Human Tim8a, Tim8b and Tim13 are auxiliary assembly factors of mature Complex IV.

Human Tim8a and Tim8b are paralogous intermembrane space proteins of the small TIM chaperone family. Yeast small TIMs function in the trafficking of proteins to the outer and inner mitochondrial membranes. This putative import function for hTim8a and hTim8b has been challenged in human models, but their precise molecular function(s) remains undefined. Likewise, the necessity for human cells to encode two Tim8 proteins and whether any potential redundancy exists is unclear. We demonstrate that hTim8a and hTim8b function in the assembly of cytochrome c oxidase (Complex IV). Using affinity enrichment mass spectrometry, we define the interaction network of hTim8a, hTim8b and hTim13, identifying subunits and assembly factors of the Complex IV COX2 module. hTim8-deficient cells have a COX2 and COX3 module defect and exhibit an accumulation of the Complex IV S2 subcomplex. These data suggest that hTim8a and hTim8b function in assembly of Complex IV via interactions with intermediate-assembly subcomplexes. We propose that hTim8-hTim13 complexes are auxiliary assembly factors involved in the formation of the Complex IV S3 subcomplex during assembly of mature Complex IV.

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

Filtered Dark Matter at a First Order Phase Transition.

We describe a new mechanism of dark matter production. If dark matter particles acquire mass during a first order phase transition, it is energetically unfavorable for them to enter the expanding bubbles. Instead, most of them are reflected and quickly annihilate away. The bubbles eventually merge as the phase transition completes and only the dark matter particles that have entered the bubbles survive to constitute the observed dark matter today. This mechanism can produce dark matter with masses from the TeV scale to above the PeV scale, surpassing the Griest-Kamionkowski bound.

Stress-induced OMA1 activation and autocatalytic turnover regulate OPA1-dependent mitochondrial dynamics.

The dynamic network of mitochondria fragments under stress allowing the segregation of damaged mitochondria and, in case of persistent damage, their selective removal by mitophagy. Mitochondrial fragmentation upon depolarisation of mitochondria is brought about by the degradation of central components of the mitochondrial fusion machinery. The OMA1 peptidase mediates the degradation of long isoforms of the dynamin-like GTPase OPA1 in the inner membrane. Here, we demonstrate that OMA1-mediated degradation of OPA1 is a general cellular stress response. OMA1 is constitutively active but displays strongly enhanced activity in response to various stress insults. We identify an amino terminal stress-sensor domain of OMA1, which is only present in homologues of higher eukaryotes and which modulates OMA1 proteolysis and activation. OMA1 activation is associated with its autocatalyic degradation, which initiates from both termini of OMA1 and results in complete OMA1 turnover. Autocatalytic proteolysis of OMA1 ensures the reversibility of the response and allows OPA1-mediated mitochondrial fusion to resume upon alleviation of stress. This differentiated stress response maintains the functional integrity of mitochondria and contributes to cell survival.

Loss of the m-AAA protease subunit AFG3L2 causes mitochondrial transport defects and tau hyperphosphorylation.

The m-AAA protease subunit AFG3L2 is involved in degradation and processing of substrates in the inner mitochondrial membrane. Mutations in AFG3L2 are associated with spinocerebellar ataxia SCA28 in humans and impair axonal development and neuronal survival in mice. The loss of AFG3L2 causes fragmentation of the mitochondrial network. However, the pathogenic mechanism of neurodegeneration in the absence of AFG3L2 is still unclear. Here, we show that depletion of AFG3L2 leads to a specific defect of anterograde transport of mitochondria in murine cortical neurons. We observe similar transport deficiencies upon loss of AFG3L2 in OMA1-deficient neurons, indicating that they are not caused by OMA1-mediated degradation of the dynamin-like GTPase OPA1 and inhibition of mitochondrial fusion. Treatment of neurons with antioxidants, such as N-acetylcysteine or vitamin E, or decreasing tau levels in axons restored mitochondrial transport in AFG3L2-depleted neurons. Consistently, tau hyperphosphorylation and activation of ERK kinases are detected in mouse neurons postnatally deleted for Afg3l2. We propose that reactive oxygen species signaling leads to cytoskeletal modifications that impair mitochondrial transport in neurons lacking AFG3L2.

A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection.

