Search

In recent years, the outbreaks of porcine epidemic diarrhea (PED) caused by the highly virulent porcine epidemic diarrhea virus (PEDV) variants occurred frequently in China, resulting in severe economic impacts to the pork industry. In this study, we selected and analyzed the genetic evolution of 15 PEDV representative strains that were identified in fecal samples of diarrheic piglets in 10 provinces and cities during 2011–2017. The phylogenetic analysis indicated that all the 15 PEDV isolates clustered into G2 genotype associated with the current circulating strains. Compared with the genome of the prototype strain CV777, these strains had 103–120 amino acid mutations in their S proteins, most of which were in the N terminal domain of S1 (S1-NTD). We also found 37 common mutations in all these 15 strains, although these strains shared 96.9–99.7% nucleotide homology and 96.3–99.8% amino acid homology in the S protein compared with the other original pandemic strains. Computational analysis showed that these mutations may lead to remarkable changes in the conformational structure and asparagine (N)-linked glycosylation sites of S1-NTD, which may be associated with the altered pathogenicity of these variant PEDV strains. We evaluated the pathogenicity of the PEDV strain FJzz1 in piglets through oral and intramuscular infection routes. Compared with oral infection, intramuscular infection could also cause typical clinical signs but with a slightly delayed onset, confirming that the variant PEDV isolate FJzz1 was highly pathogenic to suckling piglets. In conclusion, we analyzed the genetic variation and pathogenicity of the emerging PEDV isolates of China, indicating that G2 variant PEDV strains as the main prevalent strains that may mutate continually. This study shows the necessity of monitoring the molecular epidemiology and the etiological characteristics of the epidemic PEDV isolates, which may help better control the PED outbreaks., Highlights • G2 variant PEDV strains as the main prevalent strains continue to mutate along with time. • The isolated strains were prone to mutate in the S protein with many common amino acid mutations. • These mutations may lead to remarkable changes in the conformational structure of S1-NTD and the pathogenicity of PEDV. • Both oral and intramuscular routes caused typical clinical signs to piglets.

One proposed toxic mechanism of Bacillus thuringiensis Cry δ-endotoxins involves pore formation in target membranes by the α4-α5 transmembrane hairpin constituting their pore-forming domain. Here, nine selected charged and uncharged polar residues in the pore-lining α4 of the Cry4Aa mosquito-active toxin were substituted with Ala. All mutant toxins, i.e., D169A, R171A, Q173A, H178A, Y179A, H180A, Q182A, N183A and E187A, were over-expressed in Escherichia coli as 130-kDa protoxin inclusions at levels comparable to the wild-type toxin. Bioassays against Aedes aegypti larvae revealed that only H178A and H180A mutants displayed a drastic reduction in biotoxicity, albeit almost complete insolubility observed for H178A, but not for H180A inclusions. Further mutagenic analysis showed that replacements of His180 with charged (Arg, Lys, Asp, Glu), small uncharged polar (Ser, Cys) or small non-polar (Gly, Val) residues severely impaired the biotoxicity, unlike substitutions with relatively large uncharged (Asn, Gln, Leu) or aromatic (Phe, Tyr, Trp) residues. Similar to the trypsin-activated wild-type toxin, both bio-active and -inactive H180 mutants were still capable of releasing entrapped calcein from lipid vesicles and producing cation-selective channels with ~130-pS maximum conductance. Analysis of the Cry4Aa structure revealed the existence of a hydrophobic cavity near the critical His180 side-chain. Analysis of simulated structures revealed that His180-to-smaller residue conversions create a gap disrupting such cavity's hydrophobicity and hence structural arrangements of the α4-α5 hairpin. Altogether, our data disclose a critical involvement in Cry4Aa-biotoxicity of His180 exclusively present in the lumen-facing α4 for providing proper environment for the α4-α5 hairpin prior to membrane-inserted pore formation.

