Effects of Spatial Expression of Activating Transcription Factor 4 on the Pathogenicity of Two Phenotypes of Bovine Viral Diarrhea Virus by Regulating the Endoplasmic Reticulum-Mediated Autophagy Process.

The endoplasmic reticulum (ER) stress response is a highly conserved stress-defense mechanism and activates the adaptive unfolded protein response (UPR) to mitigate imbalance. The ER stress-activated signaling pathways can also trigger autophagy to facilitate cellular repair. Bovine viral diarrhea virus (BVDV) utilizes the host cellular ER as the primary site of the life cycle. However, the interplay between cellular ER stress and BVDV replication remains unclear. This report reveals that cytopathic (cp) and noncytopathic (ncp) BVDV have distinct strategies to regulate UPR mechanisms and ER stress-mediated autophagy for their own benefit. Immunoblot analysis revealed that cp and ncp BVDV differentially regulated the abundance of ER chaperone GRP78 for viral replication, while the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α)-activating transcription factor 4 (ATF4) pathway of the UPR was switched on at different stages of infection. Pretreatment with ER stress inducer promoted virion replication, but RNA interference (RNAi) knockdown of ATF4 in BVDV-infected cells significantly attenuated BVDV infectivity titers. More importantly, the effector ATF4 activated by cp BVDV infection translocated into the nucleus to mediate autophagy, but ATF4 was retained in the cytoplasm during ncp BVDV infection. In addition, we found that cp BVDV core protein was localized in the ER to induce ER stress-mediated autophagy. Overall, the potential therapeutic target ATF4 may contribute to the global eradication campaign of BVDV. IMPORTANCE The ER-tropic viruses hijack the host cellular ER as the replication platform of the life cycle, which can lead to strong ER stress. The UPR and related transcriptional cascades triggered by ER stress play a crucial role in viral replication and pathogenesis, but little is known about these underlying mechanisms. Here, we report that cytopathic and noncytopathic BVDV use different strategies to reprogram the cellular UPR and ER stress-mediated autophagy for their own advantage. The cytopathic BVDV unconventionally downregulated the expression level of GRP78, creating perfect conditions for self-replication via the UPR, and the noncytopathic BVDV retained ATF4 in the cytoplasm to provide an advantage for its persistent infection. Our findings provide new insights into exploring how BVDV and other ER-tropic viruses reprogram the UPR signaling pathway in the host cells for replication and reveal the attractive host target ATF4 for new antiviral agents.

Development and application of an indirect ELISA for the serological detection of bovine viral diarrhea virus infection based on the protein E2 antigen.

BACKGROUND: Bovine viral diarrhea virus (BVDV) causes continuous economic losses to the livestock industry. Monitoring antibodies with enzyme-linked immunosorbent assay (ELISA) is a valuable tool to ensure the purification of BVDV in cattle. However, currently available ELISA kits based on the whole BVDV virion are both costly and time-consuming. The E2 protein has good immunogenicity, induces the secretion of neutralizing antibodies and is an essential immunogen for serological detection. METHODS AND RESULTS: We developed a novel recombinant E2 protein-based indirect ELISA (rE2-iELISA) and conducted a serological survey for BVDV antibodies in 2021-2022 in Beijing, China. The results showed that E2 protein was successfully expressed with high immunogenicity and the optimal rE2-iELISA displayed high sensitivity, reproducibility and specificity. Clinical testing of 566 serum specimens indicated that 318 BVDV positive samples and 194 BVDV negative samples were tested by rE2-iELISA and the IDEXX BVDV ELISA-Ab kit, with a positive coincidence rate of 93.3%, a negative coincidence rate of 86.3%, and an overall coincidence rate of 90.5%. CONCLUSION: This study established an rE2-iELISA method, which is a highly sensitive, specific and robust ELISA-test validated to detect anti-BVDV antibodies. These findings indicate that the newly developed rE2-iELISA method has the potential to be used as a rapid, reliable and cost-effective screening tool for BVDV infection and provides technical support for the evaluation of vaccine efficacy in cattle herds in the future.

