Yangtze River, and the urbanization process of coastal cities expanded from Shanghai as the center to the north and south with high-intensity urbanization cities concentrated in Shanghai and its surrounding cities and low-intensity urbanization cities distributed in the western part of the urban agglomeration; (3) The Coupling Coordination Degree (CCD) between urbanization and EEQ in the YRDUA has continuously improved with an increase of 28.57% in the past 21 years, and the number of cities with high level coupling continues to rise, while the number of medium level coupling cities and low level coupling cities has declined. As a large-scale and long-term analysis of changes in the EEQ and the urbanization process, this study can provide theoretical support for policymakers to formulate mesoscale development planning, EEQ monitoring, and environmental protection policies.

The effect of reservoir expansion from underground karst cave to surface reservoir on water quality in southwestern China.

Due to the excessive exploitation of traditional energy sources, the attention paid to water energy has increased in recent years. As an important means to effectively utilize water energy, reservoirs play an important role in drinking water, irrigation, flood control, and drought resistance. However, utilizing reservoirs often led to water quality issues resulting from the interaction of nutrients and hydrological conditions, especially due to the special structure of karst areas. Because of the change of hydrological conditions by the effect of dam construction, the dynamic of water quality will be more obvious in karst areas with a fast exchange of water and contaminants between underground and surface streams. In the present study, the change in water quality of a karst reservoir, the Muzhu Reservoir in the Houzhai Catchment, was studied. Long-term monitored datasets (1981-2002) and water quality datasets of more recent years were used to assess the effect on the water quality of reservoir expansion from the underground reservoir to the surface reservoir in a karst area. Long-term series datasets had shown that the hydro-chemistry type had been changed from HCO3-·SO42--Ca2+·Mg2+ type to HCO3--Ca2+ type in the short term after the reservoir's expansion. The chemical components of water originating from a rock background reduced markedly after the reservoir's expansion, whereas the content of the anthropogenic contribution in the water decreased after the expansion, except in April and May. Isotopic characteristics showed that δ15N-NO3- and δ18O-NO3- values were positively correlated before and after the reservoir expansion, but the slope of the linear regression before the expansion was 0.34, while the slope of the linear regression before the expansion was close to 0.7. This indicated that although denitrification and assimilation may occur simultaneously after the reservoir's expansion, the role of denitrification on nitrate removal decreased, which resulted in nitrate accumulation in the karst reservoir. The results highlighted that nitrate accumulation in karst reservoirs should be monitored to decrease nitrate concentration in the future.

Hsp70 Inhibits the Replication of Fowl Adenovirus Serotype 4 by Suppressing Viral Hexon with the Assistance of DnaJC7.

Fowl adenovirus serotype 4 (FAdV-4) infection results in serious hepatitis-hydropericardium syndrome (HHS) in broilers, which has caused great economic losses to the poultry industry; however, the specific host responses to FAdV-4 remain unknown. In this study, we identified 141 high-confidence protein-protein interactions (PPIs) between the main viral proteins (Hexon, Fiber 1, Fiber 2, and Penton bases) and host proteins via a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. We found that heat shock protein 70 (Hsp70), the protein with the highest score, and its cofactor DnaJ heat shock protein 40 family member C7 (DnaJC7) could negatively regulate the replication of FAdV-4. Furthermore, the nucleotide binding domain (NBD) of Hsp70 and the J domain of DnaJC7 were necessary for inhibiting FAdV-4 replication. We verified that DnaJC7 as a bridge could bind to Hsp70 and Hexon, assisting the indirect interaction between Hsp70 and Hexon. In addition, we found that FAdV-4 infection strongly induced the expression of autophagy proteins and cellular Hsp70 in a dose-dependent manner. Blockage of Hexon by Hsp70 overexpression was significantly reduced when the autophagy pathway was blocked by the specific inhibitor chloroquine (CQ). Our results showed that Hsp70 was co-opted by DnaJC7 to interact with viral Hexon and inhibited Hexon through the autophagy pathway, leading to a considerable restriction of FAdV-4 replication. IMPORTANCE FAdV-4, as the main cause of HHS, has quickly spread all over the world in recent years, seriously threatening the poultry industry. The aim of this study was to identify the important host proteins that have the potential to regulate the life cycle of FAdV-4. We found that Hsp70 and DnaJC7 played crucial roles in regulating the amount of viral Hexon and extracellular viral titers. Moreover, we demonstrated that Hsp70 interacted with viral Hexon with the assistance of DnaJC7, followed by suppressing Hexon protein through the autophagy pathway. These results provide new insight into the role of the molecular chaperone complex Hsp70-DnaJC7 in FAdV-4 infection and suggest a novel strategy for anti-FAdV-4 drug development by targeting the specific interactions among Hsp70, DnaJC7 and Hexon.

