Genetic Diversity and Population Structure Analyses of Wild Relatives and Cultivated Cowpea (Vigna unguiculata (L.) Walp.) from Senegal Using Simple Sequence Repeat Markers

Cowpea (Vigna unguiculata (L.)) is an important crop for food security in Senegal; therefore, understanding the genetic diversity of local germplasm is relevant for crop improvement and genetic maintenance in the era of climate change. For this purpose, 15 microsatellite markers were used to estimate the genetic diversity of Senegalese cowpea germplasm, including 671 accessions grown in eight regions and 66 wild relatives and intermediate forms (weedy). For the cultivated, the main expected heterozygosity (mHe) ranged between 0.317 (Fatick) and 0.439 (South). A narrow genetic variation between accessions from the different regions was observed with genetic similarity ranging from 0.861 to 0.965 and genetic differentiation indices (Fst) between 0.018 and 0.100. The accessions from southern Senegal (Kédougou, Sédhiou, and Kolda regions) are more diverse than the others. However, the accessions from the North (Saint-Louis) are genetically different from other regions. The diversity analysis in wild relatives from Senegal, which had never been performed before, revealed that the wild/weedy forms remain more diverse than the cultivated with genetic diversity values (He) of 0.389 and 0.480, respectively. STRUCTURE software divided the Senegalese germplasm into five subpopulations. Three of them (i, ii, and iii) included only cultivated accessions from several regions, one (v) mainly from Saint-Louis, and one (iv) the wild/weedy with some cultivated accessions. Our results support the hypothesis that Vigna unguiculata var. spontanea is the wild progenitor of cowpea. The accessions from the South, the northern recession accessions, and the wild/weedy could serve as sources of new genes for the genetic improvement of cowpea in Senegal.

     

Micro-Geographical Heterogeneity in Schistosoma mansoni and S. haematobium Infection and Morbidity in a Co-Endemic Community in Northern Senegal

Background

Schistosoma mansoni and S. haematobium are co-endemic in many areas in Africa. Yet, little is known about the micro-geographical distribution of these two infections or associated disease within such foci. Such knowledge could give important insights into the drivers of infection and disease and as such better tailor schistosomiasis control and elimination efforts.

Methodology

In a co-endemic farming community in northern Senegal (346 children (0–19 y) and 253 adults (20–85 y); n = 599 in total), we studied the spatial distribution of S. mansoni and S. haematobium single and mixed infections (by microscopy), S. mansoni-specific hepatic fibrosis, S. haematobium-specific urinary tract morbidity (by ultrasound) and water contact behavior (by questionnaire). The Kulldorff''s scan statistic was used to detect spatial clusters of infection and morbidity, adjusted for the spatial distribution of gender and age.

Principal Findings

Schistosoma mansoni and S. haematobium infection densities clustered in different sections of the community (p = 0.002 and p = 0.023, respectively), possibly related to heterogeneities in the use of different water contact sites. While the distribution of urinary tract morbidity was homogeneous, a strong geospatial cluster was found for severe hepatic fibrosis (p = 0.001). Particularly those people living adjacent to the most frequently used water contact site were more at risk for more advanced morbidity (RR = 6.3; p = 0.043).

Conclusions/Significance

Schistosoma infection and associated disease showed important micro-geographical heterogeneities with divergent patterns for S. mansoni and S. haematobium in this Senegalese community. Further in depth investigations are needed to confirm and explain our observations. The present study indicates that local geospatial patterns should be taken into account in both research and control of schistosomiasis. The observed extreme focality of schistosomiasis even at community level, suggests that current strategies may not suffice to move from morbidity control to elimination of schistosomiasis, and calls for less uniform measures at a finer scale.     

The Role of Programmed Cell Death Ligand-1 (PD-L1/CD274) in the Development of Graft versus Host Disease

Programmed cell death ligand-1 (PD-L1/CD274) is an immunomodulatory molecule involved in cancer and complications of bone marrow transplantation, such as graft rejection and graft-versus-host disease. The present study was designed to assess the dynamic expression of this molecule after hematopoietic stem cell transplantation in relation to acute graft-versus-host disease. Female BALB/c mice were conditioned with busulfan and cyclophosphamide and transplanted with either syngeneic or allogeneic (male C57BL/6 mice) bone marrow and splenic cells. The expression of PD-L1 was evaluated at different time points employing qPCR, western blot and immunohistochemistry. Allogeneic- but not syngeneic-transplanted animals exhibited a marked up-regulation of PD-L1 expression in the muscle and kidney, but not the liver, at days 5 and 7 post transplantation. In mice transplanted with allogeneic bone marrow cells, the enhanced expression of PD-L1 was associated with high serum levels of IFNγ and TNFα at corresponding intervals. Our findings demonstrate that PD-L1 is differently induced and expressed after allogeneic transplantation than it is after syngeneic transplantation, and that it is in favor of target rather than non-target organs at the early stages of acute graft-versus-host disease. This is the first study to correlate the dynamics of PD-L1 at the gene-, protein- and activity levels with the early development of acute graft-versus-host disease. Our results suggest that the higher expression of PD-L1 in the muscle and kidney (non-target tissues) plays a protective role in skeletal muscle during acute graft-versus-host disease.     

