Cardiomyocyte-specific perilipin 5 overexpression leads to myocardial steatosis and modest cardiac dysfunction

Presence of ectopic lipid droplets (LDs) in cardiac muscle is associated to lipotoxicity and tissue dysfunction. However, presence of LDs in heart is also observed in physiological conditions, such as when cellular energy needs and energy production from mitochondria fatty acid β-oxidation are high (fasting). This suggests that development of tissue lipotoxicity and dysfunction is not simply due to the presence of LDs in cardiac muscle but due at least in part to alterations in LD function. To examine the function of cardiac LDs, we obtained transgenic mice with heart-specific perilipin 5 (Plin5) overexpression (MHC-Plin5), a member of the perilipin protein family. Hearts from MHC-Plin5 mice expressed at least 4-fold higher levels of plin5 and exhibited a 3.5-fold increase in triglyceride content versus nontransgenic littermates. Chronic cardiac excess of LDs was found to result in mild heart dysfunction with decreased expression of peroxisome proliferator-activated receptor (PPAR)α target genes, decreased mitochondria function, and left ventricular concentric hypertrophia. Lack of more severe heart function complications may have been prevented by a strong increased expression of oxidative-induced genes via NF-E2-related factor 2 antioxidative pathway. Perilipin 5 regulates the formation and stabilization of cardiac LDs, and it promotes cardiac steatosis without major heart function impairment.     

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells

In this study we used differentiated adult human upcyte^® cells for the in vitro generation of liver organoids. Upcyte^® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte^® process). Proliferating upcyte^® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte^® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.     

SAMM50 acts with p62 in piecemeal basal- and OXPHOS-induced mitophagy of SAM and MICOS components

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.     

Primate Tourism,Range Restriction,and Infant Risk Among <Emphasis Type="Italic">Macaca thibetana</Emphasis> at Mt. Huangshan,China

Primate tourism is a growing trend in habitat countries, but few assessments of its impact on primate groups are available. We compare infant mortality in a group of Tibetan macaques (Macaca thibetana) 6 yr before the government translocated the group and subsequently used them for tourism (1986–91), 12 yr during management for tourism (1992–2002, 2004), and 1 yr when management was temporarily suspended (2003). We also compare aggression rates among adults before and during management, and test several hypotheses about specific factors (numbers of tourists, degree of range restriction, demographic changes, changes in α-males) that may have harmed infants. Infant mortality was significantly higher during management than before, but it was similar before management vs. during its suspension. After management began, serious attacks on infants occurred shortly before they died, and many infant corpses had bite wounds. Typically, infants sustained wounds after aggression broke out among adults in the provisioning area used for tourist viewing. Adult aggression rates in the provisioning area correlated positively with infant mortality over time. Range restriction accounted for 54.5% of the variation in infant mortality, and was more closely associated with both mortality and aggression than any other specific factor examined. We hypothesize that range restriction led to increased infant mortality by raising aggression levels in the provisioning area. We conclude that infant mortality is useful as an indicator of the impact of tourism on primate groups, and that range restriction is an inappropriate tourism management practice.     

Comparison of Biofilm Formation between Major Clonal Lineages of Methicillin Resistant Staphylococcus aureus

Objectives

Epidemic methicillin-resistant S. aureus (MRSA) clones cause infections in both hospital and community settings. As a biofilm phenotype further facilitates evasion of the host immune system and antibiotics, we compared the biofilm-forming capacities of various MRSA clones.

Methods

Seventy-six MRSA classified into 13 clones (USA300, EMRSA-15, Hungarian/Brazilian etc.), and isolated from infections or from carriers were studied for biofilm formation under static and dynamic conditions. Static biofilms in microtitre plates were quantified colorimetrically. Dynamic biofilms (Bioflux 200, Fluxion, USA) were studied by confocal laser-scanning and time-lapse microscopy, and the total volume occupied by live/dead bacteria quantified by Volocity 5.4.1 (Improvision, UK).