D2I and F9Y Mutations in the NS1 Protein of Influenza A Virus Affect Viral Replication via Regulating Host Innate Immune Responses.

Influenza A viruses (IAV) modulate host antiviral responses to promote viral growth and pathogenicity. The non-structural (NS1) protein of influenza A virus has played an indispensable role in the inhibition of host immune responses, especially in limiting interferon (IFN) production. In this study, random site mutations were introduced into the NS1 gene of A/WSN/1933 (WSN, H1N1) via an error prone PCR to construct a random mutant plasmid library. The NS1 random mutant virus library was generated by reverse genetics. To screen out the unidentified NS1 functional mutants, the library viruses were lung-to-lung passaged in mice and individual plaques were picked from the fourth passage in mice lungs. Sanger sequencing revealed that eight different kinds of mutations in the NS1 gene were obtained from the passaged library virus. We found that the NS1 F9Y mutation significantly enhanced viral growth in vitro (MDCK and A549 cells) and in vivo (BALB/c mice) as well as increased virulence in mice. The NS1 D2I mutation attenuated the viral replication and pathogenicity in both in vitro and in vivo models. Further studies demonstrated that the NS1 F9Y mutant virus exhibited systematic and selective inhibition of cytokine responses as well as inhibited the expression of IFN. In addition, the expression levels of innate immunity-related cytokines were significantly up-regulated after the rNS1 D2I virus infected A549 cells. Collectively, our results revealed that the two mutations in the N-terminal of the NS1 protein could alter the viral properties of IAV and provide additional evidence that the NS1 protein is a critical virulence factor. The two characterized NS1 mutations may serve as potential targets for antiviral drugs as well as attenuated vaccine development.

M2 microglia-derived extracellular vesicles promote white matter repair and functional recovery via miR-23a-5p after cerebral ischemia in mice.

Rationale: White matter repair is critical for the cognitive and neurological functional recovery after ischemic stroke. M2 microglia are well-documented to enhance remyelination and their extracellular vesicles (EVs) mediate cellular function after brain injury. However, whether M2 microglia-derived EVs could promote white matter repair after cerebral ischemia and its underlying mechanism are largely unknown. Methods: EVs were isolated from IL-4 treated microglia (M2-EVs) and untreated microglia (M0-EVs). Adult ICR mice subjected to 90-minute transient middle cerebral artery occlusion received intravenous EVs treatment for seven consecutive days. Brain atrophy volume, neurobehavioral tests were examined within 28 days following ischemia. Immunohistochemistry, myelin transmission electron microscope and compound action potential measurement were performed to assess white matter structural remodeling, functional repair and oligodendrogenesis. The effects of M2-EVs on oligodendrocyte precursor cells (OPCs) were also examined in vitro. EVs' miRNA sequencing, specific miR-23a-5p knockdown in M2-EVs and luciferase reporter assay were used to explore the underlying mechanism. Results: M2-EVs reduced brain atrophy volume, promoted functional recovery, oligodendrogenesis and white matter repair in vivo, increased OPC proliferation, survival and differentiation in vitro. miR-23a-5p was enriched in M2-EVs and could promote OPC proliferation, survival and maturation, while knocking down miR-23a-5p in M2-EVs reversed the beneficial effects of M2-EVs both in vitro and in vivo. Luciferase reporter assay showed that miR-23a-5p directly targeted Olig3. Conclusion: Our results demonstrated that M2 microglia could communicate to OPCs through M2-EVs and promote white matter repair via miR-23a-5p possibly by directly targeting Olig3 after ischemic stroke, suggesting M2-EVs is a novel and promising therapeutic strategy for white matter repair in stroke and demyelinating disease.