We previously demonstrated that the ~130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was acylated at Lys983 and thus activated its hemolytic activity. Here, attempts were made to provide greater insights into such toxin activation via fatty-acyl modification by CyaC-acyltransferase. Non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaC were separately expressed in E. coli and subsequently purified by FPLC to near homogeneity. When effects of acyl-chain length were comparatively evaluated through CyaC-esterolysis using various p-nitrophenyl (pNP) derivatives, Michaelis-Menten steady-state kinetic parameters (KM and kcat) of CyaC-acyltransferase revealed a marked preference for myristoyl (C14:0) and palmitoyl (C16:0) substrates of which catalytic efficiencies (kcat/KM) were roughly the same (~1.5 × 103 s−1mM−1). However, pNP-palmitate (pNPP) gave the highest hemolytic activity of NA/CyaA-Hly after being acylated in vitro with a range of acyl-donor substrates. LC-MS/MS analysis confirmed such CyaC-mediated palmitoylation of CyaA-Hly occurring at Lys983, denoting no requirement of an acyl carrier protein (ACP). A homology-based CyaC structure inferred a role of a potential catalytic dyad of conserved Ser30 and His33 residues in substrate esterolysis. CyaC-ligand binding analysis via molecular docking corroborated high-affinity binding of palmitate with its carboxyl group oriented toward such a dyad. Ala-substitutions of each residue (S30A or H33A) caused a drastic decrease in kcat/KM of CyaC toward pNPP, and hence its catalytic malfunction through palmitoylation-dependent activation of NA/CyaA-Hly. Altogether, our present data evidently provide such preferential palmitoylation of CyaA-Hly by CyaC-acyltransferase through the enzyme Ser30-His33 nucleophile-activation dyad in esterolysis of palmitoyl-donor substrate, particularly devoid of a natural acyl-ACP donor.

Vacuolating cytotoxin A (VacA) is a highly polymorphic virulence protein produced by the human gastric pathogen Helicobacter pylori which can cause gastritis, peptic ulcer and gastric cancer. Here, we present an optimized protein preparation of the mature full-length VacA variants (m1-and m2-types) and their 33-kDa N-terminal and 55/59-kDa C-terminal domains as biologically active recombinant proteins fused with an N-terminal His(6) tag. All recombinant VacA constructs were over-expressed in Escherichia coli as insoluble inclusions which were soluble when phosphate buffer (pH 7.4) was supplemented with 5–6 M urea. Upon immobilized-Ni2+ affinity purification under 5-M urea denaturing conditions, homogenous products (>95% purity) of 55/59-kDa domains were consistently obtained while only ~80% purity of both mature VacA variants and the 33-kDa truncate was achieved, thus requiring additional purification by size-exclusion chromatography. After successive refolding via optimized stepwise dialysis, all refolded VacA proteins were proven to possess both cytotoxic and vacuolating activity against cultured human gastric epithelial cells albeit the activity observed for VacA-m2 was lower than the m1-type variant. Such an optimized protocol described herein was effective for production of high-purity recombinant VacA proteins in large amounts (~30–40 mg per liter culture) that would pave the way for further studies on sequence-structure and function relationships of different VacA variants.

Background This study investigated suicide risk and its correlates among major affective disorder patients in China and examined possible risk factors for future suicide among individuals with major affective disorder to inform appropriate interventions and management approaches to minimize and prevent suicide. Methods A total of 1478 major affective disorder patients were consecutively examined in 13 mental health centers in China. The patients' socio-demographic and clinical characteristics were recorded using a standardized protocol and data collection procedure. DSM-IV diagnoses were established using the Mini International Neuropsychiatric Interview (MINI), and suicide risk was assessed by the suicide risk module of the MINI. Results Of the patients, 963 (65.2%) were in the nonsuicidal risk group and 515 (34.8%) were in the suicidal risk group. Compared to major depressive disorder patients, bipolar disorder patients had higher suicide risk levels ( χ 2=10.0, df=1, P =0.002); however, there were no statistically significant differences ( χ 2=2.6, df=1, P =0.1) between bipolar disorder-I and bipolar disorder-II patients. Suicide risk factors were associated with 6 variables in major affective disorder patients, as follows: male gender, unemployed, more frequent depressive episodes (>4 in the past year), depressive episodes with suicidal ideation and attempts, depressive episodes with psychotic symptoms, and no current antidepressant use. Limitations Most of the data were retrospectively collected and, therefore, subject to recall bias. Conclusions This study suggested that bipolar disorder patients have a higher suicide risk than major depressive disorder patients. The factors that were significantly associated with suicide risk may aid in identifying major affective disorder patients who are at risk for future suicidal behavior.