Results

MRSA harbouring SCCmec IV produced significantly more biomass under static conditions than SCCmec I–III (P = 0.003), and those harbouring SCCmec II significantly less than those harbouring SCCmec I or III (P<0.001). In the dynamic model, SCCmec I–III harbouring MRSA were significantly better biofilm formers than SCCmec IV (P = 0.036). Only 16 strains successfully formed biofilms under both conditions, of which 13 harboured SCCmec IV and included all tested USA300 strains (n = 3). However, USA300 demonstrated remarkably lower percentages of cell-occupied space (6.6%) compared to the other clones (EMRSA-15 = 19.0%) under dynamic conditions. Time-lapse microscopy of dynamic biofilms demonstrated that USA300 formed long viscoelastic tethers that stretched far from the point of attachment, while EMRSA-15 consisted of micro-colonies attached densely to the surface.

Conclusions

MRSA harbouring SCCmec types IV and I–III demonstrate distinct biofilm forming capacities, possibly owing to their adaptation to the community and hospital settings, respectively. USA300 demonstrated abundant biofilm formation under both conditions, which probably confers a competitive advantage, contributing to its remarkable success as a pathogen.     

Confocal Laser Endomicroscopy for the Morphometric Evaluation of Microvessels in Human Colorectal Cancer Using Targeted Anti-CD31 Antibodies

Introduction

Numerous anti-angiogenic agents are currently developed to limit tumor growth and metastasis. While these drugs offer hope for cancer patients, their transient effect on tumor vasculature is difficult to assess in clinical settings. Confocal laser endomicroscopy (CLE) is a novel endoscopic imaging technology that enables histological examination of the gastrointestinal mucosa. The aim of the present study was to evaluate the feasibility of using CLE to image the vascular network in fresh biopsies of human colorectal tissue. For this purpose we have imaged normal and malignant biopsy tissue samples and compared the vascular network parameters obtained with CLE with established histopathology techniques.

Materials and Methods

Fresh non-fixed biopsy samples of both normal and malignant colorectal mucosa were stained with fluorescently labeled anti-CD31 antibodies and imaged by CLE using a dedicated endomicroscopy system. Corresponding biopsy samples underwent immunohistochemical staining for CD31, assessing the microvessel density (MVD) and vascular areas for comparison with CLE data, which were measured offline using specific software.

Results

The vessels were imaged by CLE in both normal and tumor samples. The average diameter of normal vessels was 8.5±0.9 µm whereas in tumor samples it was 13.5±0.7 µm (p = 0.0049). Vascular density was 188.7±24.9 vessels/mm² in the normal tissue vs. 242.4±16.1 vessels/mm² in the colorectal cancer samples (p = 0.1201). In the immunohistochemistry samples, the MVD was 211.2±42.9/mm² and 351.3±39.6/mm² for normal and malignant mucosa, respectively. The vascular area was 2.9±0.5% of total tissue area for the normal mucosa and 8.5±2.1% for primary colorectal cancer tissue.

Conclusion

Selective imaging of blood vessels with CLE is feasible in normal and tumor colorectal tissue by using fluorescently labeled antibodies targeted against an endothelial marker. The method could be translated into the clinical setting for monitoring of anti-angiogenic therapy.     

Induction of the permeability transition pore in cells depleted of mitochondrial DNA