Changes in microRNA Expression Profiles in Diabetic Cardiomyopathy Rats Following H3 Relaxin Treatment.

ABSTRACT: MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the mechanisms of diabetic cardiomyopathy (DCM); however, whether human recombinant relaxin-3 (H3 relaxin) inhibits myocardial injury in DCM rats and the underlying mechanisms involving miRNAs remain unknown. miRNA expression profiles were detected using miRNA microarray and bioinformatics analyses of myocardial tissues from control, DCM, and H3 relaxin-administered DCM groups, and the regulatory mechanisms of the miRNAs were investigated. A total of 5 miRNAs were downregulated in the myocardial tissues of DCM rats and upregulated in H3 relaxin-treated DCM rats, and 1 miRNA (miRNA let-7d-3p) was increased in the myocardial tissue of DCM rats and decreased in H3 relaxin-treated DCM rats as revealed by miRNA microarray and validated by real-time polymerase chain reaction. Important signaling pathways were found to be triggered by the differentially expressed miRNAs, including metabolism, cancer, Rap1, PI3K-Akt, and MAPK signaling pathways. The study revealed that H3 relaxin improved glucose uptake in DCM rats, potentially via the regulation of miRNA let-7d-3p.

Stiff-PINN: Physics-Informed Neural Network for Stiff Chemical Kinetics.

The recently developed physics-informed neural network (PINN) has achieved success in many science and engineering disciplines by encoding physics laws into the loss functions of the neural network such that the network not only conforms to the measurements and initial and boundary conditions but also satisfies the governing equations. This work first investigates the performance of the PINN in solving stiff chemical kinetic problems with governing equations of stiff ordinary differential equations (ODEs). The results elucidate the challenges of utilizing the PINN in stiff ODE systems. Consequently, we employ quasi-steady-state assumption (QSSA) to reduce the stiffness of the ODE systems, and the PINN then can be successfully applied to the converted non-/mild-stiff systems. Therefore, the results suggest that stiffness could be the major reason for the failure of the regular PINN in the studied stiff chemical kinetic systems. The developed stiff-PINN approach that utilizes QSSA to enable the PINN to solve stiff chemical kinetics shall open the possibility of applying the PINN to various reaction-diffusion systems involving stiff dynamics.

Cortistatin ameliorates Ang II-induced proliferation of vascular smooth muscle cells by inhibiting autophagy through SSTR3 and SSTR5.

AIMS: Vascular smooth muscle cell (VSMC) proliferation plays a significant role in the development of various vascular disorders. However, the effect of cortistatin (CST) on VSMC proliferation remains unclear. Therefore, the purpose of our research aimed to study whether CST protected VSMCs from angiotensin II (Ang II)-induced proliferation and which mechanisms participated in the process. MAIN METHODS: Cultured rat VSMCs were treated with Ang II with or without CST for 24 h. Cell proliferation rate was measured by cell counting kit-8 (CCK8) assay. The expressions of CST and its receptors were assessed by quantitative real-time PCR (qRT-PCR). The protein expression levels were analyzed by western blots. Immunofluorescence and transmission electron microscopy (TEM) were used to observe autophagy. KEY FINDINGS: Our results showed that different concentrations of CST alleviated the Ang II-induced VSMC proliferation. The autophagy and reactive oxygen species (ROS) stimulated by Ang II were attenuated by CST. Furthermore, when the autophagy inhibitor 3-methyladenine (3-MA) was added, it exerted similar inhibition effects like CST, but didn't augment the protective role of CST on Ang II-induced VSMC autophagy and proliferation. Moreover, blocking somatostatin receptor 3 and 5 (SSTR3 and SSTR5) partially abrogated the suppressive effect of CST on Ang II-stimulated VSMC proliferation and autophagy. SIGNIFICANCE: This study indicated that CST could ameliorate Ang II-stimulated VSMC proliferation by inhibiting autophagy partially through its receptors SSTR3 and SSTR5, providing a reasonable evidence for CST as a novel perspective therapeutic target of vascular diseases.