Previously, we investigated transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and mimics the action of exogenous supplied CK in delaying postharvest senescence of broccoli. Here, we used proteomics and transcriptomics to compare the mechanisms of ipt-transgenic and N6-benzylaminopurine (BA) CK treatment of broccoli during postharvest storage. The 2 treatments conferred common and distinct mechanisms. BA treatment decreased the quantity of proteins involved in energy and carbohydrate metabolism and amino acid metabolism, and ipt-transgenic treatment increased that of stress-related proteins and molecular chaperones and slightly affected levels of carbohydrate metabolism proteins. Both treatments regulated genes involved in CK signaling, sugar transport, energy and carbohydrate metabolism, amino acid metabolism and lipid metabolism, although ipt-transgenic treatment to a lesser extent. BA treatment induced genes encoding molecular chaperones, whereas ipt-transgenic treatment induced stress-related genes for cellular protection during storage. Both BA and ipt-transgenic treatments acted antagonistically on ethylene functions. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. Biological significance Transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and N6-benzylaminopurine (BA) CK treated broccoli both showed retardation of postharvest senescence during storage. The mechanisms underlying the two treatments were compared. The combination of proteomic and transcriptomic evidences revealed that the 2 treatments conferred common and distinct mechanisms in delaying senescence of broccoli florets. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. This article is part of a Special Issue entitled: Translational Plant Proteomics.

The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

Background Bipolar disorder (BD) is often misdiagnosed as a major depressive disorder (MDD). This study aimed to test the usefulness of the screening tool, the Mood Disorders Questionnaire (MDQ), to identify BD patients misdiagnosed as MDD in Chinese clinical settings. Methods A total of 1487 patients treated for MDD were consecutively examined in 13 mental health centers in China. The patients’ socio-demographic and clinical characteristics were recorded using a standardized protocol and data collection procedure. The MDQ was completed by patients to identify symptoms characteristic of BD. DSM-IV diagnoses were established using the Mini International Neuropsychiatric Interview (MINI). Results Three hundred-nine of the 1487 patients (20.8%) satisfied DSM-IV criteria for BD; 118 (7.9%) for BD-I and 191 (12.8%) for BD-II on the MINI. The optimal cut-off point on the MDQ to differentiate BD from MDD was 3, while cut-off points of 5 and 3 differentiated BD-I and BD-II from MDD, respectively. The maximum sensitivity was 0.31, 0.45 and 0.22 for differentiating BD, BD-I and BD-II from MDD, respectively. Conclusions The optimal cutoff points of the MDQ for screening BD in Chinese patients originally diagnosed as MDD were considerably lower than those in earlier studies. The routine clinical use of the MDQ as a screening scale for BD in Chinese patients treated for MDD does not seem to be justified.

