The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63‐specific IFN‐γ‐secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA‐A*0201 restriction of ten predicted MPT63‐derived CD8 ⁺ T‐cell epitopes was assessed on the basis of T2 cell line and HLA‐A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN‐γ enzyme‐linked immunospot assay. It was found that five peptides bound to HLA‐A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA‐A*0201. Five immunogenic peptides (MPT63_18–26, MPT63_29–37, MPT63_20–28, MPT63_5–14 and MPT63_10–19) elicited large numbers of cytotoxic IFN‐γ‐secreting T cells in HLA‐A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA‐A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T‐SPOT.TB assay (based on ESAT‐6 and CFP‐10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T‐SPOT.TB assay reached 100%. These MPT63‐derived HLA‐A*0201‐restricted CD8 ⁺ T‐cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine.     

Yangtze finless porpoises along the main channel of Poyang Lake,China: Implications for conservation

Poyang Lake is the largest freshwater lake in China and flows into the Yangtze River. It is a traditional habitat for the endangered Yangtze finless porpoise, which has not been well investigated. To reveal the distribution of the porpoise in Poyang Lake, 12 passive acoustic surveys were conducted along 123 km of the main channel of the lake during different seasons (spring transition season, wet season, autumn transition season, and dry season) from 2008 to 2012. We counted the number of phonating porpoises encountered and calculated the detection rate (encountered individuals detected per kilometer). The median porpoise detection rates ranged from 0 to 0.65 individuals per kilometer during the different surveys. The highest median detection rate of 0.50 was detected in the autumn transition season. The seasonal shrinking of the lake during the dry season may cause a concentration of porpoises in the narrow channels and a high incidence of collisions with cargo ships and fishing boats. Conservation actions should be focused on the main channel of the lake during the dry and transition seasons. In addition, the expansion of the existing reserve to include areas with high porpoise detection rates is necessary.     

Urotensin-II Receptor Stimulation of Cardiac L-type Ca2+ Channels Requires the βγ Subunits of Gi/o-protein and Phosphatidylinositol 3-Kinase-dependent Protein Kinase C β1 Isoform

Recent studies have demonstrated that urotensin-II (U-II) plays important roles in cardiovascular actions including cardiac positive inotropic effects and increasing cardiac output. However, the mechanisms underlying these effects of U-II in cardiomyocytes still remain unknown. We show by electrophysiological studies that U-II dose-dependently potentiates L-type Ca²⁺ currents (I_Ca,L) in adult rat ventricular myocytes. This effect was U-II receptor (U-IIR)-dependent and was associated with a depolarizing shift in the voltage dependence of inactivation. Intracellular application of guanosine-5′-O-(2-thiodiphosphate) and pertussis toxin pretreatment both abolished the stimulatory effects of U-II. Dialysis of cells with the QEHA peptide, but not scrambled peptide SKEE, blocked the U-II-induced response. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin as well as the class I PI3K antagonist {"type":"entrez-nucleotide","attrs":{"text":"CH132799","term_id":"45009640","term_text":"CH132799"}}CH132799 blocked the U-II-induced I_Ca,L response. Protein kinase C antagonists calphostin C and chelerythrine chloride as well as dialysis of cells with 1,2bis(2aminophenoxy)ethaneN,N,N′,N′-tetraacetic acid abolished the U-II-induced responses, whereas PKCα inhibition or PKA blockade had no effect. Exposure of ventricular myocytes to U-II markedly increased membrane PKCβ₁ expression, whereas inhibition of PKCβ₁ pharmacologically or by shRNA targeting abolished the U-II-induced I_Ca,L response. Functionally, we observed a significant increase in the amplitude of sarcomere shortening induced by U-II; blockade of U-IIR as well as PKCβ inhibition abolished this effect, whereas Bay K8644 mimicked the U-II response. Taken together, our results indicate that U-II potentiates I_Ca,L through the βγ subunits of G_i/o-protein and downstream activation of the class I PI3K-dependent PKCβ₁ isoform. This occurred via the activation of U-IIR and contributes to the positive inotropic effect on cardiomyocytes.     

Regulation of nuclear–cytoplasmic shuttling and function of Family with sequence similarity 13, member A (Fam13a), by B56-containing PP2As and Akt

Recent genome-wide association studies reveal that the FAM13A gene is associated with human lung function and a variety of lung diseases, including chronic obstructive pulmonary disease, asthma, lung cancer, and pulmonary fibrosis. The biological functions of Fam13a, however, have not been studied. In an effort to identify novel substrates of B56-containing PP2As, we found that B56-containing PP2As and Akt act antagonistically to control reversible phosphorylation of Fam13a on Ser-322. We show that Ser-322 phosphorylation acts as a molecular switch to control the subcellular distribution of Fam13a. Fam13a shuttles between the nucleus and cytoplasm. When Ser-322 is phosphorylated by Akt, the binding between Fam13a and 14-3-3 is enhanced, leading to cytoplasmic sequestration of Fam13a. B56-containing PP2As dephosphorylate phospho–Ser-322 and promote nuclear localization of Fam13a. We generated Fam13a-knockout mice. Fam13a-mutant mice are viable and healthy, indicating that Fam13a is dispensable for embryonic development and physiological functions in adult animals. Intriguingly, Fam13a has the ability to activate the Wnt pathway. Although Wnt signaling remains largely normal in Fam13a-knockout lungs, depletion of Fam13a in human lung cancer cells causes an obvious reduction in Wnt signaling activity. Our work provides important clues to elucidating the mechanism by which Fam13a may contribute to human lung diseases.