Immunization with recombinant Erns-LTB fusion protein elicits protective immune responses against bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV), a major infectious pathogen and is associated with major economic losses and significant impact on animal welfare worldwide. Here, recombinant Erns-LTB protein vaccine containing MF59 adjuvant was prepared and assessed using a mouse model. The recombinant plasmid (pET32a-Erns-LTB) was constructed and transformed into BL21 (DE3) cells to produce Erns-LTB protein. The Erns-LTB protein was formulated with MF59 adjuvant, when delivered intraperitoneally in mice, exhibited higher immunogenic and induced superior levels of anti-BVDV IgG compared with the MF59 adjuvanted Erns protein. Importantly, after challenged with different BVDV BJ175170 and BJ1305 isolate strains, mice inoculated with Erns-LTB protein displayed alleviated pathological damage and decreased plasma virus shedding compared with mice inoculated with Erns protein. The enhanced protection from Erns-LTB protein is mediated by T cell immunity and primarily based on CD4+ T helper (Th) and CD8+ cytotoxic T lymphocyte (CTL), these results suggest that Erns-LTB protein has potential to protect against a broad range of BVDV strains thereby providing a novel direction for developing broadly protective vaccines.

[Preparation of MgO Modified Lotus Shell Biochar and Its Phosphorus Adsorption Characteristics].

To study the potential application characteristics of biochar as a phosphate adsorbent, nano-MgO-biochar was prepared by rapid pyrolysis of a mixture of MgO and lotus shells. The physicochemical properties were characterized by XRD, BET, SEM, and TEM, and adsorption experiments were conducted. The results showed that MgO was mainly supported on the surface of carbon in the form of flakes and granules, which increased the adsorption active site, and the adsorption amount of MgO-biochar MBC3 was 14 times higher than that of biochar MBC1 without MgO. The adsorption capacity of MBC9, which was prepared by rapid pyrolysis under 10% CO2 atmosphere, was further increased 16 times higher than that of MBC1. The adsorption kinetics followed a pseudo-second-order model, which indicated the adsorption of phosphate on MgO-biochar was dominated by chemical adsorption. According to the Langmuir equation, the maximum adsorption capacity of MBC3 and MBC9 could reach 283.26 mg·g-1 and 297.96 mg·g-1, respectively. MgO-biochar is a high-efficiency phosphate adsorbent, which can be used to control the eutrophication of water.

Molecular Recognition and Chirality Sensing of Epoxides in Water Using Endo-Functionalized Molecular Tubes.

Chiral epoxides are important intermediates in chemistry and biology. The high-throughput screening of asymmetric epoxidation conditions requires fast determination of the absolute configurations and ee values of chiral epoxides. Herein, we report molecular recognition and chiroptical sensing of epoxides in water using endo-functionalized molecular tubes. The absolute configurations and ee values were simultaneously determined by circular dichroism spectroscopy. In addition, real-time monitoring as well as the application to real asymmetric epoxidation was demonstrated. The method is simple, environmentally friendly, and amenable to high-throughput screening.

Selective Recognition of Highly Hydrophilic Molecules in Water by Endo-Functionalized Molecular Tubes.

Selective recognition of neutral hydrophilic molecules in water is a challenge for supramolecular chemistry but commonplace in nature. By mimicking the binding pocket of natural receptors, endo-functionalized molecular tubes are proposed to meet this challenge. We found that two molecular tubes with inwardly directed hydrogen-bond donors recognize highly hydrophilic solvent molecules in water with high selectivity. In the complexes, hydrogen bonding occurs in the deep and hydrophobic cavity. The cooperative action between hydrogen bonding and hydrophobic effects accounts for the high affinity and selectivity. The molecular receptor is fluorescent and can detect concentrations of 1,4-dioxane-a known carcinogen and persistent environmental contaminant-in water at a limit of 119 ppb. The method simplifies the analytic procedure for this highly hydrophilic molecule.

Resveratrol attenuates inflammation and oxidative stress in epididymal white adipose tissue: implications for its involvement in improving steroidogenesis in diet-induced obese mice.