Coxiella burnetii, the causative agent of Q fever, establishes a unique lysosome-derived intracellular niche termed the Coxiella-containing vacuole (CCV). The Dot/Icm-type IVB secretion system is essential for the biogenesis of the CCV and the intracellular replication of Coxiella Effector proteins, translocated into the host cell through this apparatus, act to modulate host trafficking and signaling processes to facilitate CCV development. Here we investigated the role of CBU0077, a conserved Coxiella effector that had previously been observed to localize to lysosomal membranes. CBU0077 was dispensable for the intracellular replication of Coxiella in HeLa and THP-1 cells and did not appear to participate in CCV biogenesis. Intriguingly, native and epitope-tagged CBU0077 produced by Coxiella displayed specific punctate localization at host cell mitochondria. As such, we designated CBU0077 MceA (mitochondrial Coxiellaeffector protein A). Analysis of ectopically expressed MceA truncations revealed that the capacity to traffic to mitochondria is encoded within the first 84 amino acids of this protein. MceA is farnesylated by the host cell; however, this does not impact mitochondrial localization. Examination of mitochondria isolated from infected cells revealed that MceA is specifically integrated into the mitochondrial outer membrane and forms a complex of approximately 120 kDa. Engineering Coxiella to express either MceA tagged with 3×FLAG or MceA tagged with 2×hemagglutinin allowed us to perform immunoprecipitation experiments that showed that MceA forms a homo-oligomeric species at the mitochondrial outer membrane during infection. This research reveals that mitochondria are a bona fide target of Coxiella effectors and MceA is a complex-forming effector at the mitochondrial outer membrane during Coxiella infection.

Dark Matter Decay between Phase Transitions at the Weak Scale.

We propose a new alternative to the weakly interacting massive particle paradigm for dark matter. Rather than being determined by thermal freeze-out, the dark matter abundance in this scenario is set by dark matter decay, which is allowed for a limited amount of time just before the electroweak phase transition. More specifically, we consider fermionic singlet dark matter particles coupled weakly to a scalar mediator S_{3} and to auxiliary dark sector fields, charged under the standard model gauge groups. Dark matter freezes out while still relativistic, so its abundance is initially very large. As the Universe cools down, the scalar mediator develops a vacuum expectation value (VEV), which breaks the symmetry that stabilizes dark matter. This allows dark matter to mix with charged fermions and decay. During this epoch, the dark matter abundance is reduced to give the value observed today. Later, the SM Higgs field also develops a VEV, which feeds back into the S_{3} potential and restores the dark sector symmetry. In a concrete model we show that this "VEV flip-flop" scenario is phenomenologically successful in the most interesting regions of its parameter space. We also comment on detection prospects at the LHC and elsewhere.

α-1-antitrypsin variants and the proteinase/antiproteinase imbalance in chronic obstructive pulmonary disease.

The excessive activities of the serine proteinases neutrophil elastase and proteinase 3 are associated with tissue damage in chronic obstructive pulmonary disease. Reduced concentrations and/or inhibitory efficiency of the main circulating serine proteinase inhibitor α-1-antitrypsin result from point mutations in its gene. In addition, α-2-macroglobulin competes with α-1-antitrypsin for proteinases, and the α-2-macroglobulin-sequestered enzyme can retain its catalytic activity. We have studied how serine proteinases partition between these inhibitors and the effects of α-1-antitrypsin mutations on this partitioning. Subsequently, we have developed a three-dimensional reaction-diffusion model to describe events occurring in the lung interstitium when serine proteinases diffuse from the neutrophil azurophil granule following degranulation and subsequently bind to either α-1-antitrypsin or α-2-macroglobulin. We found that the proteinases remained uninhibited on the order of 0.1 s after release and diffused on the order of 10 µm into the tissue before becoming sequestered. We have shown that proteinases sequestered to α-2-macroglobulin retain their proteolytic activity and that neutrophil elastase complexes with α-2-macroglobulin are able to degrade elastin. Although neutrophil elastase is implicated in the pathophysiology of emphysema, our results highlight a potentially important role for proteinase 3 because of its greater concentration in azurophil granules, its reduced association rate constant with all α-1-antitrypsin variants studied here, its greater diffusion distance, time spent uninhibited following degranulation, and its greater propensity to partition to α-2-macroglobulin where it retains proteolytic activity.

The mitochondrial amidoxime-reducing component (mARC1) is a novel signal-anchored protein of the outer mitochondrial membrane.

