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.

Liquorice-induced severe hypokalemic rhabdomyolysis with Gitelman syndrome and diabetes: A case report.

BACKGROUND: Licorice-induced severe hypokalemic rhabdomyolysis is clinically rare. Gitelman syndrome (GS) is the most common inherited renal tubular disease, while diabetes is one of the most prevalent diseases in the world. Recently, some studies have found that GS patients had higher diabetic morbidity. However, the coexistence of these three diseases has yet to be reported. CASE SUMMARY: We report the case of a 62-year-old Chinese man who was admitted with weakness in the extremities, muscle pain, and dark-colored urine. He had consumed liquorice water daily for seven days prior to admission. The laboratory tests revealed a serum potassium level of 1.84 mmol/L, magnesium 0.68 mmol/L, creatinine phosphokinase (CK) 10117 IU/L, and marked hemoglobinuria. Fractional chloride excretion and fractional magnesium excretion were increased. Plasma renin activity and aldosterone concentration were within the normal ranges. Sequence analysis of the SLC12A3 gene revealed that he had compound heterozygous mutations. The diagnosis of liquorice-induced severe hypokalemic rhabdomyolysis with GS and diabetes was thus genetically confirmed. Serum potassium and CK quickly improved with potassium replacement therapy, hydration, and discontinuation of liquorice ingestion. Upon follow-up at 3 mo, the levels of CK, myoglobin, and potassium remained normal, and magnesium was above 0.6 mmol/L. CONCLUSION: This case emphasizes that liquorice consumption and GS should be considered causes of hypokalemia and that the diabetic status of GS patients should be noted in the clinic.

A new porous magnetic chitosan modified by melamine for fast and efficient adsorption of Cu(II) ions.

A new porous magnetic chitosan modified by melamine (MA-CS/Fe3O4) was synthesized. The compositions and surface topographies were characterized by infrared (IR) spectroscopy, X-ray diffraction (XRD) analysis, thermogravimetric (TG) analysis and scanning electron microscope (SEM), respectively. The results of adsorption kinetics showed the adsorption behavior could be better described by the pseudo-second-order equation (R>0.999). The adsorption isotherm was well fitted by the Langmuir equation (R>0.999), and the values of separation factors were in the range of 0-1.0. The maximum adsorption capacity for Cu(II) was 2.58mmolg(-1) at the optimal experimental conditions, which were pH=5.5, t=25min, C0=5.0mmolL(-1). The rate-controlling step was supposed to be chemical adsorption rather than mass transport. The adsorbent still exhibited high adsorption capacity after five regeneration cycles. The adsorption mechanism was due to coordination between Cu(II) and N atoms.

[A highly sensitive enzyme-linked immunosorbent assay for measurement of leptin secretion in human adipocytes].

OBJECTIVE: To establish a highly sensitive and specific ELISA method for measurement of leptin and further to study the secretion of leptin during human preadipocytes differentiation and effects of troglitazone. METHODS: Rabbits Balb/c mice were immunized by recombinant human leptin and Balb/c mice were immunized by human leptin so as to produce rabbit anti-human leptin polyclonal antibodies (PAb) and mouse anti-human leptin monoclonal antibodies (MAb). Combination of the PAb as coating antibody, with a carefully paired biotinylated MAb as detector, and the avidin-horseradish peroxidase as the amplifier of detecting signals, a sandwich method, biotin-avidin ELISA (BA-ELISA) was established. Human omental preadipocytes were cultured, introduced to differentiate, and treated with 10 micromol/L troglitazone; the leptin secretion in the supernatant was detected by BA-ELISA. Peripheral blood samples were collected from 114 healthy persons and the serum leptin was detected by BA-ELISA. RESULTS: The sensitivity of BA-ELISA was 0.03 ng/ml with a working range of 0.05 - 5 ng/ml and a exogenous leptin recovery rate of 97.8%, and the intra- and interassay coefficients of variation (CVs) were less than 7.4% and 9.3% respectively. The assay detected only a single free leptin peak in gel chromatographic fractions from the mixed human sera or adipocytes culture media. The leptin secretion level detected by BA-ELISA showed that the leptin secretion of the preadipocytes increased strongly when the cells differentiated into mature adipocytes. The peak leptin secretion level of the troglitazone treated group was 2 times as that of the control group. The leptin concentration of women was than 7.6 ng/ml, significantly higher than that of the men (3.2 ng/ml, P < 0.001), and the serum leptin level was significantly correlated with body mass index both for men (r = 0.67, P < 0.001) and for women (r = 0.61, P < 0.001). CONCLUSION: A highly sensitive BA-ELISA specific for free leptin has been developed that is especially suited for the accurate measurement of the rather low leptin levels of clinical blood specimens and for basic research use.

