Strenuous exercise in warm environment is associated with improved microvascular function in sickle cell trait.

PURPOSE: Sickle cell trait is characterized by the presence of both normal and abnormal haemoglobin in red blood cells. The rate of exertional collapse is increased in athletes and military recruits who carry the trait, particularly in stressful environmental conditions. The aim of the present study was to investigate microvascular function and its determinants in response to intense exercise at control and warm environmental temperatures in carriers (AS) and non-carriers (AA) of sickle cell trait. METHODS: Nine AS and 11 AA, all healthy physically active young men, randomly participated in four experimental sessions (rest at 21 °C and 31 °C and cycling at 21 °C and 31 °C). All participants performed three exercises bouts as follows: 18-min submaximal exercise; an incremental test to exhaustion; and three 30-s sprints spaced with 20-s resting intervals. RESULTS: Skin Blood Flow (SkBF) was similar at rest between AA and AS. SkBF for all participants was higher at 31 °C than 21 °C. It was significantly higher in the AS group compared to the AA group immediately after exercise, regardless of the environmental conditions. No significant differences in hemorheological parameters, muscle damage or cardiac injury biomarkers were observed between the two groups. Our data also suggest higher oxidative stress for the AS group, with high superoxide dismutase (P = 0.044 main group effect). CONCLUSION: A specific profile is identified in the AS population, with increased microvascular reactivity after maximal exercise in stressful environment and slight pro-/antioxidant imbalance.

A day and night difference in the response of the hepatic transcriptome to cyclophosphamide treatment.

Application of omics-based technologies is a widely used approach in research aiming to improve testing strategies for human health risk assessment. In most of these studies, however, temporal variations in gene expression caused by the circadian clock are a commonly neglected pitfall. In the present study, we investigated the impact of the circadian clock on the response of the hepatic transcriptome after exposure of mice to the chemotherapeutic agent cyclophosphamide (CP). Analysis of the data without considering clock progression revealed common responses in terms of regulated pathways between light and dark phase exposure, including DNA damage, oxidative stress, and a general immune response. The overall response, however, was stronger in mice exposed during the day. Use of time-matched controls, thereby eliminating non-CP-responsive circadian clock-controlled genes, showed that this difference in response was actually even more pronounced: CP-related responses were only identified in mice exposed during the day. Only minor differences were found in acute toxicity pathways, namely lymphocyte counts and kidney weights, indicating that gene expression is subject to time of day effects. This study is the first to highlight the impact of the circadian clock on the identification of toxic responses by omics approaches.

Molecular analyses of the LRRK2 gene in European and North African autosomal dominant Parkinson's disease.

BACKGROUND: Mutations in the leucine-rich-repeat kinase 2 (LRRK2) gene have been identified in families with autosomal dominant Parkinson's disease (ADPD), the most common of which is the p.G2019S substitution that has been found at varying frequencies worldwide. Because of the size of the LRRK2 gene, few studies have analysed the entire gene in large series of ADPD families. METHODS: We performed extensive mutation analyses of all 51 coding exons of the LRRK2 gene in index cases from 226 Parkinson's disease families compatible with autosomal dominant inheritance, mostly from France (n = 182) and North Africa (n = 14). RESULTS: We found 79 sequence variants, 29 of which were novel. Eight potentially or proven pathogenic mutations were found in 22 probands (9.7%). There were four novel amino acid substitutions that are potentially pathogenic (p.S52F, p.N363S, p.I810V, p.R1325Q) and two novel variants, p.H1216R and p.T1410M, that are probably not causative. The common p.G2019S mutation was identified in 13 probands (5.8%) including six from North Africa (43%). The known heterozygous p.R1441H and p.I1371V mutations were found in two probands each, and the p.E334K variant was identified in one single patient. Most potentially or proven pathogenic mutations were located in the functional domains of the Lrrk2 protein. CONCLUSION: This study leads us to conclude that LRRK2 mutations are a common cause of autosomal dominant Parkinson's disease in Europe and North Africa.

Improvement of a stereoselective biocatalytic synthesis by substrate and enzyme engineering: 2-hydroxy-(4'-oxocyclohexyl)acetonitrile as the model.

Even if biocatalysis is finding increasing application, it still has to gain widespread use in synthetic chemistry. Reasons for this are limitations that enzymes have with regard to substrate range, reaction scope, and insufficient selectivity with unnatural compounds. These shortcomings can be challenged by enzyme and/or substrate engineering, which are employed to alter substrate specificity and enhance the enzyme selectivity toward unnatural substrates. Herein, these two approaches are coupled to improve the hydroxynitrile lyase catalyzed synthesis of 2-hydroxy-(4'-oxocyclohexyl)acetonitrile (4). The ketone functionality is masked as an enol ether, and the oxynitrilase of Hevea brasiliensis is engineered towards this masked substrate to give the product with a high optical purity and to drastically lower the amount of enzyme needed.