Chronic, low-grade systemic inflammation has been shown to play an important role in the development of obesity-related complications. Epididymal white adipose tissue (WAT) can influence testicular function through its endocrine function. The purpose of this study was to assess the effects of resveratrol on the epididymal WAT inflammatory response and on testicular steroidogenesis in obese individuals. Seven-week-old male C57BL/6J mice were fed a high-calorie and high-cholesterol diet (HCD group) or HCD supplemented with resveratrol (HCD+Res group) for 18 weeks. As we previously showed that resveratrol protects against Leydig cell steroidogenesis in HCD-induced obese mice, this study assessed macrophage infiltration in fat depots by measuring crown-like structure (CLS) density. Histological analysis showed that adipocyte size was significantly smaller and CLSs were less numerous in the HCD+Res group than the HCD group (P < 0.01). Additionally, resveratrol supplementation decreased Nfkb1 expression (P < 0.01) and increased the IκB-α protein abundance (P < 0.01) in epididymal WAT. Consistent with this alteration in NF-κB signaling, the expression of two classic proinflammatory cytokines, TNF-α (Tnfa) and IL-1ß (Il1b), were significantly decreased in the HCD+Res group compared with the HCD group (P < 0.01). Significant differences were also found in the expression of sirtuin1 (Sirt1) (P < 0.01) and manganese superoxide dismutase (Sod2) (P < 0.01) between the HCD and HCD+Res groups. Our data suggest that resveratrol can attenuate obesity-induced inflammation and oxidative stress in epididymal WAT, which partly accounts for its beneficial effects in testicular steroidogenesis.

A simple solution hybridization rapidly detects DNA by fluorescent probe.

Traditional Southern blot, as the golden standard to detect DNA, is widely used in molecular biology. However, its operation process has some significant disadvantages, such as radioactive contamination, complex procedures, time-consuming. Some new methods developed in recent years need expensive equipments and also have the defects of complex procedures and time-consuming. The paper described a method to detect DNA quickly by liquid hybridization. The probe used is a fragment of DNA sequence labeled by FITC-12-dUTP. The whole process includes four steps: probe preparation, hybridization, electrophoresis and signal detection. The comparative experiments using the single strand DNA probe and the double strand DNA probe indicated that the sensitivity of single strand DNA probes, which could detect 0.38 µg (1.82 x 10(-13) mol) plasmid, is 2.1 times than that of the double strand DNA probes which could detect 0.8 µg (3.64 x 10(-13) mol) plasmid. The total procedure is simple and can be completed in 3 h.

Cloning, expression, and purification of a Cu/Zn superoxide dismutase from Jatropha curcas.

We report cDNA cloning, expression, purification, and characterization of a novel Cu/ Zn superoxide dismutase (SOD) from Jatropha curcas leaves. The full-length cDNA of this SOD contained a 496-bp open-reading frame (ORF) encoding 162 amino acid residues. The recombinant plasmid containing the SOD coding sequence was introduced into Escherichia coli, and the SOD was expressed as a fusion protein. The recombinant SOD was purified from a high-density fed-batch culture using a combination of immobilized metal ion affinity chromatography (IMAC) and Sephadex G25 desalting chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis indicated that the recombinant SOD was a monomeric protein with a molecular mass of approximately 16.4 kDa. Isoelectric focusing showed that this SOD was a basic protein with pI values of 7.04, 7.33, 8.62, and 8.77. The activity of the SOD was stable at 70 degrees C for 10 min, and in a broad pH range from 4 to 9. The presence of urea (up to 8 M), guanidinium chloride (up to 6 M), and 2-mercaptoethanol (up to 8 mM) had little effect on the activity. The activity decreased gradually with increasing concentrations of imidazole, hydrogen peroxide, and ethylenediaminetetraacetic acid (EDTA). Atomic absorption spectrometry showed the presence of 0.239 copper and 0.258 zinc atoms, respectively, in the SOD polypeptide.

Identification of novel acyl-ACP thioesterase gene ClFATB1 from Cinnamomum longepaniculatum.