The mitochondrial amidoxime-reducing component (mARC) was recently discovered as the fifth eukaryotic molybdenum cofactor-containing enzyme. The human genome encodes two mARC proteins, mARC1 and mARC2, sharing significant homologies with respect to sequence and function. Whereas mARC2 was identified as a mitochondrial enzyme, the subcellular localization of mARC1 has remained uncharacterized, although the similarity of both proteins suggested identical subcellular localizations. In addition, neither mARC1 nor mARC2 could be attributed unambiguously to one of the four mitochondrial subcompartments. Accordingly, mechanisms triggering the subcellular distribution of both enzymes have been unexplored so far. Here, we shed light on the subcellular localization of mARC1 and demonstrate that it is integrated into the outer mitochondrial membrane. The C-terminal catalytic domain of the protein remains exposed to the cytosol and confers an N((in))-C((out)) membrane orientation of mARC1. This localization is triggered by the N terminus of the enzyme, being composed of a weak N-terminal mitochondrial targeting signal and a downstream transmembrane helix. We demonstrate the transmembrane domain of mARC1 to be sufficient for mitochondrial targeting and the N-terminal targeting signal to function as a supportive receptor for the outer mitochondrial membrane. According to its localization and targeting mechanism, we classify mARC1 as a novel signal-anchored mitochondrial protein. During mitochondrial import, mARC1 is not processed, and membrane integration proceeds membrane potential independently but requires external ATP, which finally results in the assembly of mARC1 into high oligomeric protein complexes.

An explanation for why it is difficult to form slush nitrogen from liquid nitrogen used previously for this purpose.

Slush nitrogen (SN) is used to avoid the Leidenfrost effect, which is problematic when using liquid nitrogen (LN). Slush nitrogen's usefulness has been demonstrated by its requirement for the successful cryopreservation of insect embryos. To convert LN to SN, typically, the pressure above a Dewar of LN is reduced, using a vacuum pump in a sealed system until conversion occurs. It has been observed that LN from a fresh tank will readily produce SN; however, repeated use of the same LN results in the inability to form SN in subsequent trials. The current experiments were designed to identify the cause of this phenomenon. The hypothesis is that gaseous oxygen from the surrounding, ambient air condenses and mixes with the LN to form a mixture with a lower freezing point and; therefore, prevents the formation of SN. The hypothesis was tested and found to be true.

Suction-modified needle biopsy technique for the human soleus muscle.

INTRODUCTION: The needle biopsy technique for the soleus muscle is of particular interest because of the muscle's unique fiber type distribution, contractile properties, and sensitivity to unloading. Unlike other commonly biopsied muscles, the soleus is not fully superficial and is in close proximity to neurovascular structures, resulting in a more challenging biopsy. Because of this, a standardized protocol for performing needle biopsies on the human soleus muscle that is safe, reliable, and repeatable is presented. METHODS: Ultrasonography was used on an initial set of 12 subjects to determine the optimal biopsy zone, thereby guiding the location of the incision site. There were 45 subjects recruited who attended 2 separate biopsy sessions. Each biopsy session incorporated 3 passes of the biopsy needle proximal, posterior, and distal using suction from a portable vacuum source producing 3 separate muscle specimens. RESULTS: There were 84 soleus muscle biopsy procedures which were successfully conducted yielding 252 total samples without complication. Ultrasonography was used to confirm biopsy needle infiltration of the soleus muscle. Average sample weight obtained per pass was 61.5 +/- 15.7 mg. Histochemistry and molecular analyses demonstrated a considerably higher amount of slow type I MHC in comparison to the vastus lateralis, providing verification for the successful sampling of the soleus muscle. DISCUSSION: The procedure presented consists of a detailed protocol to accurately and consistently obtain muscle biopsy samples from the human soleus muscle. We have demonstrated that the human soleus biopsy is a safe, reliable, and repeatable procedure providing ample tissue for multiple types of analyses.

The Plant Defensin Ppdef1 Is a Novel Topical Treatment for Onychomycosis.

