Life-threatening lactic acidosis occurring in adults with mitochondrial disorders.

Although relatively common in children, severe acute lactic acidosis is rare in adults with mitochondrial myopathies. We report here three cases, aged 27, 32 and 32 years, who developed life-threatening metabolic crisis with severe lactic acidosis, requiring hospitalisation in intensive care unit. Plasma lactates were elevated 10 to 15 fold normal values, necessitating extra-renal dialysis. By contrast CK levels were moderately increased (3 to 5N). No triggering factor was identified, but retrospectively all patients reported long-lasting mild muscle fatigability and weakness before their acute metabolic crisis. All of them recovered after prolonged intensive care but resting lactate levels remained elevated. Muscle biopsy showed ragged-red and COX-negative fibers in two patients and mild lipidosis in the third one. Heteroplasmic pathogenic point mutations were detected in MT-TL1 (m.3280G>A;m.3258C>T) and MT-TK (m.8363A>G). Life-threatening lactic acidosis may thus be a major inaugural clinical manifestation in adults with mitochondrial myopathies. Prolonged intensive care may lead to a dramatic and sustained improvement and is mandatory in such cases.

Culture-free Antibiotic-susceptibility Determination From Single-bacterium Raman Spectra.

Raman spectrometry appears to be an opportunity to perform rapid tests in microbiological diagnostics as it provides phenotype-related information from single bacterial cells thus holding the promise of direct analysis of clinical specimens without any time-consuming growth phase. Here, we demonstrate the feasibility of a rapid antibiotic-susceptibility determination based on the use of Raman spectra acquired on single bacterial cells. After a two-hour preculture step, one susceptible and two resistant E. coli strains were incubated, for only two hours, in the presence of different bactericidal antibiotics (gentamicin, ciprofloxacin, amoxicillin) in a range of concentrations that included the clinical breakpoints used as references in microbial diagnostic. Spectra were acquired and processed to isolate spectral modifications associated with the antibiotic effect. We evidenced an "antibiotic effect signature" which is expressed with specific Raman peaks and the coexistence of three spectral populations in the presence of antibiotic. We devised an algorithm and a test procedure that overcome single-cell heterogeneities to estimate the MIC and determinate the susceptibility phenotype of the tested bacteria using only a few single-cell spectra in four hours only if including the preculture step.

Assessment of terbium (III) as a luminescent probe for the detection of tuberculosis biomarkers.

A detection method for nicotinic acid, a specific metabolite marker of Mycobacterium tuberculosis present in cultures and patients' breath, is studied in complex solutions containing other metabolites and in biological media such as urine, saliva and breath condensate. The method is based on the analysis of the luminescence increase of Tb(3+) complexes in the presence of nicotinic acid due to the energy transfer from the excited ligand to the lanthanide ion. It is shown that other potential markers found in M. tuberculosis culture supernatant, such as methyl phenylacetate, p-methyl anisate, methyl nicotinate and 2-methoxy biphenyl, can interfere with nicotinic acid via a competitive absorption of the excitation photons. A new strategy to circumvent these interferences is proposed with an upstream trapping of volatile markers preceding the detection of nicotinic acid in the liquid phase via the luminescence of Tb(3+) complexes. The cost of the method is evaluated and compared with the Xpert MTB/RIF test endorsed by the World Health Organization.

Within-species variation in long-term trajectories of growth, fecundity and mortality in the Collembola Folsomia candida.

Senescence--the progressive deterioration of organisms with age--affects many traits of which survival and reproduction are the most commonly studied. Recent comparative studies have revealed a remarkable amount of variation in the patterns of ageing across the tree of life. This between-species diversity raises many questions about the evolution of senescence and of the shapes of the life-history age trajectories. Here, we study how the different components of the shapes of these life-history age trajectories can vary within a single species to shed light on the possible constraints involved in their evolution. To do so, we closely followed in controlled laboratory conditions, and for more than 450 days, the mortality, body length and fecundity of small cohorts of two clonal lineages of the Collembola Folsomia candida. We studied three components of the adult mortality trajectory: the baseline mortality, onset and speed of senescence. We found that they can differ between strains of a single species in such a way that, remarkably, an increased life expectancy is not synonymous with a delayed senescence: the strain that grows bigger has the longest life expectancy but suffers from a precocious senescence. We observed marked differences between the strains in the asymptotic body length and reproductive investment. More generally, our results highlight the importance of finely describing the long-term trajectories of several life-history traits in order to better understand how the patterns of senescence have been shaped by natural selection.