Criteria for the multiple use of isolated perfused hearts in electrophysiological and metabolic experiments.

Both ethical and economic restrictions limit the availability of porcine hearts for in vitro perfusion experiments. Therefore, we tested the feasibility of multiple use of in vitro perfused working hearts for electrophysiological and metabolic investigations. Pig hearts (n=7) rejected for originally planned haemodynamic measurements because of exclusion criteria were perfused in a four-chamber working heart mode. All hearts were kept in steady-state conditions on a low haemodynamic level over 2 h during 75-channel ECG recordings and NADH fluorescence measurements before and after norepinephrine (NE) was administered. QRS and QT interval durations were in a range comparable to in vitro studies and, like QRS and T amplitudes, were found to be sensitive markers of the changing condition of the isolated heart preparation, as myocardial oedema leads to prolonged QRS and QT intervals and declining ECG voltage amplitudes. A change in NADH fluorescence following NE administration was observed in the first 150 min of perfusion, but not later. Considering a time frame of 120 min, multiple use of isolated perfused porcine hearts with low-level haemodynamics may allow a broad spectrum of investigations and could therefore represent a possibility of overcoming the restricted availability of porcine hearts.

High levels of human gamma-globin gene expression in adult mice carrying a transgene of deletion-type hereditary persistence of fetal hemoglobin.

Persistent expression of the gamma-globin genes in adults with deletion types of hereditary persistence of fetal hemoglobin (HPFH) is thought to be mediated by enhancer-like effects of DNA sequences at the 3' breakpoints of the deletions. A transgenic mouse model of deletion-type HPFH was generated by using a DNA fragment containing both human gamma-globin genes and HPFH-2 breakpoint DNA sequences linked to the core sequences of the locus control region (LCR) of the human beta-globin gene cluster. Analysis of gamma-globin expression in six HPFH transgenic lines demonstrated persistence of gamma-globin mRNA and peptides in erythrocytes of adult HPFH transgenic mice. Analysis of the hemoglobin phenotype of adult HPFH transgenic animals by isoelectric focusing showed the presence of hybrid mouse alpha2-human gamma2 tetramers as well as human gamma4 homotetramers (hemoglobin Bart's). In contrast, correct developmental regulation of the gamma-globin genes with essentially absent gamma-globin gene expression in adult erythroid cells was observed in two control non-HPFH transgenic lines, consistent with autonomous silencing of normal human gamma-globin expression in adult transgenic mice. Interestingly, marked preferential overexpression of the LCR-distal (A)gamma-globin gene but not of the LCR-proximal (G)gamma-globin gene was observed at all developmental stages in erythroid cells of HPFH-2 transgenic mice. These findings were also associated with the formation of a DNase I-hypersensitive site in the HPFH-2 breakpoint DNA of transgenic murine erythroid cells, as occurs in normal human erythroid cells in vivo. These results indicate that breakpoint DNA sequences in deletion-type HPFH-2 can modify the developmentally regulated expression of the gamma-globin genes.

Investigation of the relationship between type 2 diabetes and osteoporosis using Bayesian inference.

