Biological and mathematical modeling of melanocyte development

We aim to evaluate environmental and genetic effects on the expansion/proliferation of committed single cells during embryonic development, using melanoblasts as a paradigm to model this phenomenon. Melanoblasts are a specific type of cell that display extensive cellular proliferation during development. However, the events controlling melanoblast expansion are still poorly understood due to insufficient knowledge concerning their number and distribution in the various skin compartments. We show that melanoblast expansion is tightly controlled both spatially and temporally, with little variation between embryos. We established a mathematical model reflecting the main cellular mechanisms involved in melanoblast expansion, including proliferation and migration from the dermis to epidermis. In association with biological information, the model allows the calculation of doubling times for melanoblasts, revealing that dermal and epidermal melanoblasts have short but different doubling times. Moreover, the number of trunk founder melanoblasts at E8.5 was estimated to be 16, a population impossible to count by classical biological approaches. We also assessed the importance of the genetic background by studying gain- and loss-of-function β-catenin mutants in the melanocyte lineage. We found that any alteration of β-catenin activity, whether positive or negative, reduced both dermal and epidermal melanoblast proliferation. Finally, we determined that the pool of dermal melanoblasts remains constant in wild-type and mutant embryos during development, implying that specific control mechanisms associated with cell division ensure half of the cells at each cell division to migrate from the dermis to the epidermis. Modeling melanoblast expansion revealed novel links between cell division, cell localization within the embryo and appropriate feedback control through β-catenin.     

A Randomized Controlled Trial on the Effect of Needle Gauge on the Pain and Anxiety Experienced during Radial Arterial Puncture

Background

Arterial punctures for assessment of arterial blood-gases can be a painful procedure. Lidocaine can be used to reduce pain prior to needle insertion but it is not a widely accepted practice. The purpose of this study was to determine whether a large size needle induces more pain compared to a smaller size needle for radial arterial puncture and to assess the anxiety associated with radial arterial punctures.

Methods

We conducted a prospective, double-blind, randomized, controlled, monocentric study including all outpatients who had a planned assessment of arterial blood gas analysis. Patients were randomized to have the arterial puncture performed with a 23 or a 25 G needle. The main judgement criteria was pain during arterial puncture. Visual analogue scale for pain (VAS-P) and visual analogue scale for anxiety (VAS-A) were used to assess pain and anxiety during radial arterial puncture.

Results

Two hundred consecutive patients were randomized. The 25 G needle was as painful as the 23 G needle (6.63 mm [0–19 mm] vs. 5.21 mm [0–18.49 mm], respectively, p = 0.527). Time for arterial puncture was longer with the 25 G needle than with the 23 G needle (42 s [35–55 s] vs. 33 s [24.5–35 s], respectively, p = 0.002). There was a correlation between the level of anxiety prior to the arterial puncture and the pain experienced by the patients (p: 0.369, p<0.0001). There was a correlation between the pain experienced by patients and the anxiety experienced in anticipation of another arterial puncture (p: 0.5124, p<0.0001).

Conclusions

The use of 23 G needle allows quicker arterial sampling and is not associated with increased pain and symptoms. Anxiety was correlated with the pain experienced by patients during arterial punctures.

Trial Registration

Clinicaltrials.gov: NCT02320916     

Determinants of Maternal Near-Miss in Morocco: Too Late,Too Far,Too Sloppy?

Background

In Morocco, there is little information on the circumstances surrounding maternal near misses. This study aimed to determine the incidence, characteristics, and determinants of maternal near misses in Morocco.

Method

A prospective case-control study was conducted at 3 referral maternity hospitals in the Marrakech region of Morocco between February and July 2012. Near-miss cases included severe hemorrhage, hypertensive disorders, and prolonged obstructed labor. Three unmatched controls were selected for each near-miss case. Three categories of risk factors (sociodemographics, reproductive history, and delays), as well as perinatal outcomes, were assessed, and bivariate and multivariate analyses of the determinants were performed. A sample of 30 near misses and 30 non-near misses was interviewed.

Results

The incidence of near misses was 12‰ of births. Hypertensive disorders during pregnancy (45%) and severe hemorrhage (39%) were the most frequent direct causes of near miss. The main risk factors were illiteracy [OR = 2.35; 95% CI: (1.07–5.15)], lack of antenatal care [OR = 3.97; 95% CI: (1.42–11.09)], complications during pregnancy [OR = 2.81; 95% CI:(1.26–6.29)], and having experienced a first phase delay [OR = 8.71; 95% CI: (3.97–19.12)] and a first phase of third delay [OR = 4.03; 95% CI: (1.75–9.25)]. The main reasons for the first delay were lack of a family authority figure who could make a decision, lack of sufficient financial resources, lack of a vehicle, and fear of health facilities. The majority of near misses demonstrated a third delay with many referrals. The women’s perceptions of the quality of their care highlighted the importance of information, good communication, and attitude.