Calcium-Sensing Receptor on Neutrophil Promotes Myocardial Apoptosis and Fibrosis After Acute Myocardial Infarction via NLRP3 Inflammasome Activation.

BACKGROUND: The infiltration of neutrophils aggravates inflammatory response in acute myocardial infarction (AMI), and the role of calcium-sensing receptor (CaSR) in neutrophil-associated inflammation is largely unknown. The aim of this study was to evaluate the regulatory effects of CaSR on nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome in neutrophils and to explore its role in AMI-related ventricular remodelling. METHODS: The expression of CaSR, NLRP3 inflammasome, and interleukin 1ß (IL-1ß) in peripheral blood and infiltrating neutrophils in patients and rats with AMI was detected by western blotting and immunofluorescence. Cardiomyocyte apoptosis was detected by western blotting and transmission electron microscopy. The degree of fibrosis was evaluated by Masson staining and western blotting. RESULTS: We found upregulation of CaSR, NLRP3 inflammasome, Caspase-1, and IL-1ß in peripheral neutrophils from patients with AMI compared with matched healthy controls, peaking on day 1 and decreasing gradually till 7 days. Peripheral and infiltrating neutrophils from rats with AMI showed the same trend. Calindol enhanced NLRP3 inflammasome activation and IL-1ß release in neutrophils from healthy volunteers, which was blocked by inhibitors of the PLC-IP3 pathway and ER-Ca2+ release. Calhex-231 decreased NLRP3 inflammasome activation and IL-1ß release in neutrophils from patients with AMI. The calindol-stimulated neutrophils from healthy rats promoted cardiomyocyte apoptosis and fibrosis of cardiac fibroblasts from healthy rats, which were inhibited by calhex-231. CONCLUSION: The results suggest that CaSR activates NLRP3 inflammasome in neutrophils, contributing to ventricular remodelling after AMI. CaSR inhibition may be a potential therapeutic target for heart failure in AMI.

Chemical constituents of fungus F03 belonging to Basidiomycota.

Filamentous fungus F03 belonging to Basidiomycota was obtained and identified as Phlebiopsis crassa based on ITS sequence when Morchella. sp was isolated from the wild fruiting body by spores releasing method. Chemical constituents were separated by gel chromatography, HPLC and recrystallization. Structures of compounds were confirmed by NMR data. Four products orsellinic acid (1), α-nigerose (2), uridine (3), N-(4-hydroxyphenyl)acetamide (4) were identified and all compounds were isolated from the genus Phlebiopsis for the first time.

Genome Characteristics and Evolution of Pseudorabies Virus Strains in Eastern China from 2017 to 2019.

Since late 2011, outbreaks of pseudorabies virus (PRV) have occurred in southern China causing major economic losses to the pig industry. We previously reported that variant PRV forms and recombination in China could be the source of continued epidemics. Here, we analyzed samples from intensive pig farms in eastern China between 2017 and 2019, and sequenced the main glycoproteins (gB, gC, gD, and gE) to study the evolution characteristics of PRV. Based on the gC gene, we found that PRV variants belong to clade 2 and detected a founder effect during by the PRV epidemic. In addition, we detected inter- and intra-clade recombination; in particular, inter-clade recombination in the gB genes of strains FJ-ZXF and FJ-W2, which were recombinant with clade 1 strains. We also found specific amino-acid changes and positively selected sites, possibly associated with functional changes. This analysis of the emergence of PRV in China illustrates the need for continuous monitoring and the development of vaccines against specific variants of PRV.

NLRP3 Inflammasome Is Involved in Calcium-Sensing Receptor-Induced Aortic Remodeling in SHRs.

Increasing evidence suggests that the NLRP3 (nucleotide oligomerization domain-like receptor family, pyrin domain containing 3) inflammasome participates in cardiovascular diseases. However, its role and activation mechanism during hypertension remains unclear. In this study, we tested the role and mechanism of calcium-sensing receptor (CaSR) in NLRP3 inflammasome activation during hypertension. We observed that the expressions of CaSR and NLRP3 were increased in spontaneous hypertensive rats (SHRs) along with aortic fibrosis. In vascular smooth muscle cells (VSMCs), the activation of NLRP3 inflammasome associated with CaSR and collagen synthesis was induced by angiotensin II (Ang II). Furthermore, inhibition of CaSR and NLRP3 inflammasome attenuated proinflammatory cytokine release, suggesting that CaSR-mediated activation of the NLRP3 inflammasome may be a therapeutic target in aortic dysfunction and vascular inflammatory lesions.

