Parasexuality and Ploidy Change in Candida tropicalis

Candida species exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation. Candida albicans, the most studied Candida species and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes in Candida tropicalis, a closely related species to C. albicans that was recently revealed to undergo sexual mating. C. tropicalis diploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) of C. tropicalis tetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels of C. tropicalis diploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cells in vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur in C. tropicalis and indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.     

The species identity and biogeography of Blanus (Amphisbaenia: Blanidae) in Lebanon

The genus Blanus Wagler, 1830 represents limbless, burrowing reptiles of the family Blanidae with disjunct circum-Mediterranean distribution. The recently described species Blanus alexandri Sindaco, Kornilios, Sacchi & Lymberakis, 2014 is known from south-eastern Turkey with a presumed occurrence in the Levant and Iraq. We provide here records from Lebanon and confirm the affiliation of Lebanese populations to this species by mitochondrial and nuclear DNA. Blanus alexandri comprises at least seven deeply evolved phylogenetic clades with up to 11% of uncorrected p-distances in their mitochondrial DNA. This suggests a probably older than the Miocene origin of some of these clades. Populations from Lebanon form a different clade with a genetic diversity that is close to populations from southern Turkey.     

Fine tuning of TCR signaling by CD5

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5(-/-) mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5(-/-)) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5(-/-) phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.     

Glucose Modulates Respiratory Complex I Activity in Response to Acute Mitochondrial Dysfunction

Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an “emergency shutdown” system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases.     

A study of a decade of road traffic accidents in Benghazi-Libya: 2001 to 2010

This paper aims to observe and to study the trends of road traffic accidents (RTA's) for the past ten years in Benghazi-Libya. A retrospective analysis was done using the patient records of Al-Jalaa hospital (the main trauma center in Benghazi) from over 21,753 RTA cases. The annual data were compared to each other and changes of trends were observed. RTA's represented an increasing percentage of Al-Jalaa's case load across the years. Around 41% of these cases needed to undergo surgery. The younger age group (20-29 years of age) formed the majority of cases while there was a trend towards an increasing average age of patients involved in an accident. Male patients were found to be younger than their female counterparts. Males comprised 81.5% while females formed 18.5% of RTA patients. In terms of inpatient duration, most patients stayed in the hospital for less than 1 week. Vehicle occupants (drivers and passengers) were admitted more often than pedestrians. There was a trend across the years towards an increased involvement of vehicle occupants and decrease in the proportion of pedestrians that had to be hospitalized. Additionally, there was a decrease in the fatalities of pedestrians. Overall, most RTA patients were discharged and made to follow-up in outpatient clinics however there was a startling trend towards increased LAMA and absconded patients. There were both encouraging findings as well as points that needed further emphasis and action. Public education, life support training and diversification of transport (apart from the use of the roads) should be looked into, as possible means of improving the current situation.     

Incidence,severity and preventability of medication-related visits to the emergency department: a prospective study

Background

Medication-related visits to the emergency department are an important but poorly understood phenomenon. We sought to evaluate the frequency, severity and preventability of drug-related visits to the emergency department.

Methods

We performed a prospective observational study of randomly selected adults presenting to the emergency department over a 12-week period. Emergency department visits were identified as drug-related on the basis of assessment by a pharmacist research assistant and an emergency physician; discrepancies were adjudicated by 2 independent reviewers.

Results

Among the 1017 patients included in the study, the emergency department visit was identified as drug-related for 122 patients (12.0%, 95% confidence interval [CI] 10.1%–14.2%); of these, 83 visits (68.0%, 95% CI 59.0%–76.2%) were deemed preventable. Severity was classified as mild in 15.6% of the 122 cases, moderate in 74.6% and severe in 9.8%. The most common reasons for drug-related visits were adverse drug reactions (39.3%), nonadherence (27.9%) and use of the wrong or suboptimal drug (11.5%). The probability of admission was significantly higher among patients who had a drug-related visit than among those whose visit was not drug-related (OR 2.18, 95% CI 1.46–3.27, p < 0.001), and among those admitted, the median length of stay was longer (8.0 [interquartile range 23.5] v. 5.5 [interquartile range 10.0] days, p = 0.06).