Conclusion

Women and newborns with serious obstetric complications have a greater chance of successful outcomes if they are immediately directed to a functioning referral hospital and if the providers are responsive.     

Treatment of hypoxia‐dependent cardiovascular diseases by myo‐inositol trispyrophosphate (ITPP)‐enhancement of oxygen delivery by red blood cells

Heart failure is a consequence of progression hypoxia‐dependent tissue damages. Therapeutic approaches to restore and/or protect the healthy cardiac tissue have largely failed and remain a major challenge of regenerative medicine. The myo‐inositol trispyrophosphate (ITPP) is a modifier of haemoglobin which enters the red blood cells and modifies the haemoglobin properties, allowing for easier and better delivery of oxygen by the blood. Here, we show that this treatment approach in an in vivo model of myocardial infarction (MI) results in an efficient protection from heart failure, and we demonstrate the recovery effect on post‐MI left ventricular remodelling in the rat model. Cultured cardiomyocytes used to study the molecular mechanism of action of ITPP in vitro displayed the fast stimulation of HIF‐1 upon hypoxic conditions. HIF‐1 overexpression was prevented by ITPP when incorporated into red blood cells applied in a model of blood‐perfused cardiomyocytes coupling the dynamic shear stress effect to the enhanced O₂ supply by modification of haemoglobin ability to release O₂ in hypoxia. ITPP treatment appears a breakthrough strategy for the efficient and safe treatment of hypoxia‐ or ischaemia‐induced injury of cardiac tissue.     

A novel role for central ACBP/DBI as a regulator of long‐chain fatty acid metabolism in astrocytes

Acyl‐CoA‐binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl‐CoA esters. Several studies have suggested that ACBP acts as an acyl‐CoA pool former and regulates long‐chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam‐Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABA_A receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism‐related gene expression using ACBP‐deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA‐CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes.

     

Mutations in CAPN1 Cause Autosomal-Recessive Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a genetically and clinically heterogeneous disease characterized by spasticity and weakness of the lower limbs with or without additional neurological symptoms. Although more than 70 genes and genetic loci have been implicated in HSP, many families remain genetically undiagnosed, suggesting that other genetic causes of HSP are still to be identified. HSP can be inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner. In the current study, we performed whole-exome sequencing to analyze a total of nine affected individuals in three families with autosomal-recessive HSP. Rare homozygous and compound-heterozygous nonsense, missense, frameshift, and splice-site mutations in CAPN1 were identified in all affected individuals, and sequencing in additional family members confirmed the segregation of these mutations with the disease (spastic paraplegia 76 [SPG76]). CAPN1 encodes calpain 1, a protease that is widely present in the CNS. Calpain 1 is involved in synaptic plasticity, synaptic restructuring, and axon maturation and maintenance. Three models of calpain 1 deficiency were further studied. In Caenorhabditis elegans, loss of calpain 1 function resulted in neuronal and axonal dysfunction and degeneration. Similarly, loss-of-function of the Drosophila melanogaster ortholog calpain B caused locomotor defects and axonal anomalies. Knockdown of calpain 1a, a CAPN1 ortholog in Danio rerio, resulted in abnormal branchiomotor neuron migration and disorganized acetylated-tubulin axonal networks in the brain. The identification of mutations in CAPN1 in HSP expands our understanding of the disease causes and potential mechanisms.     

Caffeoyl coenzyme A O-methyltransferase down-regulation is associated with modifications in lignin and cell-wall architecture in flax secondary xylem

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT, EC 2.1.1.104) down-regulated-flax (Linum usitatissimum) plants were generated using an antisense strategy and functionally characterized. Chemical analyses (acetyl bromide and thioacidolysis) revealed that the lignin quantity was reduced and that the Syringyl/Guaïacyl (S/G) lignin monomer ratio was modified in the non-condensed lignin fraction of two independent down-regulated lines. These modifications were associated with altered xylem organization (both lines), reduced cell-wall thickness (one line) and the appearance of an irregular xylem (irx) phenotype (both lines). In addition UV microspectroscopy also indicated that CCoAOMT down-regulation induced changes in xylem cell-wall structure and the lignin fractions. Microscopic examination also suggested that CCoAOMT down-regulation could influence individual xylem cell size and identity. As a first step towards investigating the cellular mechanisms responsible for the unusual structure of flax lignin (G-rich, condensed), recombinant flax CCoAOMT protein was produced and its affinity for different potential substrates evaluated. Results indicated that the preferred substrate was caffeoyl coenzyme A, followed by 5-hydroxyconiferaldehyde suggesting that flax CCoAOMT possesses a small, but probably significant 5′ methylating activity, in addition to a more usual 3′ methylating activity.     

