Measuring mitochondrial respiration in intact single muscle fibers

Measurement of mitochondrial function in skeletal muscle is a vital tool for understanding regulation of cellular bioenergetics. Currently, a number of different experimental approaches are employed to quantify mitochondrial function, with each involving either mechanically or chemically induced disruption of cellular membranes. Here, we describe a novel approach that allows for the quantification of substrate-induced mitochondria-driven oxygen consumption in intact single skeletal muscle fibers isolated from adult mice. Specifically, we isolated intact muscle fibers from the flexor digitorum brevis muscle and placed the fibers in culture conditions overnight. We then quantified oxygen consumption rates using a highly sensitive microplate format. Peak oxygen consumption rates were significantly increased by 3.4-fold and 2.9-fold by simultaneous stimulation with the uncoupling agent, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and/or pyruvate or palmitate exposure, respectively. However, when calculating the total oxygen consumed over the entire treatment, palmitate exposure resulted in significantly more oxygen consumption compared with pyruvate. Further, as proof of principle for the procedure, we isolated fibers from the mdx mouse model, which has known mitochondrial deficits. We found significant reductions in initial and peak oxygen consumption of 51% and 61% compared with fibers isolated from the wild-type (WT) animals, respectively. In addition, we determined that fibers isolated from mdx mice exhibited less total oxygen consumption in response to the FCCP + pyruvate stimulation compared with the WT mice. This novel approach allows the user to make mitochondria-specific measures in a nondisrupted muscle fiber that has been isolated from a whole muscle.     

Yersinia pseudotuberculosis effector YopJ subverts the Nod2/RICK/TAK1 pathway and activates caspase-1 to induce intestinal barrier dysfunction

Yersinia pseudotuberculosis is an enteropathogenic bacteria that disrupts the intestinal barrier and invades its host through gut-associated lymphoid tissue and Peyer's patches (PP). We show that the Y.?pseudotuberculosis effector YopJ induces intestinal barrier dysfunction by subverting signaling of the innate immune receptor Nod2, a phenotype that can be reversed by pretreating with the Nod2 ligand muramyl-dipeptide. YopJ, but not the catalytically inactive mutant YopJ(C172A), acetylates critical sites in the activation loops of the RICK and TAK1 kinases, which are central mediators of Nod2 signaling, and decreases the affinity of Nod2 for RICK. Concomitantly, Nod2 interacts with and activates caspase-1, resulting in increased levels of IL-1β. Finally, IL-1β within PP plays an essential role in inducing intestinal barrier dysfunction. Thus, YopJ alters intestinal permeability and promotes the dissemination of Yersinia as well as commensal bacteria by exploiting the mucosal inflammatory response.     

Carbon monoxide improves cardiac function and mitochondrial population quality in a mouse model of metabolic syndrome

Aims

Metabolic syndrome induces cardiac dysfunction associated with mitochondria abnormalities. As low levels of carbon monoxide (CO) may improve myocardial and mitochondrial activities, we tested whether a CO-releasing molecule (CORM-3) reverses metabolic syndrome-induced cardiac alteration through changes in mitochondrial biogenesis, dynamics and autophagy.

Methods and Results

Mice were fed with normal diet (ND) or high-fat diet (HFD) for twelve weeks. Then, mice received two intraperitoneal injections of CORM-3 (10 mg.kg⁻¹), with the second one given 16 hours after the first. Contractile function in isolated hearts and mitochondrial parameters were evaluated 24 hours after the last injection. Mitochondrial population was explored by electron microscopy. Changes in mitochondrial dynamics, biogenesis and autophagy were assessed by western-blot and RT-qPCR. Left ventricular developed pressure was reduced in HFD hearts. Mitochondria from HFD hearts presented reduced membrane potential and diminished ADP-coupled respiration. CORM-3 restored both cardiac and mitochondrial functions. Size and number of mitochondria increased in the HFD hearts but not in the CORM-3–treated HFD group. CORM-3 modulated HFD-activated mitochondrial fusion and biogenesis signalling. While autophagy was not activated in the HFD group, CORM-3 increased the autophagy marker LC3-II. Finally, ex vivo experiments demonstrated that autophagy inhibition by 3-methyladenine abolished the cardioprotective effects of CORM-3.

Conclusion

CORM-3 may modulate pathways controlling mitochondrial quality, thus leading to improvements of mitochondrial efficiency and HFD-induced cardiac dysfunction.     

Photodegradation of the Mycobacterium ulcerans toxin, mycolactones: considerations for handling and storage

Background

Mycolactones are toxins secreted by M. ulcerans, the etiological agent of Buruli ulcer. These toxins, which are the main virulence factors of the bacilli, are responsible for skin lesions. Considering their specificity for M. ulcerans and their presence in skin lesions even at early stages, mycolactones are promising candidates for the development of a diagnostic tool for M. ulcerans infection. Stability of purified mycolactones towards light and heat has not yet been investigated, despite the importance of such parameters in the selection of strategies for a diagnosis tool development. In this context, the effects of UV, light and temperature on mycolactone stability and biological activity were studied.