Respiratory complexes are believed to play a role in the function of the mitochondrial permeability transition pore (PTP), whose dysregulation affects the process of cell death and is involved in a variety of diseases, including cancer and degenerative disorders. We investigated here the PTP in cells devoid of mitochondrial DNA (ρ(0) cells), which lack respiration and constitute a model for the analysis of mitochondrial involvement in several pathological conditions. We observed that mitochondria of ρ(0) cells maintain a membrane potential and that this is readily dissipated after displacement of hexokinase (HK) II from the mitochondrial surface by treatment with either the drug clotrimazole or with a cell-permeant HK II peptide, or by placing ρ(0) cells in a medium without serum and glucose. The PTP inhibitor cyclosporin A (CsA) could decrease the mitochondrial depolarization induced by either HK II displacement or by nutrient depletion. We also found that a fraction of the kinases ERK1/2 and GSK3α/β is located in the mitochondrial matrix of ρ(0) cells, and that glucose and serum deprivation caused concomitant ERK1/2 inhibition and GSK3α/β activation with the ensuing phosphorylation of cyclophilin D, the mitochondrial target of CsA. GSK3α/β inhibition with indirubin-3'-oxime decreased PTP-induced cell death in ρ(0) cells following nutrient ablation. These findings indicate that ρ(0) cells are equipped with a functioning PTP, whose regulatory mechanisms are similar to those observed in cancer cells, and suggest that escape from PTP opening is a survival factor in this model of mitochondrial diseases. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Dominance Style Among Macaca thibetana on Mt. Huangshan, China

The dominance style concept has proven useful for understanding covariation patterns in relationship qualities, particularly among macaques. However, the dominance styles of many macaques, including Tibetan macaques (Macaca thibetana), have not been examined in detail. We describe patterns of bidirectionality of aggression, postconflict affiliation and kin bias in a group of wild, but provisioned Tibetan macaques over a 2-yr period in order make an initial assessment of their dominance style. Bidirectional aggression, including percentage of counteraggression (1.9%), and conciliatory tendencies (6.4%) were consistently low across partner combinations, seasons and locations (forest vs. provisioning area). In addition, females consistently displayed high levels of kin bias in affiliation and tolerance. Compared with macaque species with better known dominance styles, the Tibetan data generally fell within the range for despotic species and outside the range for relaxed species. Although other researchers have tentatively classified them as tolerant or relaxed, we conclude that Tibetan macaques display a despotic dominance style. This conclusion poses complications to explanations based both on phylogenetic inertia and socio-ecological models.     

Effects of Acute Sepsis on Cellular Dynamics and Amyloid Formation in a Mouse Model of Alzheimer’s Disease

Our objective was to investigate how sepsis influences cellular dynamics and amyloid formation before and after plaque formation. As such, APP-mice were subjected to a polymicrobial abdominal infection resulting in sepsis at 2 (EarlySepsis) and 4 (LateSepsis) months of age. Behavior was tested before sepsis and at 5 months of age. We could not detect any short-term memory or exploration behavior alterations in APP-mice that were subjected to Early or LateSepsis. Immunohistochemical analysis revealed a lower area of NeuN⁺ and Iba1⁺ signal in the cortex of Late compared with EarlySepsis animals (p = 0.016 and p = 0.01), with an increased astrogliosis in LateSepsis animals compared with WT-Sepsis (p = 0.0028), EarlySepsis (p = 0.0032) and the APP-Sham animals (p = 0.048). LateSepsis animals had larger areas of amyloid compared with both EarlySepsis (p = 0.0018) and APP-Sham animals (p = 0.0024). Regardless of the analyzed markers, we were not able to detect any cellular difference at the hippocampal level between groups. We were able to detect an increased inflammatory response around hippocampal plaques in LateSepsis compared with APP-Sham animals (p = 0.0003) and a decrease of AQP4 signal far from Sma⁺ vessels. We were able to show experimentally that an acute sepsis event before the onset of plaque formation has a minimal effect; however, it could have a major impact after its onset.     

The mitochondrial chaperone TRAP1 regulates F-ATP synthase channel formation

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes bioenergetic and proteostatic adaptations of cells, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Electrophysiological measurements indicate that TRAP1 directly inhibits a channel activity of purified F-ATP synthase endowed with the features of the permeability transition pore (PTP) and that it reverses PTP induction by CyPD, antagonizing PTP-dependent mitochondrial depolarization and cell death. Conversely, CyPD outcompetes the TRAP1 inhibitory effect on the channel. Our data identify TRAP1 as an F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.Subject terms: Cancer metabolism, Ion channels, Metabolic pathways, Chaperones