Characteristics and Dynamics of Salmonella Contamination along the Coast of Agadir,Morocco

The occurrence of Salmonella enterica in the environment of tropical and desert regions has remained largely uninvestigated in many areas of the world, including Africa. In the present study, we investigated the presence of Salmonella spp. along 122 km of the coastline of Agadir (southern Morocco) in relation to environmental parameters. A total of 801 samples of seawater (243), marine sediment (279), and mussels (279) were collected from six sites between July 2004 and May 2008. The overall prevalence of Salmonella spp. was 7.1%, with the highest occurrence in mussels (10%), followed by sediment (6.8%) and seawater (4.1%). Only three serotypes were identified among the 57 Salmonella sp. strains isolated. S. enterica serotype Blockley represented 43.8% of all Salmonella strains and was identified in mussel and sediment samples. S. enterica serotype Kentucky (29.8%) was found almost exclusively in mussels, whereas S. enterica serotype Senftenberg (26.3%) was detected in sediment and seawater. Statistical analysis using generalized additive models identified seawater temperature, environmental temperature, rainfall, and solar radiation as significant factors associated with the presence of Salmonella. Rainfall was the only variable showing a linear positive effect on the presence of Salmonella in the sea, whereas the remaining variables showed more complex nonlinear effects. Twenty-eight (49.1%) Salmonella isolates displayed resistance to ampicillin (22 isolates), nalidixic acid (9 isolates), sulfonamide compounds (2 isolates), and tetracycline (1 isolate), with six of these isolates displaying multiple resistance to two of these antimicrobial agents. Pulsed-field gel electrophoresis analysis revealed homogenous restriction patterns within each serotype that were uncorrelated with the resistance pattern profiles.Salmonella enterica bacteria are one of the most frequent causes of food-borne infections transmitted to humans, mainly from animal products (9). In addition to health concerns, the presence of Salmonella contamination in the food chain has serious economic consequences related to the costs of medical care and lost productivity (36). Thus, studies aimed at examining the capacity of Salmonella spp. to survive in different habitats are critical for controlling contamination and understanding the routes of colonization of new hosts (40).Salmonella bacteria display a high degree of resistance to a large variety of stress factors, which provides them with an enhanced capacity to persist in changing environments (40). However, the persistence of the organism outside of the host is not uniform among the different serogroups (3, 4, 13, 24, 32, 34). About 50 of the more than 2,500 different serotypes of Salmonella included in the current classification scheme (29) are dominantly identified in human and animal sources (34). Information about groups that predominate in a given environment and their relationship to potential human or animal origins remains scarce. In recent years, some studies carried out in aquatic environments have provided new insights into the ecological preferences of different Salmonella serogroups in these environments. These studies have revealed the presence of specific patterns of contamination in different geographical areas in association with environmental and oceanographic variables (13, 24, 32) and have identified major factors and conditions that favor the presence of the contaminating bacteria. Identification of the different Salmonella strains present in the environment at serotype level is an essential preliminary step in discriminating potentially clinically important strains among the Salmonella present in contaminated areas, providing invaluable information about the nature of the contamination and allowing the inference of potential routes of dissemination through microbial source tracking (31). All of this information is critical for an improved assessment of the potential risks to public health associated with Salmonella and for the evaluation of the ecological preferences of the diverse and heterogeneous group of organisms which comprise the genus Salmonella (26).The lack of information regarding the epidemiology, contamination, and potential routes of transmission of Salmonella is of particular concern in many regions of the world, such as Africa and Central America, where gastrointestinal diseases continue to be a major cause of illness, primarily due to poor sanitary conditions and nutritional deficiencies. In the present study, we investigated the dynamics of Salmonella contamination in the coastal areas of Agadir, a populous region of southern Morocco where shellfish production and maritime tourism are important to the local economy. Information concerning the biological characteristics of the isolates was correlated with environmental data in order to evaluate the climatic conditions that favor contamination of this region by this pathogen and to identify the potential sources of contamination.