Calpain-6 Deficiency Promotes Skeletal Muscle Development and Regeneration

Calpains are Ca²⁺-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6''s in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.     

Nocturnal Blood Pressure Pattern Affects Left Ventricular Remodeling and Late Gadolinium Enhancement in Patients with Hypertension and Left Ventricular Hypertrophy

Background

Left ventricular hypertrophy (LVH) is an independent predictor of cardiac mortality, regardless of its etiology. Previous studies have shown that high nocturnal blood pressure (BP) affects LV geometry in hypertensive patients. It has been suggested that continuous pressure overload affects the development of LVH, but it is unknown whether persistent pressure influences myocardial fibrosis or whether the etiology of LVH is associated with myocardial fibrosis. Comprehensive cardiac magnetic resonance (CMR) including the late gadolinium enhancement (LGE) technique can evaluate both the severity of changes in LV geometry and myocardial fibrosis. We tested the hypothesis that the nocturnal non-dipper BP pattern causes LV remodeling and fibrosis in patients with hypertension and LVH.

Methods

Forty-seven hypertensive patients with LVH evaluated by echocardiography (29 men, age 73.0±10.4 years) were examined by comprehensive CMR and 24-h ambulatory blood pressure monitoring (ABPM).

Results and Conclusions

Among the 47 patients, twenty-four had nocturnal non-dipper BP patterns. Patients with nocturnal non-dipper BP patterns had larger LV masses and scar volumes independent of etiologies than those in patients with dipper BP patterns (p = 0.035 and p = 0.015, respectively). There was no significant difference in mean 24-h systolic BP between patients with and without nocturnal dipper BP patterns (p = 0.367). Among hypertensive patients with LVH, the nocturnal non-dipper blood pressure pattern is associated with both LV remodeling and myocardial fibrosis independent of LVH etiology.     

Controlled Cre/loxP Site-Specific Recombination in the Developing Brain in Medaka Fish,Oryzias latipes

Background

Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.

Methodology/Principal Findings

To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0–1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2–3 dpf embyos compared with 0–1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.

Conclusions/Significance

We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.     

Relationships between Values of Antibodies to Several Connective Tissue Disease Autoantigens and Pulmonary Function in a Japanese General Population: The Takahata Study

Background

Accumulating evidence suggests the involvement of an autoimmune mechanism in the pathogenesis of respiratory dysfunction. The aim of this study was to investigate the relationship between pulmonary function and serum antibodies to several connective tissue disease autoantigens (ACTDA) levels, which has not been investigated in a general population.

Methods

Blood sampling and spirometry were performed for subjects (n = 3,257) aged ≥40 years who participated in a community-based annual health check in Takahata, Japan, from 2004 to 2006. ACTDA was measured by enzyme immunoassay, and subjects with ACTDA values ≥20 were defined as positive.

Results

In males, there were significant inverse relationships between logarithmically transformed ACTDA values and spirometric parameters, including % predicted values for forced expiratory volume in 1 s (FEV₁) and maximal midexpiratory flow (MMF) as well as FEV₁/forced vital capacity (FVC). Multiple linear regression analysis revealed that except for the relationship between ACTDA and FEV₁/FVC, these relationships were still significant after adjustment for Brinkman index (a measure of inhaled cigarette consumption). The prevalence of positive ACTDA was greater in male never-smokers with mixed ventilation disorders and relatively severe airflow obstruction (% predicted FEV₁ below the median value).

Conclusions

Autoimmunity may be involved in the mechanism of impaired pulmonary function in the general population.     

Chitosan Nanoparticle Encapsulated Hemagglutinin-Split Influenza Virus Mucosal Vaccine

Subunit/split influenza vaccines are less reactogenic compared with the whole virus vaccines. However, their immunogenicity is relatively low and thus required proper adjuvant and/or delivery vehicle for immunogenicity enhancement. Influenza vaccines administered intramuscularly induce minimum, if any, mucosal immunity at the respiratory mucosa which is the prime site of the infection. In this study, chitosan (CS) nanoparticles were prepared by ionic cross-linking of the CS with sodium tripolyphosphate (TPP) at the CS/TPP ratio of 1:0.6 using 2 h mixing time. The CS/TPP nanoparticles were used as delivery vehicle of an intranasal influenza vaccine made of hemagglutinin (HA)-split influenza virus product. Innocuousness, immunogenicity, and protective efficacy of the CS/TPP-HA vaccine were tested in influenza mouse model in comparison with the antigen alone vaccine. The CS/TPP-HA nanoparticles had required characteristics including nano-sizes, positive charges, and high antigen encapsulation efficiency. Mice that received two doses of the CS/TPP-HA vaccine intranasally showed no adverse symptoms indicating the vaccine innocuousness. The animals developed higher systemic and mucosal antibody responses than vaccine made of the HA-split influenza virus alone. The CS/TPP-HA vaccine could induce also a cell-mediated immune response shown as high numbers of IFN-γ-secreting cells in spleens while the HA vaccine alone could not. Besides, the CS nanoparticle encapsulated HA-split vaccine reduced markedly the influenza morbidity and also conferred 100% protective rate to the vaccinated mice against lethal influenza virus challenge. Overall results indicated that the CS nanoparticles invented in this study is an effective and safe delivery vehicle/adjuvant for the influenza vaccine.     

Complete genome sequence of the bacterium Porphyromonas gingivalis TDC60, which causes periodontal disease

Porphyromonas gingivalis is a black-pigmented asaccharolytic anaerobe and a major causative agent of periodontitis. Here, we report the complete genome sequence of P. gingivalis strain TDC60, which was recently isolated from a severe periodontal lesion in a Japanese patient.     

LIGHT/TNFSF14 enhances adipose tissue inflammatory responses through its interaction with HVEM

Obesity-induced adipose tissue inflammation is characterized by increased macrophage infiltration and cytokine production, and is associated with metabolic disorders. LIGHT/TNFSF14, a member of the TNF superfamily, plays a role in the development of various inflammatory diseases. The purpose of this study was to examine the involvement of soluble LIGHT (sLIGHT) in obesity-induced adipose tissue inflammatory responses. LIGHT gene expression on macrophages/adipocytes was upregulated by treatment with obesity-related factors. sLIGHT displayed chemotactic activity for macrophages and T cells, and enhanced inflammatory cytokine release from macrophages, adipocytes, and adipose tissue-derived SVF cells. The sLIGHT-induced inflammatory responses were blunted by neutralizing anti-HVEM antibody or knockout of HVEM, a receptor for sLIGHT. These findings indicate that sLIGHT enhances adipose tissue inflammatory responses through its interaction with HVEM.