Methodology/Principal Findings

To investigate the effect of these physical parameters, mycolactones were exposed to different wavelengths in several solvents and temperatures. Structural changes and biological activity were monitored. Whilst high temperature had no effect on mycolactones, UV irradiation (UV-A, UV-B and UV-C) and sunlight exposure caused a considerable degradation, as revealed by LC-MS and NMR analysis, correlated with a loss of biological activity. Moreover, effect of UVs on mycolactone caused a photodegradation rather than a phototransformation due to the identification of degradation product.

Conclusion/Significance

This study demonstrates the high sensitivity of mycolactones to UVs as such it defines instructions for storage and handling.     

Cysteine scanning mutagenesis and disulfide mapping analysis of arrangement of GspC and GspD protomers within the type 2 secretion system

The type II secretion system (T2SS) secretes enzymes and toxins across the outer membrane of Gram-negative bacteria. The precise assembly of T2SS, which consists of at least 12 core-components called Gsp, remains unclear. The outer membrane secretin, GspD, forms the channels, through which folded proteins are secreted, and interacts with the inner membrane component, GspC. The periplasmic regions of GspC and GspD consist of several structural domains, HR(GspC) and PDZ(GspC), and N0(GspD) to N3(GspD), respectively, and recent structural and functional studies have proposed several interaction sites between these domains. We used cysteine mutagenesis and disulfide bonding analysis to investigate the organization of GspC and GspD protomers and to map their interaction sites within the secretion machinery of the plant pathogen Dickeya dadantii. At least three distinct GspC-GspD interactions were detected, and they involve two sites in HR(GspC), two in N0(GspD), and one in N2(GspD). None of these interactions occurs through static interfaces because the same sites are also involved in self-interactions with equivalent neighboring domains. Disulfide self-bonding of critical interaction sites halts secretion, indicating the transient nature of these interactions. The secretion substrate diminishes certain interactions and provokes an important rearrangement of the HR(GspC) structure. The T2SS components OutE/L/M affect various interaction sites differently, reinforcing some but diminishing the others, suggesting a possible switching mechanism of these interactions during secretion. Disulfide mapping shows that the organization of GspD and GspC subunits within the T2SS could be compatible with a hexamer of dimers arrangement rather than an organization with 12-fold rotational symmetry.     

Placental Growth Factor for the Prediction of Adverse Outcomes in Patients with Suspected Preeclampsia or Intrauterine Growth Restriction

Background

The circulating concentration of PlGF is reported to be lower in patients experiencing preeclampsia and patients delivering a small for gestational age (SGA) neonate. To evaluate the predictive value of circulating PlGF for preeclampsia and adverse outcome in patients with suspected preeclampsia or intrauterine growth restriction (IUGR).

Methodology/Principal Findings

A double blind prospective study. We enrolled 96 women for suspected preeclampsia or IUGR, and measured plasma levels of PlGF (Triage®) at enrolment. We defined adverse outcome as severe preeclampsia, SGA neonate (<10^th centile) or elective delivery for maternal or fetal complication. Severe adverse outcome was studied among patients included <34 weeks gestation (WG) and defined as eclampsia, HELLP syndrome, very SGA (<3^rd centile) or elective delivery <34 WG. The mean logtransformed PlGF level was lower for women who experienced preeclampsia (2.9 vs 3.7, p = 0.02), and was markedly lower for patients who experienced adverse outcome (2.9 vs 4.3, p<0.001). The odds of presenting an adverse outcome were higher for the lowest tertile of PlGF compared to the higher (OR = 13 , 95% CI [3–50]). For severe adverse outcome, odds were respectively for the lowest and intermediate tertile as compared with the higher tertile : OR = 216, 95% CI [18–2571]; and OR = 17, 95% CI [3–94]. When included <34 WG, patients with a PlGF level <12 pg/ml experienced a severe adverse outcome in 96% of cases (24/25), and only 1 of 20 patients with a PlGF level >5^th centile experienced a severe adverse outcome within 15 days (5%).

Conclusions/Significance

Among women with suspected preeclampsia or IUGR, PlGF helps identify women who will experience an adverse outcome and those who will not within a time period of 15 days.     

Cis-regulatory properties of medaka synexpression groups

During embryogenesis, tissue specification is triggered by the expression of a unique combination of developmental genes and their expression in time and space is crucial for successful development. Synexpression groups are batteries of spatiotemporally co-expressed genes that act in shared biological processes through their coordinated expression. Although several synexpression groups have been described in numerous vertebrate species, the regulatory mechanisms that orchestrate their common complex expression pattern remain to be elucidated. Here we performed a pilot screen on 560 genes of the vertebrate model system medaka (Oryzias latipes) to systematically identify synexpression groups and investigate their regulatory properties by searching for common regulatory cues. We find that synexpression groups share DNA motifs that are arranged in various combinations into cis-regulatory modules that drive co-expression. In contrast to previous assumptions that these genes are located randomly in the genome, we discovered that genes belonging to the same synexpression group frequently occur in synexpression clusters in the genome. This work presents a first repertoire of synexpression group common signatures, a resource that will contribute to deciphering developmental gene regulatory networks.     

Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI.     

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation

Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120-160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway.