Tuning the geometries of a de novo blue copper protein by axial interactions

The axial interactions of Cu(2+) in type 1 copper proteins control the physical characteristics of the proteins. We tuned the geometries of a de novo designed blue copper protein with a four-helical bundle structure. The designed protein axially bound various ligands, such as chloride, phosphate, sulfate, acetate, azide, and imidazole, to Cu(2+), exhibiting a blue or green color. The UV-vis spectral bands were observed at approximately 600?nm and approximately 450?nm, with the A (~450)/A (~600) ratios between 0.14 and 1.58. The stronger axial interaction shifted the geometry of the type 1 copper site from trigonal planar geometry (blue copper) toward a tetrahedral-like geometry (green copper). Resonance Raman spectral analyses showed that the phosphate-bound type had the highest-strength Cu-S bond, similar to that of plastocyanin. The chloride-bound type exhibited features similar to those of stellacyanin and nitrite reductase, and the imidazole-bound type exhibited features similar to those of azurin M121E mutant.     

p130Cas-dependent actin remodelling regulates myogenic differentiation

Actin dynamics are implicated in various cellular processes, not only through the regulation of cytoskeletal organization, but also via the control of gene expression. In the present study we show that the Src family kinase substrate p130Cas (Cas is Crk-associated substrate) influences actin remodelling and concomitant muscle-specific gene expression, thereby regulating myogenic differentiation. In C2C12 myoblasts, silencing of p130Cas expression by RNA interference impaired F-actin (filamentous actin) formation and nuclear localization of the SRF (serum-response factor) co-activator MAL (megakaryocytic acute leukaemia) following the induction of myogenic differentiation. Consequently, formation of multinucleated myotubes was abolished. Re-introduction of wild-type p130Cas, but not its phosphorylation-defective mutant, into p130Cas-knockdown myoblasts restored F-actin assembly, MAL nuclear localization and myotube formation. Depletion of the adhesion molecule integrin β3, a key regulator of myogenic differentiation as well as actin cytoskeletal organization, attenuated p130Cas phosphorylation and MAL nuclear localization during C2C12 differentiation. Moreover, knockdown of p130Cas led to the activation of the F-actin-severing protein cofilin. The introduction of a dominant-negative mutant of cofilin into p130Cas-knockdown myoblasts restored muscle-specific gene expression and myotube formation. The results of the present study suggest that p130Cas phosphorylation, mediated by integrin β3, facilitates cofilin inactivation and promotes myogenic differentiation through modulating actin cytoskeleton remodelling.     

7-Hydroperoxycholesterol as a marker of oxidative stress in rat kidney induced by paraquat

The in vivo paraquat-induced oxidative stress in rat tissue was studied by analyzing cholesterol-derived hydroperoxide as an index of lipid peroxidation. Paraquat (10 mg/kg) was administered i.p. to rats. Rats were sacrificed and lung, liver, and kidney were collected 2, 24 h, and 5 d after paraquat injection. Lipids were extracted and analyzed by HPLC with post-column chemiluminescence. We found that two cholesterol-derived hydroperoxides, 7α-hydroperoxycholest-5-en-3β-ol (7α-OOH) and 7β-hydroperoxycholest-5-en-3β-ol (7β-OOH) were present in lungs of control animals (0.06 and 0.06 nmol/g, respectively), in livers (6.5 and 15.8 nmol/g, respectively) and in kidneys (3.7 and 8.9 nmol/g, respectively). In liver paraquat increased lipid peroxidation approximately by 60% over the levels of control animals only at 2 h after paraquat treatment. In kidney, augmented lipid peroxidation, 7α-OOH and 7β-OOH (by 70% and 147%, respectively) above levels was found at 2 h after paraquat treatment. Interestingly, these increase remained in kidney of rats 5 d after a single dose of paraquat. In contrast, cholesterol-derived hydroperoxides were not affected in lung of paraquat dosed rats. This is the first report on 7α-OOH and 7β-OOH accumulations in rat liver and kidney, and it seems to reflect greater oxidative stress in the pathology of kidney of rats treated with acute paraquat at low dose.     