Onychomycosis, or fungal nail infection, causes not only pain and discomfort but can also have psychological and social consequences for the patient. Treatment of onychomycosis is complicated by the location of the infection under the nail plate, meaning that antifungal molecules must either penetrate the nail or be applied systemically. Currently, available treatments are limited by their poor nail penetration for topical products or their potential toxicity for systemic products. Plant defensins with potent antifungal activity have the potential to be safe and effective treatments for fungal infections in humans. The cystine-stabilized structure of plant defensins makes them stable to the extremes of pH and temperature as well as digestion by proteases. Here, we describe a novel plant defensin, Ppdef1, as a peptide for the treatment of fungal nail infections. Ppdef1 has potent, fungicidal activity against a range of human fungal pathogens, including Candida spp., Cryptococcus spp., dermatophytes, and non-dermatophytic moulds. In particular, Ppdef1 has excellent activity against dermatophytes that infect skin and nails, including the major etiological agent of onychomycosis Trichophyton rubrum. Ppdef1 also penetrates human nails rapidly and efficiently, making it an excellent candidate for a novel topical treatment of onychomycosis.

"Be rational!" Epistemic aims and socio-cognitive tension in argumentation about dietary choices.

Argumentation is a social practice that can lead to epistemic outcomes, that is, to the construction of knowledge. Recent research in collaborative learning has pointed out the significance of affective and motivational aspects, as well as the influence of socio-relational concerns, which have been found to frequently take priority over epistemic ones. Our research objective is to investigate how the epistemic and socio-relational dimensions of students' argumentative interactions are intertwined. We apply discourse analysis to examine the interactions in a small group of four 11th-graders evaluating the nutritional acceptability of omnivorous and vegetarian diets. The epistemic dimension is analyzed in terms of the aims pursued by the participants and the epistemic outcomes achieved. The socio-relational dimension is analyzed in terms of fluctuations of interpersonal tensions and their relaxations. The results show a convergence of participants' epistemic aims and the epistemic statuses of the options. Most of the epistemic outcomes are produced in sequences in which socio-cognitive tension arises and then relaxes. Enduring high socio-cognitive tension and overcoming conflict seem to have encouraged the adoption of epistemic aims. Moreover, our findings suggest that driven by epistemic aims in high socio-cognitive tensed contexts, students can refine the conditions by which they engage in argumentation. These results call for further investigating on what constitutes an appropriate or productive level of interpersonal tension for learning. Educational implications are related to the design of argumentative learning environments promoting epistemic aims and outcomes through the encouragement of suitable socio-cognitive climates leading to them.

Social sensitivity: a manifesto for CSCL research.

Technologies for computer-supported collaborative learning (CSCL) are playing an increasingly prominent role in educational contexts, especially as teachers and students strive to deal with pandemic-related constraints. However, the technologies being used for collaboration on a daily basis are not sufficiently equipped to promote collaborative learning as both a cognitive and a socio-emotional process. They may even run the risk of hindering the constructive exchange of ideas and provoking disputes and negative encounters. In this squib, we argue that the field of CSCL is failing to address this risk, because our research efforts are far too scattered and siloed. We introduce a manifesto of social sensitivity: increasing interdisciplinary efforts to enhance constructively critical, respectful, and cohesive collaborations in technology-supported environments. We call for concrete actions in CSCL research that ultimately contribute to more democratic and equitable collaborations.

Defining emergency telehealth.

The American College of Emergency Physicians Emergency Telehealth Section was charged with development of a working definition of emergency telehealth that aligns with the College's definition of emergency medicine. A modified Delphi method was used by the section membership who represented telehealth providers in both private and public health-care delivery systems, academia and industry, rural and urban settings. Presented in this manuscript is the final definition of emergency telehealth developed with an additional six clarifying statements to address the context of the definition. Emergency telehealth is a core domain of emergency medicine and is inclusive of remotely providing all types of care for acute conditions of any kind requiring expeditious care irrespective of any prior relationship. The development of this definition is important to the global community of emergency physicians and all patients seeking acute care to ensure that appropriately trained clinicians are providing the highest quality of emergency services via the telehealth modality. We recommend implementing emergency telehealth in a manner that ensures appropriate qualifications of providers, appropriate/parity reimbursement for telehealth services and, most importantly, the delivery of quality care to patients in a safe, efficient, timely and cost-effective manner.

Argumentation, Eureka and emotion: An analysis of group projects in creative design training.

Creativity training has been generally based on avoiding critique during idea generation, although benefits of argumentation have been shown during idea selection and elaboration. The research reported here aims to understand how argumentative interactions involving role-play, with subsequent group reflection on them, contribute to collaborative creative design projects. The study was carried within a specialised Masters course at the Royal College of Art (London), organised jointly with Imperial College London, and focuses on analysing group reflection sessions of two groups of students whose on-going project was initially defined as "communication by touch". Results showed that although students reported difficulties in playing argumentative roles that were not aligned with their personal views, their debates enabled them to arrive at "Eureka!" moments with respect to better grounded and precise definitions of their project concepts. We highlight the complex ways in which emotions circulate with respect to "Eureka!" moments, role-play and grounding. Given differences in ways that groups played out their assigned argumentative roles, we conclude that role play debate and group reflection on it need to be applied and considered as a whole in creative design training.