A putative fatty acyl-acyl carrier protein (acyl-ACP) thioesterase (thioesterase) full-length cDNA sequence named as ClFATB1 was obtained from the seed cDNA library of Cinnamomum longepaniculatum by the SMART-RACE method. The novel gene encodes a protein of 382 amino acid residues with close homology to fatty acid thioesterase type B (FATB) enzymes of other plants, with two essential residues (His285 and Cys320) for thioesterase catalytic activity. The gene was transcribed in all tissues of C. longepaniculatum, the highest being in seeds. Recombinant ClFATB1 in Escherichia coli had higher specific activities against saturated 16:0- and 18:0-ACPs than on unsaturated 18:1-ACP. Overexpression of ClFATB1 in transgenic tobaccos upregulated thioesterase activities of crude proteins against 16:0-ACP and 18:0-ACP by 20.3 and 5.7%, respectively, and resulted in an increase in the contents of palmitic and stearic acids by 15.4 and 10.5%, respectively. However, ectopic expression of this gene decreased the substrate specificities of crude proteins to unsaturated 18:1-ACP by 12.7% in transgenic tobacco and lowered the contents of oleic, linoleic, and linolenic acids in transgenic leaves. So ClFATB1 would potentially upregulate the synthesis of saturated fatty acids and downregulate unsaturated ones in the fatty acid synthesis pathway of plants.

Alternatively spliced transcripts of group 3 late embryogenesis abundant protein from Pogonatherum paniceum confer different abiotic stress tolerance in Escherichia coli.

The gene encoding a group 3 late embryogenesis abundant (LEA) protein was cloned from callus of the drought-tolerant grass Pogonatherum paniceum. Three alternatively spliced transcripts of this gene were amplified by RT-PCR. According to the bioinformatics analysis, the gene contained three exons and two introns. The PpLEA3.1 transcript which was the most abundant one in P. paniceum contained all three exons, and the PpLEA3.2 transcript lacked fragments of the first two exons which encoded a 41-amino acid-long region of the PpLEA3 protein. The PpLEA3.3 transcript retained the second intron. The three splicing patterns resulted in changes in the number of repeats of an 11-amino acid motif, hydropathy and the predicted 3-dimensional structure. When expressed in Escherichia coli, the three proteins differentially affected growth responses to salt, cold and heat stress. These results confirmed that the complete motif repeat structures and an appropriate hydrophilic/hydrophobic balance are important for the LEA protein in providing protection against various forms of stresses.

MicroRNA profiles and their control of male gametophyte development in rice.

Plant microRNAs (miRNAs) act as negative regulators of gene expression by slicing target transcripts or inhibiting translation. A number of miRNAs play important roles in development. In order to investigate the potential function of miRNAs during male gametogenesis in rice, we obtained both gene and small RNA expression profiles by combining microarray and high-throughput sequencing technologies. From the microarray datasets, 2,925 male gametophyte-specific genes were identified, including 107 transcription factors and three significant Argonaute genes (AGO12, AGO13, and AGO17). From the sRNA-Seq datasets, 104 unique miRNAs (miRus) were identified, including 47 known miRus and 57 novel miRus; interestingly, most of the new miRus are pollen-specific and not conserved among species. Furthermore, an interactive network of miRNA-target was constructed based on the two datasets. By employing enrichment analysis, the miRNA-regulated targets were found to be involved in both the up and down pathways, but predominantly in the down pathways, including 37 GO biological processes and 32 KEGG pathways. These findings indicate that miRNAs play a broad regulatory role during male gametophyte development in rice.

Growth and antioxidant responses in Jatropha curcas seedling exposed to mercury toxicity.

