An automated microfluidic chip system for detection of piscine nodavirus and characterization of its potential carrier in grouper farms

Groupers of the Epinephelus spp. are an important aquaculture species of high economic value in the Asia Pacific region. They are susceptible to piscine nodavirus infection, which results in viral nervous necrosis disease. In this study, a rapid and sensitive automated microfluidic chip system was implemented for the detection of piscine nodavirus; this technology has the advantage of requiring small amounts of sample and has been developed and applied for managing grouper fish farms. Epidemiological investigations revealed an extremely high detection rate of piscine nodavirus (89% of fish samples) from 5 different locations in southern Taiwan. In addition, positive samples from the feces of fish-feeding birds indicated that the birds could be carrying the virus between fish farms. In the present study, we successfully introduced this advanced technology that combines engineering and biological approaches to aquaculture. In the future, we believe that this approach will improve fish farm management and aid in reducing the economic loss experienced by fish farmers due to widespread disease outbreaks.     

Efficient identification of amino acid types for fast protein backbone assignments

We describe a procedure that allows for very efficient identification of amino acid types in proteins by selective ¹⁵N-labeling. The usefulness of selective incorporation of ¹⁵N-labeled amino acids into proteins for the backbone assignment has been recognized for several years. However, widespread use of this method has been hindered by the need to purify each selectively labeled sample and by the relatively high cost of labeling with ¹⁵N-labeled amino acids. Here we demonstrate that purification of the selectively ¹⁵N-labeled samples is not necessary and that background-free HSQC spectra containing only the peaks of the overexpressed heterologous protein can be obtained in crude lysates from as little as 100 ml cultures, thus saving time and money. This method can be used for fast and automated backbone assignment of proteins.     

Triple decoding of hepatitis C virus RNA by programmed translational frameshifting

Ribosomes can be programmed to shift from one reading frame to another during translation. Hepatitis C virus (HCV) uses such a mechanism to produce F protein from the -2/+1 reading frame. We now report that the HCV frameshift signal can mediate the synthesis of the core protein of the zero frame, the F protein of the -2/+1 frame, and a 1.5-kDa protein of the -1/+2 frame. This triple decoding function does not require sequences flanking the frameshift signal and is apparently independent of membranes and the synthesis of the HCV polyprotein. Two consensus -1 frameshift sequences in the HCV type 1 frameshift signal facilitate ribosomal frameshifts into both overlapping reading frames. A sequence which is located immediately downstream of the frameshift signal and has the potential to form a double stem-loop structure can significantly enhance translational frameshifting in the presence of the peptidyl-transferase inhibitor puromycin. Based on these results, a model is proposed to explain the triple decoding activities of the HCV ribosomal frameshift signal.     

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.     

Chloroplast DNA polymorphism in different types of cytoplasmic male sterile rice

The lengths of open reading frame (ORF)100 and ORF29-TrnC^GCA, the intronic sequence of rps16 and the transcribed spacer of TrnT^UGU-TrnL^UAA in chloroplast from different lines of cytoplasmic male sterility (CMS) rice were studied using indica types, japonica types and common wild rice as controls. The results show that the lengths of ORF100 and ORF29-TrnC^GCA in CMS lines are similar to those of typical indica. The sequences of the rps16 intron and the TrnT^UGU-TrnL^UAA spacer in sporophyte sterile types (wild-abortive type, Yinshui type and K type) are almost the same, and they also share a molecular marker of GTTGAG at nucleotide positions 220–225 in the rps16 intron. Therefore, it is speculated that the source of these three types is the same. In contrast, a gametophyte sterile type, Yuetai A does not contain such a GTTGAG sequence in the rps16 intron and has a unique G at position 595, which may works as a molecular marker distinguishing the sporophyte sterile type from the gametophyte sterile type. Based on the observation that CMS rice has much lower cytoplasmic polymorphism than indica, japonica and wild rice, it is concluded that CMS rice lack cytoplasm diversity. Therefore, it is important to introduce new sources of cytoplasm into hybrid rice.     