[Primary culture of human omental preadipocytes and study of their biological properties].

OBJECTIVE: To develop a primary culture method of human omental preadipocytes and to study their biological properties, such as hyperplasia, hypertrophy and endocrine secretion of human visceral adipose tissue. METHODS: Using enzyme-digesting method, fibroblast-like cells from the human omental adipose tissues were cultured. The morphological changes of the cultured cells were observed and the growth curve was drawn by MTT method. The intracytoplasmic lipid of the cultured cells was determined by oil red O staining. The leptin and adiponectin levels in the culture supernatants were measured by ELISA. RESULTS: The cultured fibroblast-like cells were homogeneous. Proliferation of cells began at the 3 rd day and the cell numbers increased in indicial way from the 3 rd day to the 9 th day. The doubling time of cells was about 60 hours. During the process of induction by conditional medium, the cells became round and larger, and more adipose droplets were aggregated. On the 21 st day, more than 90% of the cells became adipocytes. Leptin secretion was detected at low level in the preadipocytes and continuously increased during differentiation, with a peak on day 17. It remained constant from day 17 onward. Unlike leptin, adiponectin secretion was not detected until day 7 after induction, when differentiated adipocytes had already been observed. Its secretion increased dramatically between days 7 and 17, and reached a maximum level on day 17, but had a significant reduction on day 21. Extraction of intracytoplasmic lipid stained with oil red O and detection of leptin and adiponectin both verified the isolated cells were preadipocytes functioning actively. CONCLUSION: A human omental preadipocytes model has been established and different secretion patterns of leptin and adiponectin secretion related to preadipocyte differentiation has been characterized. Adiponectin may be proposed as a specific marker for preadipocyte differentiation.

[A comparative study of postharvest fruit senescence in different litchi cultivars].

Among the 5 tested litchi (Litchi chinensis Sonn.) cultivars ("Huaizhi", "Guiwei", "Nuomici", "Hongmili" and "Shuijingqiu", "Nuomici" became deteriorated much faster than other cultivars while "Guiwei" fruit was the slowest in the rotting process (Fig. 1A). Fruit deterioration was accompanied by fruit desiccation (Fig. 2B), but the speed of water loss was not significantly correlated to fruit deterioration rate, indicating that it was not the key factor causing the difference in postharvest performance among cultivars. Fruit deterioration rate was significantly positively correlated to membrane leakage (Fig. 2A), suggesting the capacity to maintain membrane integrity is closely related to the shelflife of litchi. Skin browning potential, uronic acid concentration, degree of methylation of pectin and soluble Ca content in pericarp as well as total Ca content in the pulp were not significantly correlated with fruit deterioration. Content of structural Ca (water-insoluble but acetic acid-soluble calcium, membrane or wall-bound Ca), the major form of Ca in the pericarp, was negatively correlated to fruit deterioration rate (Fig. 2E). The results proved that differences in fruit desiccation rate, browning potential, Ca other than structural form were not the major cause leading to difference in postharvest performance among different cultivars. "Guiwei" being more tolerant to desiccation than other cultivars is likely associated its higher structural Ca concentration in the pericarp.