Interpretation

More than 1 in 9 emergency department visits are due to drug-related adverse events, a potentially preventable problem in our health care system.Adverse drug-related events are unfavourable occurrences related to the use or misuse of medications.¹ It has been estimated that such events account for 17 million emergency department visits and 8.7 million hospital admissions annually in the United States.^2,3 Between 1995 and 2000, costs associated with adverse drug-related events rose from US$76.6 billion to over US$177.4 billion.^3,4Adverse drug-related events have recently been evaluated in ambulatory care settings and among patients admitted to hospital,^5–9 and it has been estimated that 5%–25% of hospital admissions are drug-related.^7,8 Unfortunately, emergency department visits are not reflected in most hospital studies, because patients seen in the emergency department for an adverse drug-related event are typically not admitted.¹⁰ In addition, most research evaluating drug-related visits to the emergency department has involved retrospective studies or analysis of administrative data.^11–13 Retrospective studies may underestimate the incidence of drug-related visits because information may be missing or inaccurately documented.¹⁴ Finally, studies performed to date have used variable definitions of “drug-related events,”^1,10 which limits comparative evaluation and generalizability.Despite the burden of drug-related morbidity and mortality, prospective research characterizing drug-related visits to the emergency department has been limited.^15–17 We sought to overcome some of the limitations of research in this area by using a prospective design and a comprehensive definition of adverse drug-related events. The purpose of this study was to evaluate the frequency, severity and preventability of drug-related visits to the emergency department of a large tertiary care hospital, to classify the visits by type of drug-related problem and to identify patient, prescriber, drug and system factors associated with these visits.     

Suppression of Mitochondrial DNA Instability of Autosomal Dominant Forms of Progressive External Ophthalmoplegia-Associated ANT1 Mutations in Podospora anserina