Activation of Wnt/β-Catenin Pathway in Monocytes Derived from Chronic Kidney Disease Patients

Patients with chronic kidney disease (CKD) have significantly increased morbidity and mortality resulting from infections and cardiovascular diseases. Since monocytes play an essential role in host immunity, this study was directed to explore the gene expression profile in order to identify differences in activated pathways in monocytes relevant to the pathophysiology of atherosclerosis and increased susceptibility to infections. Monocytes from CKD patients (stages 4 and 5, estimated GFR <20 ml/min/1.73 m²) and healthy donors were collected from peripheral blood. Microarray gene expression profile was performed and data were interpreted by GeneSpring software and by PANTHER tool. Western blot was done to validate the pathway members. The results demonstrated that 600 and 272 genes were differentially up- and down regulated respectively in the patient group. Pathways involved in the inflammatory response were highly expressed and the Wnt/β-catenin signaling pathway was the most significant pathway expressed in the patient group. Since this pathway has been attributed to a variety of inflammatory manifestations, the current findings may contribute to dysfunctional monocytes in CKD patients. Strategies to interfere with this pathway may improve host immunity and prevent cardiovascular complications in CKD patients.     

Barth syndrome: Cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation

Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I + III₂ + IV_n but also decreased amounts of individual complexes I and IV and supercomplexes I + III and III + IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.     

Dim light at night prior to adolescence increases adult anxiety-like behaviors

Dim light at night (dLAN) disrupts circadian organization and influences adult behavior. We examined early dLAN exposure on adult affective responses. Beginning 3 (juvenile) or 5 weeks (adolescent) of age, mice were maintained in standard light–dark cycles or exposed to nightly dLAN (5 lx) for 5 weeks, then anxiety-like and fear responses were assessed. Hypothalami were collected around the clock to assess core clock genes. Exposure to dLAN at either age increased anxiety-like responses in adults. Clock and Rev-ERB expression were altered by exposure to dLAN. In contrast to adults, dLAN exposure during early life increases anxiety and fear behavior.     

A clinical experience with perforator flaps in the coverage of extensive defects of the upper extremity

Traditional skin free flaps, such as radial arm, lateral arm, and scapular flaps, are rarely sufficient to cover large skin defects of the upper extremity because of the limitation of primary closure at the donor site. Muscle or musculocutaneous flaps have been used more for these defects. However, they preclude a sacrifice of a large amount of muscle tissue with the subsequent donor-site morbidity. Perforator or combined flaps are better alternatives to cover large defects. The use of a muscle as part of a combined flap is limited to very specific indications, and the amount of muscle required is restricted to the minimum to decrease the donor-site morbidity. The authors present a series of 12 patients with extensive defects of the upper extremity who were treated between December of 1999 and March of 2002. The mean defect was 21 x 11 cm in size. Perforator flaps (five thoracodorsal artery perforator flaps and four deep inferior epigastric perforator flaps) were used in seven patients. Combined flaps, which were a combination of two different types of tissue based on a single pedicle, were needed in five patients (scapular skin flap with a thoracodorsal artery perforator flap in one patient and a thoracodorsal artery perforator flap with a split latissimus dorsi muscle in four patients). In one case, immediate surgical defatting of a deep inferior epigastric perforator flap on a wrist was performed to immediately achieve thin coverage. The average operative time was 5 hours 20 minutes (range, 3 to 7 hours). All but one flap, in which the cutaneous part of a combined flap necrosed because of a postoperative hematoma, survived completely. Adequate coverage and complete wound healing were obtained in all cases. Perforator flaps can be used successfully to cover a large defect in an extremity with minimal donor-site morbidity. Combined flaps provide a large amount of tissue, a wide range of mobility, and easy shaping, modeling, and defatting.     

Cytochrome P450 Oxidoreductase Influences CYP2B6 Activity in Cyclophosphamide Bioactivation

Introduction

Cyclophosphamide is commonly used as an important component in conditioning prior to hematopoietic stem cell transplantation, a curative treatment for several hematological diseases. Cyclophosphamide is a prodrug activated mainly by cytochrome P450 2B6 (CYP2B6) in the liver. A high degree of inter- and intra-individual variation in cyclophosphamide kinetics has been reported in several studies.

Materials and Methods

Hydroxylation of cyclophosphamide was investigated in vitro using three microsomal batches of CYP2B6*1 with different ratios of POR/CYP expression levels. Twenty patients undergoing hematopoietic stem cell transplantation were also included in the study. All patients received an i.v. infusion of cyclophosphamide (60 mg/kg/day, for two days) as a part of their conditioning. Blood samples were collected from each patient before cyclophosphamide infusion, 6 h after the first dose and before and 6 h after the second dose. POR gene expression was measured by mRNA analysis and the pharmacokinetics of cyclophosphamide and its active metabolite were determined.

Results

A strong correlation between the in vitro intrinsic clearance of cyclophosphamide and the POR/CYP ratio was found. The apparent K _m for CYP2B6.1 was almost constant (3-4 mM), while the CL_int values were proportional to the POR/CYP ratio (3-34 μL/min/nmol CYP). In patients, the average expression of the POR gene in blood was significantly (P <0.001) up-regulated after cyclophosphamide infusion, with high inter-individual variations and significant correlation with the concentration ratio of the active metabolite 4-hydroxy-cyclophosphamide/cyclophosphamide. Nine patients were carriers for POR*28; four patients had relatively high POR expression.

Conclusions

This investigation shows for the first time that POR besides CYP2B6 can influence cyclophosphamide metabolism. Our results indicate that not only CYPs are important, but also POR expression and/or activity may influence cyclophosphamide bioactivation, affecting therapeutic efficacy and treatment related toxicity and hence on clinical outcome. Thus, both POR and CYP genotype and expression levels may have to be taken into account when personalizing treatment schedules to achieve optimal therapeutic drug plasma concentrations of cyclophosphamide.