This study aims to determine the prevalence of Type 2 diabetes in women with osteoporosis and estimate the odds ratio (OR) of osteoporosis in women with Type 2 diabetes using Bayesian inference. This is a case-control study design that looked into prevalence of diabetes among 582 female patients who had normal bone mineral density (BMD) and 598 female patients with osteoporosis. The subjects included women at least 30 yr of age who had their BMD measured in the lumbar spine and femoral neck using dual-energy X-ray absorptiometry at a tertiary referral center in Manila, Philippines. Prevalence of Type 2 diabetes in subjects with osteoporosis is 22.41%, whereas 19.07% of the subjects with normal BMD had diabetes. The odds of developing osteoporosis is 22.54% higher for Type 2 diabetic subjects. Patients with osteoporosis were older than subjects with normal BMD by almost 10 yr. Of the diabetic osteoporotic patients, 44.78% were physically active compared with 20.72% diabetics with normal BMD. Most of the diabetics (60.36%) with normal BMD were obese, whereas majority of diabetic osteoporotics (64.93%) have normal body mass index (BMI). Less than 10% of both diabetic osteoporotics and diabetics with normal BMD have ever undergone hormone replacement therapy. Of the 598 subjects with osteoporosis, 124 (20.74%) had suffered from fragility fractures. When controlling for physical activity and BMI, the odds of developing osteoporosis was 21.73% and 53.89% higher for Type 2 diabetics, respectively. In considering all possible confounders and effect modifiers (age, physical activity, BMI, and hormone replacement therapy) in the model which made use of a diffuse normal prior distribution, the estimate for OR (Model 1) is 0.67. A separate analysis excluding modifiable confounders (Model 2) gave the measure of association an equal likelihood of diabetes being a protective factor or a risk factor. The crude OR indicated that Type 2 diabetes is a risk factor for osteoporosis. However, when identified confounders were included in the model, the direction of the relationship changed. Considering the credible intervals (95% credible interval in both models), the study concluded that diabetes is indeed a protective factor for osteoporosis. Results of the study may have potential limitations. There are sources of bias that have been identified--selection bias where patients included in the study were referred by primary care givers for a specified reason as well as misclassification and recall biases on certain information such as type and duration of physical activity. Diabetes is a protective factor for osteoporosis in this referred population of women. However, with the well-known diabetes-related factors, that is, microvascular complications, visual acuity, and risk for fall, one should still strongly consider assessing and screening for osteoporosis and fracture risk reduction in diabetic patients.

Fluorimetric characterisation of metabolic activity of ex vivo perfused pig hearts.

Autofluorescence of tissues and organs is an indicator of the physiological state of cells. The aim of the study was to investigate whether fluorimetric determination of the redox state of the ex vivo perfused pig heart can provide fast online detection of progressive changes in heart muscle tissue. Measurements on six organs perfused in a four-chamber working heart model were performed using a spectroscopic method exploiting the specific and different fluorescence lifetimes of intrinsic fluorophores such as NADH and flavins and providing a means of internal signal referencing. It was shown that the redox potential of heart muscle tissue can be assessed by fluorescence measurement. In the steady-state phase of the beating heart, spectroscopic measurements revealed a change in redox state from an initial constant level to a continuous decrease, accompanied by a decrease in heart performance and indications of changes in electrolyte equilibrium (K(+) concentration). At the same time, troponin I levels in the perfusate increased. The results indicate that fluorimetric determination of heart muscle metabolic activity yields reliable information about the functional status of the ex vivo heart and may be advantageous for the optimisation of ex vivo organ models.

Vascularized periosteum associated with cancellous bone graft: an experimental study.

The association of a vascularized periosteal flap with a cancellous bone graft was studied on a group of 20 Wistar rats. Ten rats were sacrificed at 6 weeks and seven at 12 weeks (three died prematurely). The behavior of the cancellous bone graft buried in striated muscle and the osteogenic capacity of a simple vascularized periosteal flap also were observed on the same animals. Results of the study are as follows: In 14 of 17 animals, a vascularized periosteal flap wrapped around a cancellous bone graft resulted in new cortical bone formation with little resorption of the initial cancellous graft. A vascularized musculoperiosteal flap has produced a small amount of new compact bone only in 4 of 17 animals. A cancellous bone graft buried into well-vascularized muscle tissue was resorbed (15 cases) or necrotic (2 cases) at 12 weeks. In conclusion, the association of a vascularized periosteal flap and cancellous bone is a better means to produce compact bone than a vascularized periosteal flap alone or an isolated cancellous bone graft.

The medialis pedis flap: a new fasciocutaneous flap.

An anatomic study (30 fresh specimens dissected) and clinical experience (5 patients) have shown the reliability of a fasciocutaneous flap raised from the medial side of the foot. The artery that supplies the flap is issued from the medial plantar artery. The arch of rotation allows one to cover some specific areas, such as the medial malleolus, posterior aspect of the heel, and distal insertion of Achilles tendon.

Skin island flaps supplied by the vascular axis of the sensitive superficial nerves: anatomic study and clinical experience in the leg.

An anatomic study performed on 64 fresh injected legs has shown the role of the vascular axis that follows the superficial sensitive nerves in supplying the skin. Three nerves were studied: the saphenous nerve, the superficial peroneal nerve, and the sural nerve. Conclusions are the same for the three nerves: The vascular axis, which can be either a true artery or an interlacing network, ensures the vascularization of the nerves, gives off several cutaneous branches in the suprafascial course of the nerve, and anastomoses with the septocutaneous arteries issuing from a deep main vessel. The superficial nerves that course the leg can therefore be considered as vascular relays owing to their neurocutaneous arteries. The concept of a neuroskin island flap has been developed and applied to six clinical cases for coverage of some specific areas of the knee and of the lower part of the limb.