Replication of hepatitis C virus RNA on autophagosomal membranes

Previous studies indicated that hepatitis C virus (HCV) perturbs the autophagic pathway to induce the accumulation of autophagosomes in cells. To understand the role of autophagosomes in the HCV life cycle, we established a stable Huh7 hepatoma cell line that contained an HCV subgenomic RNA replicon and also expressed a GFP-LC3 fusion protein. The GFP-LC3 protein is localized to autophagosomes during autophagy and served as a convenient marker for autophagosomes. Our results indicate that the silencing of the expression of LC3 or Atg7, two protein factors critical for the formation of autophagosomes, suppresses the replication of HCV RNA. Confocal microscopy studies revealed the localization of HCV NS5A and NS5B proteins, which are two important components of the HCV RNA replication complex, and nascent HCV RNA to autophagosomes. The association of the HCV RNA replication complex with the autophagosomal membranes was further confirmed by co-immunoprecipitation and immunoelectron microscopy studies. Interestingly, inhibition of Class III PI3K activity had no effect on the autophagosomes induced by HCV. These results indicate that HCV induces autophagosomes via a Class III PI3K-independent pathway and uses autophagosomal membranes as sites for its RNA replication.     

Associations of liver dysfunction with incident dementia,cognition, and brain structure: A prospective cohort study of 431 699 adults

The relationship between liver dysfunction and dementia has been researched extensively but remains poorly understood. In this study, we investigate the longitudinal and cross-sectional associations between liver function and liver diseases and risk of incident dementia, impaired cognition, and brain structure abnormalities using Cox proportion hazard model and linear regression model. 431 699 participants with a mean of 8.65 (standard deviation [SD] 2.61) years of follow-up were included from the UK Biobank; 5542 all-cause dementia (ACD), 2427 Alzheimer's disease (AD), and 1282 vascular dementia (VaD) cases were documented. We observed that per SD decreases in alanine transaminase (ALT; hazard ratio [HR], 0.917; P_FDR <0.001) and per SD increases in aspartate aminotransferase (AST; HR, 1.048; P_FDR = 0.010), AST to ALT ratio (HR, 1.195; P_FDR <0.001), gamma-glutamyl transpeptidase (GGT; HR, 1.066; P_FDR <0.001), alcoholic liver disease (ALD; HR, 2.872; P_FDR <0.001), and fibrosis and cirrhosis of liver (HR, 2.285; P_FDR = 0.002), being significantly associated with a higher risk of incident ACD. Restricted cubic spline models identified a strong U-shaped association between Alb and AST and incident ACD (P_nonlinear <0.05). Worse cognition was positively correlated with AST, AST to ALT ratio, direct bilirubin (DBil), and GGT; negatively correlated with ALT, Alb, and total bilirubin (TBil); and ALD and fibrosis and cirrhosis of liver (P_FDR <0.05). Moreover, changes in ALT, GGT, AST to ALT ratio, and ALD were significantly associated with altered cortical and subcortical regions, including hippocampus, amygdala, thalamus, pallidum, and fusiform (P_FDR <0.05). In sensitivity analysis, metabolic dysfunction-associated steatotic liver disease (MASLD) was associated with the risk of ACD and brain subcortical changes. Our findings provide substantial evidence that liver dysfunction may be an important factor for incident dementia. Early intervention in the unhealthy liver may help prevent cognitive impairment and dementia incidence.     

Vinorelbine-Induced Oxidative Injury in Human Endothelial Cells Mediated by AMPK/PKC/NADPH/NF-κB Pathways

Vinorelbine tartrate (VNR), a semi-synthetic vinca alkaloid acquired from vinblastine, has extensively been used as an anticancer agent. However, VNR-induced oxidative damage may cause several side effects, such as venous irritation, vascular pain, and necrotizing vasculitis, thereby repressing clinical treatment efficiency. The molecular mechanisms underlying the induced oxidative stress in endothelial cells are still largely unknown. This study was designed to test the hypothesis that VNR induces oxidative injury through modulation of AMP-activated protein kinase (AMPK) and possible mechanisms were then explored. Human umbilical vein endothelial cells (HUVECs) were treated with VNR (5–0.625 μM) to produce oxidative damage. The VNR-mediated AMPK, PKC, and NADPH oxidase expressions were investigated by western blotting. Furthermore, several oxidative stress-induced oxidative damage markers as well as pro-inflammatory responses were also investigated. VNR treatment resulted in dephosphorylation of AMPK, which in turn led to an activation of NADPH oxidase by PKC; however, the phenomena were repressed by AICAR (an agonist of AMPK). Furthermore, VNR suppressed Akt/eNOS and enhanced p38 mitogen-activated protein kinase (MAPK), which in turn activated the NF-κB pathway. Furthermore, VNR facilitated several pro-inflammatory events, such as the adherence of monocytic THP-1 cells to HUVECs, pro-inflammatory cytokines release, and overexpression of adhesion molecular. Our results highlight a possible molecular mechanism for VNR-mediated endothelial dysfunction.