Aim To investigate the results of the Hypomania Checklist-32 (HCL-32) administered as a screening instrument in clinical settings to mood disorders patients in the depressive phase. Methods A total of 1487 patients diagnosed and being treated for major depressive disorder (MDD) in 13 mental health centers across China were self-rated by the HCL-32 and independently examined by the Mini International Neuropsychiatric Interview (MINI). Results After examination by the MINI, 309 (20.8%) of the 1487 patients clinically diagnosed as having MDD satisfied DSM-IV criteria for bipolar disorder (BD): 118 (7.9%) for bipolar I disorder (BD-I) and 191 (12.8%) for bipolar II disorder (BD-II). The mean HCL-32 score of the BD patients was statistically higher than that of patients with unipolar depression (UD, major depressive disorder), BD-II higher than UD, while no significant difference between BD-I and BD-II. The HCL-32 distinguished between BD and UD (best cutoff score 14), between BD-II and UD (best cutoff 12). At the optimum cutoff of 12 between BD and UD, the sensitivity was 0.86, specificity 0.69. Limitations No standardized instruments were used to measure the severity of depressive symptoms. Depressed patients with a previous history of BD were excluded from this study. Conclusions The HCL-32 results in this multicenter study of patients in the depressive phase were similar to those of earlier, generally smaller-scale studies which subjects could be in any mood phases. A score of 12 could be used as the optimum cutoff between BD and UD to improve screening for BD-II if the HCL-32 was applied in clinical settings in China.

Background Bipolar disorder (BD) is a recurrent, complex illness and often misdiagnosed and treated as a major depressive disorder (MDD). This study set out (1) to investigate the proportion of BD in patients treated for MDD using DSM-IV diagnostic criteria; (2) to test the usefulness of the screening tool — the 32-item Hypomania Checklist (HCL-32) in Chinese patients; and (3) to assess whether MDD patients with subthreshold manic features (patients who screened positive for BD on the HCL-32, but did not meet the diagnostic criteria for DSM-IV BD as measured by Mini International Neuropsychiatric Interview (MINI)) differ from those with BD, and from those suffering from MDD without manic features in terms of basic demographic and clinical variables. Methods A total of 1487 patients treated for MDD were consecutively examined in 13 mental health centers in China. The patients' socio-demographic and clinical characteristics were recorded using a standardized protocol and data collection procedure. The HCL-32 was self-completed by patients to identify hypomanic symptoms, and the MINI was used by clinicians to establish DSM-IV diagnoses. Results The proportions of undiagnosed BD (all types), BD-I and BD-II were 20.8%, 7.9% and 12.8%, respectively. The HCL-32 had low positive predictive value (0.43). Compared to MDD patients without subthreshold manic features, MDD patients with subthreshold manic features were younger at onset, less likely to be married and had more depressive episodes on a seasonal basis, and more frequent depressive episodes overall. Compared to BD patients, MDD patients with subthreshold manic features had an older age at onset and less frequent depressive episodes and less family history of psychiatric disorders, appetite, weight gain and time spent sleeping, suicide ideation and attempts and psychotic symptoms. Conclusions At least one fifth of Chinese patients treated for MDD may have an undiagnosed BD. The HCL-32 is useful to identify broader subthreshold bipolar features. The findings need to be confirmed by longitudinal studies using more comprehensive, standardized instruments.

The aim of this work was to evaluate the genetic variations of the sweet potato by a random amplified polymorphic DNA (RAPD) assay with emphasis on correlations with morphological traits. Cuttings of superior and inferior lines derived from tissue culture-regenerated plants and asexually propagated plants of the Tainung 57 (TN57) and Tainung 66 (TN66) sweet potato were planted in the field. Three important agronomic traits, top weight, root weight, and root numbers, were measured 3 months after planting. The RAPD-PCR (polymerase chain reaction) technique was used for detecting genomic variations within and between varieties. Of the 160 primers tested in this study, 38 revealed clear and repeatable RAPD polymorphisms. Among the 38 primers, 8 showed consistent amplified band patterns among the plants with variations within and between varieties, while the others indicated polymorphisms within or between varieties. RAPD markers demonstrated a clear association with root weight. However, the presence and absence of these genetic markers did not correspond well with either the top weight or root number. Genetic instability revealed in the selection stress of superior and inferior plants was found to be independent of variety. In addition, both asexual propagated and tissue culture-induced somaclonal variations were observed in this study. Our results show that RAPD is a useful tool for detecting somaclonal variations from varietal and intra-varietal sweet potato for monitoring of DNA changes in somaclonal variants. The identification of regions which are associated with the character of root weight by RAPD markers enables us to use these markers as selection tools to improve root weight in sweet potato.