Jatropha curcas seedlings were exposed to varying concentrations of mercury in order to investigate mercury accumulation, and the changes in growth and antioxidant enzyme activities using in vitro embryo germination and culture. Our results showed that mercury is readily accumulated by germinating embryos and growing seedlings, and its content was greater in the radicles than those of in the cotyledons and hypocotyls. This accumulation was directly correlated with an increase in tested mercury concentrations in the medium. Biomass in the cotyledons, hypocotyls and radicles increased gradually with increasing mercury concentrations, peaking in seedlings exposed to mercury concentration of 50 microM, and then decreased. Superoxide dismutase activities in the cotyledons, hypocotyls and radicles showed largest increment at mercury concentration of 100 microM. Peroxidase activities in the cotyledons and hypocotyls reached peaks at mercury concentration of 200 microM, and the highest activity in the radicles was observed at 100 microM. Catalase activities in the cotyledons and hypocotyls were significantly induced, and the highest activity in the radicles was observed at mercury concentration of 200 microM. Phenylalanine ammonia-lyase activities in the hypocotyls had a positive correlation to mercury concentrations, and the highest activities in the cotyledons and radicles were found at mercury concentrations of 200 and 100 microM, respectively. Analysis of superoxide dismutase, peroxidase and catalase isoenzymes suggested that different patterns depend on mercury concentrations and tissue types, and the staining intensities of these isoenzymes are consistent with the changes of these enzyme activities assayed in solutions.

Cloning and expression analysis of carboxyltransferase of acetyl-coA carboxylase from Jatropha curcas.

A full-length cDNA of the carboxyltransferase (accA) gene of acetyl-coenzym A (acetyl-CoA) carboxylase from Jatropha curcas was cloned and sequenced. The gene with an open reading frame (ORF) of 1149 bp encodes a polypeptide of 383 amino acids, with a molecular mass of 41.9 kDa. Utilizing fluorogenic real-time polymerase chain reaction (RT-PCR), the expression levels of the accA gene in leaves and fruits at early, middle and late stages under pH 7.0/8.0 and light/darkness stress were investigated. The expression levels of the accA gene in leaves at early, middle and late stages increased significantly under pH 8.0 stress compared to pH 7.0. Similarly, the expression levels in fruits showed a significant increase under darkness condition compared to the control. Under light stress, the expression levels in the fruits at early, middle and late stages showed the largest fluctuations compared to those of the control. These findings suggested that the expression levels of the accA gene are closely related to the growth conditions and developmental stages in the leaves and fruits of Jatropha curcas.

[Design, optimization and quality evaluation of curcumin self-emulsifying drug delivery system (SEDDS)].

OBJECTIVE: To develop the self-emulsifying drug delivery system (SEDDS) of curcumin, and evaluate its quality in vitro. METHODS: The excipients of curcumin SEDDS were selected via its solubility study in various oils, surfactants and co-surfactants and the formulation was optimized using ternary phase diagram study and central composite design-response surface methodology (oil, surfactant and co-surfactant percentages as factors; solubility, droplet size, polydispersity index and emulsifying time as responses). The appearance, droplet size and polydispersity index after emulsifying and the emulsifying time of optimized curcumin SEDDS were studied. The solubility of curcumin in the solution of SEDDS was determined. RESULTS: Castor oil-(tween-80) -ethanol = 28: 55: 20 (w/ w/w) was selected for optimum curcumin SEDDS. The droplet size was 222. 2 nm, polydispersity index was 0. 171. The time of self-emulsifying was 10 s and the solubility of curcumin in SEDDS was 1.93 mg/mL. CONCLUSION: Curcumin SEDDS formulation is selected and optimized successfully, and the preparation of curcumin SEDDS is simple, the quality is stable.

[DNA methylation-mediated regulation of OsMAPK2 gene expression in rice callus formation].

A DNA segment with DNA methylation site was detected from rice callus with or without 5-azaC treatment by MSAP. This segment was located on the first exon of gene OsMAPK2 and its 5' non-coding region. Gene OsMAPK2 had a CpG island in the 5' region and was homologous to AtMAPK12. Real-time quantitative PCR and Hpa II-McrBC PCR were conducted to detect the gene expression and DNA methylation of OsMAPK2 in the process of callus formation. The results showed that the DNA methylation was able to control the expression of OsMAPK2. The additon of 2.0 mg/L 2,4-D could induce DNA demethylation in the 5' region and activate the expression of OsMAPK2 gene. However, after long-time stimulation (100 h), the gene was methylated again, and the gene expression level was decreased. The trends of DNA methylation and gene expression stimulated by low concentrations of 2,4-D (0.5 and 1.0 mg/L) were similar to 2.0 mg/L 2,4-D, but the expression levels in each time point were low. On the other hand, high concentration of 2,4-D (5.0 mg/L) completed the processes of induction and suppression of the gene in a shorter time.