The impact of aprotinin on postoperative renal dysfunction in neonates undergoing cardiopulmonary bypass: a retrospective analysis.

BACKGROUND: Recent concern about the safety of aprotinin administration to adults has led to its suspension from worldwide markets. However, few studies have examined its safety in pediatric patients. Studies in children evaluating aprotinin's safety have been hindered by the heterogeneity of pediatric patients and the inconsistency of clinical protocols. In this investigation, we retrospectively reviewed 200 neonatal cardiac surgical cases performed at our institution to examine the safety of aprotinin, focusing on postoperative renal dysfunction, using a consistent aprotinin dosing protocol. METHODS: Two-hundred consecutive neonates scheduled for palliative or corrective congenital cardiac surgery requiring cardiopulmonary bypass (CPB) from January 1, 2005 through February 28, 2007 were included in this retrospective investigation. Preoperative, intraoperative and postoperative data were collected and analyzed. Markers of safety included 72-h postoperative renal dysfunction, need for dialysis (peritoneal or hemodialysis), thrombosis and in-hospital mortality. RESULTS: Neonates were divided into those who received aprotinin (aprotinin group; n = 156) and those who did not (no aprotinin group; n = 44). Twenty-four and 72-h postoperative serum creatinine levels were significantly greater than baseline levels in both groups. The degree of change in creatinine levels was highly significant and similar between the two groups. A larger percentage of neonates in the aprotinin group developed renal dysfunction, although this difference was not statistically significant. Stepwise logistic regression, assessing the impact on renal dysfunction of all variables that indicated significance between neonates who did or did not receive aprotinin and between neonates who did or did not develop renal dysfunction, identified CPB time and age as significant predictors of postoperative renal dysfunction. All neonates who developed postoperative renal dysfunction had a CPB time of more than 100 min regardless of the use of aprotinin. Additionally, using this subset, similar percentages of renal dysfunction occurred in both groups. A second multivariable regression analysis to simultaneously account for the predictors of CPB time, age and aprotinin administration found CPB time to be the only significant predictor of renal dysfunction. Incidences of postoperative dialysis, postoperative thrombosis and in-hospital mortality were not statistically significantly different between the aprotinin and the no aprotinin groups. CONCLUSION: The occurrence of postoperative renal dysfunction in neonates was more significantly predicted by the duration of CPB than by the intraoperative administration of aprotinin. CPB times of more than 100 min appeared to be a critical marker for the development of postoperative renal dysfunction. Randomized prospective trials are needed to confirm the validity of our retrospective findings.

Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis.

Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.

Early effects of mechanical ventilation on isotonic contractile properties and MAF-box gene expression in the diaphragm.

This study aimed to determine the time-dependent effects of diaphragmatic inactivity on its maximum shortening velocity (V(max)) and the muscle atrophy F-box (MAF-box, atrogin-1) gene expression during controlled mechanical ventilation (CMV). Twenty-four New Zealand White rabbits were grouped into 1 day, 2 days, and 3 days of CMV and controls in equal numbers. The in vitro isotonic contractile properties of the diaphragm were determined. In addition, myosin heavy chain protein and mRNA, myosin light chain, MAF-box mRNA, and volume density of abnormal myofibrils were measured. Tetanic force decreased, and V(max) increased from control of 6.4 to 6.6, 7.7, and 8.1 muscle lengths per second after 1, 2, and 3 days of CMV, respectively (P < 0.02). The increased V(max) compensated for the decreased tetanic force; consequently, compared with the controls, maximum power output was unchanged after 3 days of CMV. V(max) correlated with the volume density of abnormal myofibrils [y = 0.1x + 5.7 (r = 0.87, P < 0.01)]. In the diaphragm, MAF-box was overexpressed (355% of control) after 1 day of CMV, before the evidence of structural myofibril disarray. In conclusion, CMV produced a time-dependent increase in V(max) that was associated with the degree of myofibrillar disarray and independent of changes in myosin isoform expression. Furthermore, CMV produced an increase in MAF-box mRNA levels that may be partially or completely responsible for the degree of myofibrillar disarray resulting from CMV.