Genetic diversity and evolution of two capsid protein genes of citrus tristeza virus isolates from China.

The genetic diversity and population structure of citrus tristeza virus (CTV) isolates from China were investigated based on partial sequences spanning the C-terminal end of p61 and the complete sequences of the CPm and CP genes. Phylogenetic analysis revealed five known groups (RB, T30, T36, HA and VT) and one new group (VI) consisting of only Chinese CTV isolates. Incongruent phylogenetic trees coupled with recombination analysis suggested several recombination events in the CPm gene. Positive selection was detected at codon 9 of CPm and codons 31, 41 and 68 of CP. The widespread CTV subpopulation AT-1 found in China has a unique amino acid insertion at the C-terminus of p61, which could increase CTV population complexity with implications for the evolutionary history of the virus. Our results suggest relevant roles for gene flow, purifying selection and recombination in shaping the CTV population in China.

Protein-protein interactions between proteins of Citrus tristeza virus isolates.

Citrus tristeza virus (CTV) is one of the most devastating pathogens of citrus. Its genome is organized into 12 open reading frames (ORFs), of which ten ORFs located at the 3'-terminus of the genome have multiple biological functions. The ten genes at the 3'-terminus of the genome of a severe isolate (CTV-S4) and three ORFs (CP, CPm and p20) of three other isolates (N4, S45 and HB1) were cloned into pGBKT7 and pGADT7 yeast shuttle vectors. Yeast two-hybridization (Y2H) assays results revealed a strong self-interaction for CP and p20, and a unique interaction between the CPm of CTV-S4 (severe) and CP of CTV-N4 (mild) isolates. Bimolecular fluorescence complementation also confirmed these interactions. Analysis of the deletion mutants delineated the domains of CP and p20 self-interaction. Furthermore, the domains responsible for CP and p20 self-interactions were mapped at the CP amino acids sites 41-84 and p20 amino acids sites 1-21 by Y2H. This study provided new information on CTV protein interactions which will help for further understanding the biological functions.

TDP-43 Amyloid Fibril Formation via Phase Separation-Related and -Unrelated Pathways.

Intrinsically disordered regions (IDRs) in proteins can undergo liquid-liquid phase separation (LLPS) for functional assembly, but this increases the chance of forming disease-associated amyloid fibrils. Not all amyloid fibrils form through LLPS however, and the importance of LLPS relative to other pathways in fibril formation remains unclear. We investigated this question in TDP-43, a motor neuron disease and dementia-causing protein that undergoes LLPS, using thioflavin T (ThT) fluorescence, NMR, transmission electron microscopy (TEM), and wide-angle X-ray scattering (WAXS) experiments. Using a fluorescence probe modified from ThT strategically designed for targeting protein assembly rather than ß-sheets and supported by TEM images, we propose that the biphasic ThT signals observed under LLPS-favoring conditions are due to the presence of amorphous aggregates. These aggregates represent an intermediate state that diverges from the direct pathway to ß-sheet-dominant fibrils. Under non-LLPS conditions in contrast (at low pH or at physiological conditions in a construct with key LLPS residues removed), the protein forms a hydrogel. Real-time WAXS data, ThT signals, and TEM images collectively demonstrate that the gelation process circumvents LLPS and yet still results in the formation of fibril-like structural networks. We suggest that the IDR of TDP-43 forms disease-causing amyloid fibrils regardless of the formation pathway. Our findings shed light on why both LLPS-promoting and LLPS-inhibiting mutants are found in TDP-43-related diseases.

The genotypes of citrus tristeza virus isolates from China revealed by sequence analysis of multiple molecular markers.

The genotypes of ten citrus tristeza virus (CTV) isolates from central China were determined by examining multiple molecular markers (MMMs) using 11 primer pairs. The results revealed that one isolate contained a single T30 genotype, two isolates contained a single VT genotype, and the other seven isolates were mixtures of two or more genotypes. Sequence analysis of amplified MMMs showed a high genetic diversity in Chinese CTV populations. The genotypes resembling T36, RB and B165 were identified from Chinese CTV isolates for the first time. Our results suggest that genotype assignment of CTV cannot be based solely on the amplification profiles of MMMs, and sequencing of MMMs is required.

The epitope structure of Citrus tristeza virus coat protein mapped by recombinant proteins and monoclonal antibodies.

It has been known that there exists serological differentiation among Citrus tristeza virus (CTV) isolates. The present study reports three linear epitopes (aa 48-63, 97-104, and 114-125) identified by using bacterially expressed truncated coat proteins and ten monoclonal antibodies against the native virions of CTV-S4. Site-directed mutagenesis analysis demonstrated that the mutation D98G within the newly identified epitope (97)DDDSTGIT(104) abolished its reaction to MAbs 1, 4, and 10, and the presence of G98 in HB1-CP also resulted in its failure to recognize the three MAbs. Our results suggest that the conformational differences in the epitope I (48)LGTQQNAALNRDLFLT(63) between the CPs of isolates S4 and HB1 might contribute to the different reactions of two isolates to MAbs 5 and 6. This study provides new information for the antigenic structures of CTV, and will extend the understanding of the processes required for antibody binding and aid the development of epitope-based diagnostic tools.

[Distributional characteristics of nutrients in the sea-surface microlayer and subsurface water of the Bohai and Yellow Sea in summer].

The horizontal distributional characteristics of nutrients (DIN, PO4(3-) -P, SiO3(2-) -Si) were studied in the sea-surface microlayer (SML) and subsurface water of the Bohai and Yellow Sea in June 2011, and the vertical distributions of nutrients in the transects of the Yellow Sea Cold Water Mass and the north of the Changjiang River estuary were also investigated. The results showed that the nutrient concentrations in the sea-surface microlayer and subsurface water decreased gradually from inshore to offshore, and higher values were observed in the Changjiang River estuary. A tongue-shaped distribution was observed in the south of the Yellow Sea as a result of the Changjiang diluted water. Stratification occurred in the surface layer and vertical mixing characteristics were observed in the bottom due to the influence of the Yellow Sea Cold Water Mass. In the vertical distributions, higher concentrations of DIN and SiO3(2-) -Si in the north of the Changjiang River estuary occurred in the sea surface compared with the bottom due to the Changjiang River diluted water. The enrichment results of NH(+) -N, NO2(-) -N, NO3(-)-N, PO4(3-) -P, SiO3(2-)-Si and DIN showed that the sea-surface microlayer displayed an apparent enrichment for nutrients except NO3(-) -N, and the mean enrichment factors ranged from 1.37 to 2.21.

Effective pressure and bubble generation in a microfluidic T-junction.

To improve the existing trial-and-error process in designing a microfluidic T-junction, a systematic study of the geometrical (mainly the channel length) effects on the generated bubbly/slug flow was conducted to figure out basic design guidelines based on experimental and theoretical analyses. A driving system with dual constant pressure sources, instead of the commonly used dual constant volume-rate sources (such as two syringe pumps), was chosen in this study. The newly proposed effective pressure ratio (P(e)*) has revealed its advantages in excluding the surface tension effect of fluids. All the data of generated bubbly/slug flow for a given geometry collapse excellently into the same relationship of void fraction and effective pressure ratio. This relationship is insensitive to the liquid viscosity and the operation range is strongly affected by the geometrical effect, i.e., the channel length ratio of downstream to total equivalent length of the main channel in a T-junction chip. As to the theoretical design and analysis of gas-liquid-flow characteristics in a microfluidic T-junction, which is still sporadic in the literature, the proposed semi-empirical model has successfully predicted the operation boundaries and the output flow rate of bubbly/slug flow of different investigated cases and demonstrated its usability.