[Clinical effect and mechanismn of xuefu zhuyu capsule in treating unstable angina pectoris].

OBJECTIVE: To observe the clinical effect of Xuefu Zhuyu Capsule (XFZYC) in treating unstable angina pectoris (UAP) and to investigate its mechanism of protection on vascular endothelia. METHODS: Sixty UAP patients were randomly assigned to two groups, the 30 in the treated group were treated by conventional therapy plus XFZYC, and the 30 in the control group treated by conventional therapy alone. The frequency and persistent time of angina pectois, dosage of nitroglycerin used, changes of electrocardiogram (ECG) were observed, and the plasma levels of endothelin (ET), nitric oxide (NO), von Willebrand factor (vWF), soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) were tested before and after the two-month therapeutic course. RESULTS: (1) The clinical symptoms as frequency and persistent time of angina pectoris in the treated group were bettered significantly after treatment and the dosage of nitroglycerin used decreased (all P < 0.01), showing significant difference when compared with those in the control group (P < 0.05). (2) The effective rates on UAP symptoms and ECG in the treated group were 86.7% and 76.7% respectively, which were significantly higher than those in the control group (70.0% and 63.3%, P < 0.05). (3) Laboratory examination showed in the control group, changes only displayed in the decrease of vWF and ET and increase of NO (all P < 0.05), while in the treated group, plasma levels of vWF, ET, sVCAM-1 and sICAM-1 after treatment significantly decreased and were lower than those in the control group (P < 0.01), but NO elevated significantly and was higher than that in the control group (P < 0.01). CONCLUSIONS: XFZYC is an effective Chinese patent medicine for treatment of UAP. It displays its effect on protecting vascular endothelia and anti-angina pectoris partially by decreasing the plasma levels of ET, vWF, sVCAM-1 and sICAM-1 and elevating the level of NO.

Growth and antioxidant responses in Jatropha curcas cotyledons under lead stress.

Jatropha curcas embryos were grown in vitro to observe the effects of lead on cotyledon responses. The cotyledon biomass increased initially and then decreased with increasing lead concentration. The SOD activity increased gradually up to 200 microM and then decreased. The POD activity showed a similar trend. The CAT activity was increased at all lead concentrations, the highest activity being observed at 200 microM. However, the PAL activity was inhibited significantly except for 100 microM. Anaylsis by electrophoresis suggested a significant correlation between lead concentration and patterns of SOD, POD and CAT isoenzymes, and these results were consistent with changes of the antioxidant enzyme activities as assayed in solution.

[Effect of polyethylene glycol on the regeneration of the callus of Pogonatherum paniceum under different culture conditions].

This paper studied the effect of polyethylene glycol (PEG) on regeneration and free proline accumulation of callus of Pogonatherum paniceum (Lam.) Hack. under motionless liquid culture condition and shake liquid culture condition. Callus of P. paniceum had the ability to resist the stress of PEG. The effects of PEG stress and culture conditions on the callus of P. paniceum appeared mainly in two aspects, delaying regeneration time and debasing regeneration rates. The shake liquid culture mainly delayed the regeneration time and PEG stress mainly debased the regeneration rates. Free proline accumulated in the two culture conditions, and the contents of proline were positively correlated with PEG concentrations and culture time. After stress removal, most of the callus could recover the ability of regeneration, and the free proline might pay an important part in the inhibition and recovery. So it must be chosen a more than 300 g x L(-1) PEG concentration and long than 3 weeks culture time in the selection of drought-resistant mutants of P. paniceum. The motionless liquid culture was more suitable for selection of drought-resistant mutants.