Rapid urine preparation prior to identification of uropathogens by MALDI-TOF MS.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) has been introduced in clinical routine microbiology laboratories. For the rapid diagnosis of urinary tract infections, culture-independent methods prior MALDI-mediated identification have been described. Here, we describe a comparison of three of these methods based on their performance of bacterial identification and their potential as a routine tool for microbiology labs : (i) differential centrifugation, (ii) urine filtration and (iii) a 5-h bacterial cultivation on solid culture media. For 19 urine samples, all methods were directly compared and correct bacterial species identification by MALDI was used as performance indicator. A higher percentage of correct MALDI identification was obtained after filtration (78.9 %) and the growth-based method (84.2 %) as compared to differential centrifugation (68.4 %). Additional testing of 76 mono-microbial specimens (bacteriuria > 10(5) CFU/mL) confirmed the good performance of short growth with a 90.8 % correct MALDI score, with a potentially better fit to the routine workflow of microbiology labs.

An original internal fixation technique by tension band wiring with steel wire in fractures of the coronoid process.

Fractures of the coronoid process, which is a key element in anterior elbow joint stability, represent 14% of proximal ulnar fractures. Optimal treatment should stabilize all fractures associated with elbow instability. Different techniques have been described: suture repair, screws, plates… We propose a series of 5 patients who were treated with an original, easy, tension band wiring fixation technique using steel wire with easy hardware removal.

Viability of 3h grown bacterial micro-colonies after direct Raman identification.

Clinical diagnostics in routine microbiology still mostly relies on bacterial growth, a time-consuming process that prevents test results to be used directly as key decision-making elements for therapeutic decisions. There is some evidence that Raman micro-spectroscopy provides clinically relevant information from a limited amount of bacterial cells, thus holding the promise of reduced growth times and accelerated result delivery. Indeed, bacterial identification at the species level directly from micro-colonies at an early time of growth (6h) directly on their growth medium has been demonstrated. However, such analysis is suspected to be partly destructive and could prevent the further growth of the colony needed for other tests, e.g. antibiotic susceptibility testing (AST). In the present study, we evaluated the effect of the powerful laser excitation used for Raman identification on micro-colonies probed after very short growth times. We show here, using envelope integrity markers (Syto 9 and Propidium Iodide) directly on ultra-small micro-colonies of a few tens of Escherichia coli and Staphylococcus epidermidis cells (3h growth time), that only the cells that are directly impacted by the laser lose their membrane integrity. Growth kinetics experiments show that the non-probed surrounding cells are sometimes also affected but that the micro-colonies keep their ability to grow, resulting in normal aspect and size of colonies after 15h of growth. Thus, Raman spectroscopy could be used for very early (<3h) identification of grown micro-organisms without impairing further antibiotics susceptibility characterization steps.

Reverse wedge osteotomy of the distal radius in Madelung's deformity.

Madelung's deformity results from a growth defect in the palmar and ulnar region of the distal radius. It presents as an excessively inclined radial joint surface, inducing "spontaneous progressive palmar subluxation of the wrist". The principle of reverse wedge osteotomy (RWO) consists in the reorientation of the radial joint surface by taking a circumferential bone wedge, the base of which is harvested from the excess of the radial and dorsal cortical bone of the distal radius, then turning it over and putting back this reverse wedge into the osteotomy so as to obtain closure on the excess and opening on the deficient cortical bone. RWO corrects the palmar subluxation of the carpus and improves distal radio-ulnar alignment. All five bilaterally operated patients were satisfied, esthetically and functionally. Its corrective power gives RWO a place apart among the surgical techniques currently available in Madelung's deformity.

Trabecular microarchitecture in established osteoporosis: relationship between vertebrae, distal radius and calcaneus by X-ray imaging texture analysis.