Efficiency of prenatal counselling for sickle cell disease in Guadeloupe.

As in most caribbean countries, Sickle Cell Disease (SCD) is a major public health problem in Guadeloupe. A prenatal counselling program was developed, at an early stage of pregnancy, for at-risk couples. Over a 6 year period, 144 couples at-risk of having a child with homozygous sickle cell (SS: n = 103) or sickle cell C disease (SC: n = 41) were seen for prenatal counselling. Among those belonging to the SS risk group, 64 (62%) underwent prenatal diagnosis (PND), which allowed identification of 27 SS fetuses, with an induced abortion rate of 70%. Among those of the SC risk group, 14 (34%) accepted PND and the diagnosis of SC was made in 5 cases with an induced abortion rate of 60%. Factors, appeared to play a role in seeking PND and induced abortion, were the type of risk (SS or SC), multiparity, existence of affected child in the family and gestational age at the time of counselling. Our experience reveals that, an early prospective identification of at-risk couples combined with education to increase the awareness of the problem at the individual and population level need to be achieved to further improve the efficiency of our prevention program.

Coverage of large skin defects of the pediatric upper extremity.

Reconstructive surgery of the locomotion apparatus in children has been built from several surgical advances. It is a specific activity that cannot be detached from all other orthopedic pediatric surgery. The care of problems in a child must be provided with a holistic point of view: It cannot be the addition of different techniques done by different surgeons. Bone morphology has a close relationship with the soft tissues; an injury of the envelope has consequences for growth harmony.

Management of purpura fulminans at the upper extremity.

A multidisciplinary approach often is needed, especially at the acute phase. Indeed, resuscitative treatment is first indicated. [figure: see text] Optimal time for surgery depends on the patient's status. Surgical management should be more aggressive than previously thought. Debridement of necrotic tissue must be performed when the eschar has demarcated to avoid infection. Escharectomy can improve the vitality of the distal segments by releasing the compressive mechanical effect. In the same vein, early amputations are likely to reduce the need for secondary revisional surgery. Necrotic tissue over a joint should be excised early and procedures to provide coverage by flaps should be undertaken to avoid osteoarthritis. Late sequelae are dominated by skeletal growth disturbances and require specific procedures.

[Vascularization of the skin of the limbs and surgical applications]. / Vascularisation tégumentaire des membres et applications chirurgicales.

Skin flaps for coverage of skin defects in orthopaedic surgery have been more and more used in recent years. However all the authors are not in agreement concerning the pattern of vascularization of these flaps. Two problems should be studied separately: the vascularization of the skin in situ which is already rather well known and the vascularization and the pattern of vascularization of the flaps after transfer. Several models of flaps, that is axial, septal or musculo-cutaneous, are based on the origin of skin vascularization, that is by long running arteries, septal arterioles or indirectly by arteries coming from muscles. Therefore a flap must be determined by its type of vascularization, its shape and its composition. Surgical rules concerning skin incisions and deep structures approaches are recalled.

[External supramalleolar flap in the reconstructive surgery of the foot]. / Le lambeau supramalléolaire externe dans la chirurgie réparatrice du pied.

The external supramalleolar flap is a mixed cutaneoaponeurotic flap removed from the external surface of lower part of leg and with, as its principal vascular pedicle, the anterior perforating branch of the peroneal artery. The flap can be used in two ways: as a rotation flap with distal cutaneous hinge allowing lover third of internal surface of leg and instep to be covered, or as a retrograde flux island flap by anastomosis of perforating artery to arteries of foot. The latter procedure is of interest in that it permits covering of loss of distal substance of foot in the dorsal or plantar region. Results of use of this method in 35 patients situate the place of the external supramalleolar flap in the techniques available for lower limb repair.

[Soft tissue preservation and reconstruction in non-supporting foot parts]. / Weichteilerhaltung und Rekonstruktionen an unbelasteten Fussanteilen.

The foot has two skin surfaces which differ on a morphological and functional basis. The plantar skin is a highly specialized organ which has a dash-pot-like effect in weight bearing. The skin which does not participate in weight bearing covers tendons and a very rich syndesmotic complex. The internal soft tissue is segmented in tiny compartments and therefore ist not extensible. The skin and the rather thin subcutaneous layer are not extensible either because of the proximity and the rigidity of the underlying structures. In consequence, trauma to the soft tissue of the foot may cause compartment syndromes and skin defects, or secondary unstable scars which impede function. Compartment syndromes must be recognized early and treated by immediate release. Fresh skin defects need early coverage when tendons and joints are exposed. There is a variety of local flaps which are evenly good for replacement of retracting scars.