[Comparison of the application among intensity-modulated radiotherapy, 3D-conformal radiotherapy and conventional radiotherapy for locally advanced middle-low rectal cancer].

OBJECTIVE: To compare the application among intensity-modulated radiotherapy (IMRT), three-dimensional conformal radiotherapy(3D-CRT) and conventional radiotherapy (CRT) for locally advanced middle-low rectal cancer. METHODS: From January 2015 to December 2016, 93 locally advanced middle-low rectal cancer patients with clinical stage cT3N+M0 or cT4N0/+M0 who underwent preoperative concurrent chemoradiotherapy at Department of Colorectal Surgery, the Third Affiliated Hospital of Kunming Medical University and had complete data were enrolled in this retrospective cohort study. Patients were divided into IMRT group (17 cases), 3D-CRT group (28 cases) and CRT group (48 cases) according to different radiotherapy methods. The frequency and dose of CRT were 1 time/day, 5 times/week, for a total of 5 weeks, with a single dose of 2.0 Gy, the total dose was 50 Gy. Frequency and dose of 3D-CRT and IMRT were 1 time/day, 5 times/week, for a total of 23 to 28 times, with a single dose of 1.8 to 2.0 Gy, and a total dose of 45.0 to 50.4 Gy. The chemotherapy regimen was performed with capecitabine tablets at a dose of 825 mg/m2 twice a day for 5 days every week, at the same time during radiotherapy. The efficacy, chemotherapy adverse reactions and immune function of the three groups were compared. RESULTS: There was no significant difference in the baseline data among the three groups (all P>0.05). The proportion of patients receiving permanent ostomy in the IMRT group and the 3D-CRT group was 29.4%(5/17) and 32.1%(9/28) respectively, which was lower than 58.3%(28/48) in CRT group, and the difference was statistically significant (χ²=7.982, P=0.030), while this proportion was not significantly different between IMRT and 3D-CRT group(χ²=0.037, P=0.848). The pathologic complete response(pCR) rate was 23.7%(22/93) in the whole study, and the pCR rate was 39.3%(11/28) in the 3D-CRT group, which was higher than that of CRT group and IMRT group [12.5%(6/48) and 29.4%(5/17)], and the difference was statistically significant (χ²=7.407, P=0.025), while there was no significant difference in pCR rate between CRT group and IMRT group (χ²=2.554, P=0.110). There was no adverse reaction of grade 3 or above in all three groups. No significant difference in the incidence of bone marrow suppression, abnormal liver and kidney function markers, digestive tract reaction or radiation dermatitis was found(all P>0.05). After receiving concurrent chemoradiotherapy, the proportion of CD3/CD4 cells in the IMRT group and the CRT group decreased compared with that before treatment(23.1±9.3 vs. 31.1±10.9, 27.4±10.7 vs. 33.6±7.2, respectively); the proportion of CD3/CD8 cells was up-regulated (36.1±15.2 vs. 24.8±10.9, 30.9±14.4 vs. 24.0±8.3,respectively), and the differences were statistically significant (both P<0.05), while the above indexes before and after treatment were not significantly different in the 3D-CRT group(all P>0.05). After treatment, the proportion of CD4/CD8 cells in IMRT group decreased (0.8±0.6 vs. 1.6±1.0, t=3.838, P=0.003), while this proportion was not significantly different in CRT group and 3D-CRT group(all P>0.05). CONCLUSIONS: IMRT and 3D-CRT can reduce the rate of permanent stoma. 3D-CRT can increase pCR rate. No obvious advantage is shown in IMRT as compared with 3D-CRT in the short-term efficacy. On the contrary, an immunosuppressive status may occur. Therefore, 3D-CRT is recommended as the best preoperative treatment strategy for patients with locally advanced middle-low rectal cancer, especially for those with immunosuppression status.