INTRODUCTION: Osteoporosis is an alteration of bone mass and microarchitecture leading to an increased risk of fractures. A radiograph is a 2D projection of the 3D bone network exposing a texture, that can be assessed by texture analysis. We compared the trabecular microarchitecture of the spine, radius and calcaneus in a series of osteoporotic cadavers. MATERIALS AND METHODS: Thirty-four cadavers (11 men, 23 women), mean age 85.2±2.1years, were radiographed from T4 to L5 to identify those with vertebral fractures (FV). Non-fractured vertebrae (NFV), radius and calcaneus were taken and analyzed by densitometry, radiography and texture analysis under run-length, skeletonization of the trabeculae, and fractal geometry. RESULTS: Six subjects (five women, one man) were selected, mean age 82.5±5.5years. Twelve calcanei and 10 radii were taken. Two radii were excluded. The texture of NFV was significantly correlated (P<0.01) with that of the radius for horizontal run-lengths. No relationship between the texture of NFV and calcaneus was found. DISCUSSION: In the horizontal direction (perpendicular to the stress lines), the microarchitecture of NFV and radius showed a disappearance of the transverse rods anchoring the plates. Due to its particular microarchitecture, the calcaneus is not representative of the vertebral status. CONCLUSION: Bone densitometry provides no information about microarchitecture. Texture analysis of X-ray images of the radius would be a minimally invasive tool, providing an early detection of microarchitectural alterations. LEVEL OF EVIDENCE: IV retrospective study.

[Long-term functional outcomes of digital ischemia under tourniquet: observations in three cases]. / Résultats fonctionnels à long terme d'ischémies digitales sous garrot : à propos de trois observations.

Digital tourniquet is a quick, simple and reliable method to ensure a bloodless operative field distal to the MP joint. However, a forgotten tourniquet is an exceptional but serious complication related to digital ischemia. Few cases were reported in literature without a long-term outcome. Three digits of three patients, aged 70, 49 and 14 at the time of accident, had a tourniquet left in place for 2 days for the first two and 6 days for the last one. Final assessment was carried out 3, 4 and 16 years respectively after the initial accident. All fingers survived with sequelae such as pain, cold intolerance, dysesthesia, allodynia, joint stiffness and skin and nail trophic disorders. A hypertrophic scar was still visible at the site of the tourniquet. Capillary pulse was normal in all cases. Radiological changes were visible when the tourniquet had been left for more than 2 days. The impact on professional and day life activities was considerable. No surgery for the sequelae was done. Avoiding a missed finger tourniquet requires a suitable tourniquet with a visual reminder and its removal must be considered a crucial part of the surgery.

Simplified scaphoid reconstruction technique with Zaidemberg's vascularized radial graft.

For more than 10 years, we have been using a simplified reconstruction technique for scaphoid non-unions that involves the use of a graft first described by Zaidemberg et al. [1]. This approach requires that an island bone graft harvested from the radial styloid and pedicled on the 1,2-intercompartmental supraretinacular artery be embedded into the site of the non-union. The objective of our technical modifications was to simplify the harvesting and handling of the graft and the internal fixation. This technique is only used for cases of scaphoid non-union with avascular changes in the proximal fragment, repeated non-union after bone grafting and internal fixation, chronic non-union with osteophyte formation in the dorso-radial aspect and fracture secondary to Preiser disease.

Melanic through nature or nurture: genetic polymorphism and phenotypic plasticity in Harmonia axyridis.

Individuals can adapt to heterogeneity in their environment through either local adaptation or phenotypic plasticity. Colour forms of the ladybird Harmonia axyridis are a classic example of local adaptation, in which the frequency of melanic forms varies greatly between populations. In some populations, there are also large seasonal changes in allele frequency, with melanism being costly in summer and beneficial in winter. We report that the non-melanic morph of H. axyridis dramatically increases its degree of melanization at cold temperatures. Furthermore, there is genetic variation in reaction norms, with different families responding to temperature in different ways. Variation at different spatial and temporal scales appears to have selected for either genetic or phenotypically plastic adaptations, which may be important in thermoregulation. As melanism is known to have a large effect on fitness in H. axyridis, this plasticity of melanization may have hastened its spread as an invasive species.

Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes.

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells.

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.

Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport.

Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37 degreesC, ultrastructural studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.