Tumor microenvironment disparity in multiple primary lung cancers: Impact of non-intrinsic factors,histological subtypes,and genetic aberrations

IntroductionMultiple primary lung cancers (MPLCs) occur in common carcinogenetic risks such as lifestyle, biological aging, immune responses, hormones, and metabolism. Although MPLCs harbor various genetic profiles within the same individuals, differences in the tumor microenvironment (TME) are unclear. We investigated the impact of genetic aberrations, non-intrinsic factors, and pathological subtypes on tumor immunity.Materials and MethodsIn total, 73 surgically resected specimens from 32 patients with MPLC were analyzed. PD-L1 expression in tumor cells (TCs) and immune cells (ICs), CD3-positive tumor-infiltrating lymphocytes (TILs), CD8/CD3 ratios, and FOXP3-positive TILs that compose TMEs were evaluated by immunohistochemistry and classified on a score of 0–2. 38 tumors were sequenced for somatic mutations in 409 cancer-associated genes.ResultsFemales and never or light smokers had a higher incidence of PD-L1-negative tumors and a higher concordance rate. PD-L1 positivity in TCs and ICs was significantly less frequent in EGFR-mutated than in wild-type tumors. Differences in the score of TMEs were observed between the KRAS-mutated-only tumor and the KRAS and TP53-co-mutated tumors, and between the KRAS-mutated-only tumor and the KRAS and STK11-co-mutated tumors. Significantly more FOXP3-high TILs were observed in invasive pathological subtypes than in non-invasive ones.ConclusionComparing TMEs among MPLCs revealed that non-smokers or light smokers and females were unlikely to express PD-L1 regardless of tumor site and confirmed that the EGFR mutations and co-occurring KRAS and STK11 or TP53 mutations were associated with TME. Pathological subtypes may impact the efficacy of immune therapy due to their potential correlations with regulatory T cells.     

Juvenile migration of Japanese dace Pseudaspius hakonensis revealed in a tributary of Lake Biwa with seasonal changes in abundance and stable isotope ratios

Ichthyological Research - Migration patterns of Japanese dace Pseudaspius hakonensis in and around Lake Biwa, Japan are unknown, although fluvial–lacustrine life history has been known in...     

Association between Sleep Duration and Urinary Albumin Excretion in Patients with Type 2 Diabetes: The Fukuoka Diabetes Registry

Objective

Few studies have so far investigated the impact of sleep duration on chronic kidney disease in diabetic patients. The objective of the present study was to examine the relationship between sleep duration and albuminuria in type 2 diabetic patients.

Research Design and Methods

A total of 4,870 Japanese type 2 diabetic patients ≥20 years of age were divided into six groups according to self-reported sleep duration: less than 4.5 hours, 4.5–5.4 hours, 5.5–6.4 hours, 6.5–7.4 hours, 7.5–8.4 hours and more than 8.5 hours. The association between sleep duration and urinary albumin-creatinine ratio (UACR) was examined cross-sectionally.

Results

Both short and long sleep durations were significantly associated with higher UACR levels and higher proportions of patients with albuminuria (≥30 mg/g) and macroalbuminuria (≥300 mg/g) compared with a sleep duration of 6.5–7.4 hours (P for quadratic trend <0.001). A U-shaped association between sleep duration and UACR remained significant even after adjustment for potential confounders, including age, sex, duration of diabetes, current smoking habits, former smoking habits, current drinking habits, regular exercise habits, total energy intake, total protein intake, hypnotic use and estimated glomerular filtration rate. Furthermore, the association remained substantially unchanged after additional adjustment for body mass index, hemoglobin A_1c, systolic blood pressure, renin-angiotensin system inhibitor use and depressive symptoms.

Conclusions

Our findings suggest that sleep duration has a U-shaped association with the UACR levels in type 2 diabetic patients, independent of potential confounders.