Novel protein chip for the detection of antibodies against infectious bronchitis virus.

BACKGROUND: Infectious bronchitis (IB) caused by the IB virus (IBV) can cause acute damage to chickens around the world. Therefore, rapid diagnosis and immune status determination are critical for controlling IBV outbreaks. Enzyme-linked immunosorbent assays (ELISAs) have been widely used in the detection of IBV antibodies in the early infection and continuous infection of IB because they are more sensitive and quicker than other diagnostic methods. RESULTS: We have developed two indirect microarray methods to detect antibodies against IBV: a chemiluminescent immunoassay test (CIT) and a rapid diagnostic test (RDT). IBV nonstructural protein 5 (nsp5) was expressed, purified from Escherichia coli, and used to spot the initiator integrated poly(dimethylsiloxane), which can provide a near "zero" background for serological assays. Compared with the IDEXX IBV Ab Test kit, CIT and RDT have a sensitivity and specificity of at least 98.88% and 91.67%, respectively. No cross-reaction was detected with antibodies against avian influenza virus subtypes (H5, H7, and H9), Newcastle disease virus, Marek's disease virus, infectious bursal disease virus, and chicken anemia virus. The coefficients of variation of the reproducibility of the intra- and inter-assays for CIT ranged from 0.8 to 18.63%. The reproducibility of RDT was consistent with the original results. The application of the IBV nsp5 protein microarray showed that the positive rate of the CIT was 96.77%, that of the nsp5 ELISA was 91.40%, and that of the RDT was 90.32%. Furthermore, the RDT, which was visible to the naked eye, could be completed within 15 min. Our results indicated that compared with nsp5 ELISA, the CIT was more sensitive, and the RDT had similar positive rates but was faster. Furthermore, the two proposed methods were specific and stable. CONCLUSIONS: Two microarray assays, which were rapid, specific, sensitive, and relatively simple, were developed for the detection of an antibody against IBV. These methods can be of great value for the surveillance of pathogens and monitoring the efficiency of vaccination.

Cyclosporin A induces autophagy in cardiac fibroblasts through the NRP-2/WDFY-1 axis.

Cyclosporin A (CsA) is an effective immunosuppressive agent, but its myocardial toxicity limits its widespread and long-term clinical application. In this study, CsA treatment led to damages in myocardial fiber structure, an increase in myocardial fibrosis, and changes in heart size and shape; moreover, the degree of damage was exacerbated with prolonged drug application and increases in dose. However, the mechanism is not clear; therefore, the purpose of this study was to reveal the mechanism of CsA-induced myocardial fibrosis and identify a new target for the prevention and treatment of CsA-induced myocardial injury. Cardiac fibroblasts were treated with CsA (5, 10, or 20 µg/mL) for 24 h. Autophagy was observed by electron microscopy and immunofluorescence. The expression of NRP-2/WDFY-1, autophagy-related proteins (Beclin1 and LC3B), fibrosis-related proteins (MMP2/9), and fibroblast phenotype conversion factor (α-SMA) was evaluated by Western blot. The expression of collagen I was determined by ELISA. Then, we used the gene interference technique to alter WDFY-1 expression with or without CsA or 3-MA treatment for 24 h, and the effects on autophagy and the expression of autophagy-related proteins, fibrosis-associated proteins, IFN-α, TNF-α, and IL-6 were determined. The results showed the following: (1) CsA induced fibrosis-related protein (MMP2/9), fibroblast phenotype conversion factor (α-SMA), and collagen I up-regulation in a dose-dependent manner. (2) CsA induced the formation of autophagosomes and up-regulated the expression of Beclin1, LC3B, and the ERK/MAPK pathway in cardiac fibroblasts. (3) CsA induced NRP-2 down-regulation and WDFY-1 up-regulation. (4) Depletion of WDFY-1 inhibited CsA-induced autophagy, TNF-α and IFN-α up-regulation, and fibrosis. (5) The autophagy inhibitor 3-MA inhibited CsA-induced TNF-α and IFN-α up-regulation and fibrosis. Overall, cyclosporin A induces autophagy in cardiac fibroblasts through the NRP-2/WDFY-1 axis, which promotes the progression of myocardial fibrosis.