Maintenance and expression of mitochondrial DNA (mtDNA) are essential for the cell and the organism. In humans, several mutations in the adenine nucleotide translocase gene ANT1 are associated with multiple mtDNA deletions and autosomal dominant forms of progressive external ophthalmoplegia (adPEO). The mechanisms underlying the mtDNA instability are still obscure. A current hypothesis proposes that these pathogenic mutations primarily uncouple the mitochondrial inner membrane, which secondarily causes mtDNA instability. Here we show that the three adPEO-associated mutations equivalent to A114P, L98P, and V289M introduced into the Podospora anserina ANT1 ortholog dominantly cause severe growth defects, decreased reactive oxygen species production (ROS), decreased mitochondrial inner membrane potential (Δψ), and accumulation of large-scale mtDNA deletions leading to premature death. Interestingly, we show that, at least for the adPEO-type M106P and A121P mutant alleles, the associated mtDNA instability cannot be attributed only to a reduced membrane potential or to an increased ROS level since it can be suppressed without restoration of the Δψ or modification of the ROS production. Suppression of mtDNA instability due to the M106P and A121P mutations was obtained by an allele of the rmp1 gene involved in nucleo-mitochondrial cross- talk and also by an allele of the AS1 gene encoding a cytosolic ribosomal protein. In contrast, the mtDNA instability caused by the S296M mutation was not suppressed by these alleles.THE maintenance and expression of mitochondrial DNA (mtDNA) depend on many nuclear-encoded gene products. Recent studies have shown that defects in this maintenance can have devastating consequences for the cell and the organism. In humans, these defects are an important cause of neurological diseases including autosomal dominant (or recessive) progressive external ophthalmoplegia (adPEO) (Chinnery 2003; Copeland 2008). These disorders are characterized by multiple large-scale deletions of mtDNA. Three different genes that can cause PEO with multiple mtDNA deletions have been identified: the mtDNA polymerase (POLG), the heart/muscle isoform of the adenine nucleotide translocator (ANT1), and the mitochondrial DNA helicase, Twinkle.The adenine nucleotide translocator (ANT), also known as the ADP/ATP mitochondrial translocator, is the most abundant protein in the inner mitochondrial membrane (Riccio et al. 1975; Nury et al. 2006; Klingenberg 2008). It exports ATP produced by mitochondrial oxidative phosphorylation toward the cytosol to meet the energy requirements of the cell; in exchange, it transports ADP into the mitochondrial matrix to fuel the conversion of ADP to ATP by the F₁F_O-ATP synthase. In humans, four isoforms of the ANT protein exist, and they are differently expressed in a tissue-specific manner (Stepien et al. 1992; Palmieri 2004; Dolce et al. 2005). The human ANT1 isoform is predominantly expressed in skeletal and cardiac muscle, and specific ANT1 mutations are associated with adPEO characterized by mtDNA instability (Kaukonen et al. 1999, 2000; Napoli et al. 2001; Komaki et al. 2002; Siciliano et al. 2003). In mice, Ant1 knockout induces mitochondrial myopathy (Graham et al. 1997), increased H₂O₂ production, and mtDNA damage and inhibits oxidative phosphorylation (Esposito et al. 1999). Some of these mutations were introduced in the AAC2 gene of Saccharomyces cerevisiae that encodes the major ADP/ATP mitochondrial translocator isoform in this organism. Numerous and sometimes contradictory effects have been reported depending in particular on the yeast laboratory strains examined (Kaukonen et al. 2000; Chen 2002, 2004; Fontanesi et al. 2004; Palmieri et al. 2005; Wang et al. 2008b).In an attempt to better understand how these mutations affect mitochondrial DNA stability and their functional consequences on mitochondrial metabolism, we decided to introduce them in the unique ADP/ATP translocator gene of Podospora anserina, PaAnt. Like S. cerevisiae, the filamentous fungus P. anserina is an excellent system for genetic and molecular analyses. In contrast to S. cerevisiae, it is a strict multicellular aerobe that can display heteroplasmic states in which intact and rearranged mitochondrial genomes coexist. In this organism, life span is a reflection of mtDNA stability, and death is always associated with large mtDNA rearrangements. “Natural death” or aging is accompanied by large-scale reorganizations of the mtDNA whereas a nuclear-controlled premature death syndrome is accompanied by the accumulation of site-specific mtDNA deletions (Belcour et al. 1999; Silar et al. 2001 for reviews). P. anserina therefore occupies an interesting position among model systems for studying the cellular consequences of mutations in the ADP/ATP translocase gene.We show here that the mutations M106P, A121P, and S296M, equivalent to the L98P, A114P (familial), and V289M (sporadic) human mutations, severely impair the vegetative and sexual development of the fungus and are responsible for decreased ROS production and for decreased inner membrane potential (Δψ). The severity of the phenotypes differs according to the mutation. The three mutations show mtDNA instability, which leads to premature death. All these mutated traits are dominant. Interestingly, the mtDNA instability associated with the M106P and A121P mutations depends on the rmp1 gene. This gene exists under two naturally occurring alleles, rmp1-1 and rmp1-2, which control mtDNA integrity in some genetic contexts (Belcour et al. 1991; Contamine et al. 1996, 2004). When associated with the rmp1-1 allele, the M106P and A121P mutations lead to rapid mtDNA instability whereas, in the presence of the rmp1-2 allele, mtDNA instability is suppressed, and life span is considerably increased. Surprisingly, suppression is not accompanied by a restoration of the Δψ or a modification in the ROS level, demonstrating that these parameters are not sufficient to explain the M106P and A121P mtDNA instability. Mitochondrial DNA instability due to the M106P and A121P mutations is also suppressed by a mutation in the AS1 gene encoding a ribosomal protein. The suppressor effects are not observed for the S296M mutation.     

Simultaneous presentation of coarctation of the thoracic aorta and aneurysm of the superior mesenteric artery

A 34-year-old woman presented with both coarctation of the thoracic aorta and aneurysm of the superior mesenteric artery. The former was managed by open surgical repair, the latter by stent-graft. This case illustrates the need for facility with both percutaneous and open approaches to diseases of the aorta and its branches.     

Alternative Oxidase Expression in the Mouse Enables Bypassing Cytochrome c Oxidase Blockade and Limits Mitochondrial ROS Overproduction

Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects.