Identification of a neuronal laminin receptor: an Mr 200K/120K integrin heterodimer that binds laminin in a divalent cation-dependent manner

Neuronal interactions with extracellular matrix (ECM) components are crucial for axon growth and guidance during development and nerve regeneration. Laminin (LN), a prominent ECM glycoprotein, promotes neuronal survival and axon growth. To identify neuronal receptors for LN, we looked for cell surface proteins on the neuronal cell line B50 that bind LN. An integrin alpha/beta 1 dimeric receptor was identified and purified using lectin and LN affinity chromatography. The purified integrin contains two subunits with Mrs of 200 K and 120 K that bind LN specifically in the presence, but not the absence, of divalent cations (Ca2+/Mg2+ or Ca2+/Mn2+). The Mr 120 K protein was identified as the rat integrin beta 1 subunit using two beta 1 subunit-specific antibodies, and was shown to form a noncovalent complex with the Mr 200K putative alpha subunit. Since neurons and neuronal cell lines express similar integrin beta 1-class heterodimers that mediate attachment and process outgrowth on LN, the Mr 200K/120K complex identified here is likely to be an important laminin receptor used by neurons. This integrin may also mediate binding to LN by many nonneuronal cell types.     

Active Ion Transport Across Canine Blood Vessel Walls

Experiments giving evidence of active Na and Cl ion fluxes across large canine blood vessel walls (aorta, vena cava) in vitro have been presented. The information has been obtained using ion tracer techniques after Ussing and with diffusion cells of the Hogben type. The available data suggest that the membranes are satisfactorily oxygenated by the bathing solutions saturated with oxygen at atmospheric pressure. Evidence is offered which indicates that active ion transport does occur across the aorta and vena cava in in vitro experiments. Under the conditions of the experiment net Na and Cl flux takes place from intima to adventitia across the aorta, and from adventitia to intima across the vena cava at low measured potential differences. The possible relationships of derangement of active ion transport mechanisms, produced by electric currents and tissue injury potential differences, to intravascular thrombosis are alluded to. It would appear that sodium and chloride fluxes across large blood vessel walls in vitro occur at least in part as the result of metabolic processes and cannot be explained simply on the basis of diffusion across a semipermeable membrane.     

Two novel microsatellite markers for prenatal prediction of spinal muscular atrophy (SMA)

Autosomal recessive spinal muscular atrophy (SMA) has been mapped to a 6-cM interval on chromosome 5q12–13.3, flanked proximally by locus D5S6 and distally by locus D5S112. In this study we describe the isolation of two new microsatellite markers (EF1/2a and EF13/14) near locus D5S125, which lies 2 cM distal to D5S6. We show by linkage analysis and the study of the recombinants in 55 SMA pedigrees that the disease lies in the 4-cM interval between EF1/2a and D5S112. Fluorescence in situ analysis of cosmids from D5S6, EF1/2a and D5S112 confirms the genetic order and relative distance of markers. The microsatellites EF1/2a and EF13/14 are the first highly polymorphic PCR based proximal markers in SMA to be described, and will be of value in prental prediction of the disorder.     

Prevalence and risk factors of schistosomiasis among primary school children in four selected regions of The Gambia

BackgroundThe Gambia initiated a control programme for schistosomiasis in 2015. In light of this, recent and comprehensive data on schistosomiasis is required to effectively guide the control programme. This study aimed to evaluate the prevalence and associated risk factors of schistosomiasis among primary school children in The Gambia.MethodsWe utilised data from a previous study conducted in 2015 in 4 regions of The Gambia: North Bank Region (NBR), Lower River Region (LRR), Central River Region (CRR) and Upper River Region (URR). In the parent study, ten schools were selected randomly from each region. Urine and stool samples collected from 25 boys and 25 girls (7–14 years) in each school were examined for urinary schistosomiasis (Schistosoma haematobium infection) and intestinal schistosomiasis (Schistosoma mansoni infection) using urine filtration, dipstick and Kato-Katz methods.Principal findingsUrinary schistosomiasis had an overall prevalence of 10.2% while intestinal schistosomiasis had a prevalence of 0.3% among the sampled school children. Prevalence of urinary schistosomiasis was significantly different among regions (χ 2 = 279.958, df = 3, p < 0.001), with CRR (27.6%) being the most endemic region, followed by URR (12.0%), then LRR (0.6%), and NBR (0.0%). Prevalence of intestinal schistosomiasis was also significantly variable among regions, with 4 of the 5 positive cases detected in CRR and 1 case in URR. Every school sampled in CRR had at least one student infected with S. haematobium, 50% of schools in URR had S. haematobium infection, and just one school in LRR had S. haematobium infection. While S. haematobium infection was significantly higher in boys (χ 2 = 4.440, df = 1, p = 0.035), no significant difference in infection rate was observed among age groups (χ 2 = 0.882, df = 2, p = 0.643). Two of the 5 students infected with S. mansoni were boys and 3 were girls. Four of these 5 students were in the 10–12 years age group and 1 was in the 7–9 years age group. Macrohaematuria and microhaematuria were found to be statistically associated with presence of S. haematobium eggs in urine. Being a male was a risk factor of S. haematobium infection. Bathing, playing and swimming in water bodies were found to pose less risk for S. haematobium infection, indicating that the true water contact behaviour of children was possibly underrepresented.ConclusionThe findings of this study provide invaluable information on the prevalence of schistosomiasis in The Gambia. This was useful for the schistosomiasis control efforts of the country, as it guided mass drug administration campaigns in eligible districts in the study area. More studies on S. mansoni and its intermediate snail hosts are required to establish its true status in The Gambia. As children sometimes tend to provide responses that potentially please the research or their teacher, data collection frameworks and approaches that ensure true responses in studies involving children should be devised and used.     

Cross-Species Array Comparative Genomic Hybridization Identifies Novel Oncogenic Events in Zebrafish and Human Embryonal Rhabdomyosarcoma

Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS – identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer.     

Recognition of Peroxisomes by Immunofluorescence in Transformed and Untransformed Tobacco Cells

We report the visualization of peroxisomes in tobacco (Nicotiana tabacum) leaves using fluorescently labeled antibodies to glycolate oxidase. In transgenic tobacco leaves the expression of isocitrate lyase was also visualized. In dual probing experiments both enzymes were shown to be present together in all peroxisomes in transgenic tobacco leaves.     

Association between Ocular Bacterial Carriage and Follicular Trachoma Following Mass Azithromycin Distribution in The Gambia

Background

Trachoma, caused by ocular Chlamydia trachomatis infection, is the leading infectious cause of blindess, but its prevalence is now falling in many countries. As the prevalence falls, an increasing proportion of individuals with clinical signs of follicular trachoma (TF) is not infected with C. trachomatis. A recent study in Tanzania suggested that other bacteria may play a role in the persistence of these clinical signs.

Methodology/Principal Findings

We examined associations between clinical signs of TF and ocular colonization with four pathogens commonly found in the nasopharnyx, three years after the initiation of mass azithromycin distribution. Children aged 0 to 5 years were randomly selected from 16 Gambian communitites. Both eyes of each child were examined and graded for trachoma according to the World Health Organization (WHO) simplified system. Two swabs were taken from the right eye: one swab was processed for polymerase chain reaction (PCR) using the Amplicor test for detection of C. trachomatis DNA and the second swab was processed by routine bacteriology to assay for the presence of viable Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus and Moraxella catarrhalis. Prevalence of TF was 6.2% (96/1538) while prevalence of ocular C. trachomatis infection was 1.0% (16/1538). After adjustment, increased odds of TF were observed in the presence of C. trachomatis (OR = 10.4, 95%CI 1.32–81.2, p = 0.03), S. pneumoniae (OR = 2.14, 95%CI 1.03–4.44, p = 0.04) and H. influenzae (OR = 4.72, 95% CI 1.53–14.5, p = 0.01).

Conclusions/Significance

Clinical signs of TF can persist in communities even when ocular C. trachomatis infection has been controlled through mass azithromycin distribution. In these settings, TF may be associated with ocular colonization with bacteria commonly carried in the nasopharnyx. This may affect the interpretation of impact surveys and the determinations of thresholds for discontinuing mass drug administration.     

Complement C3 and C5 Deficiency Affects Fracture Healing

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3^-/- and C5^-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3^-/-: -25%, p=0.02; C5^-/-: -20% p=0.052) and newly formed bone (C3^-/-: -38%, p=0.01; C5^-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3^-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.     

Modelling the fracture-healing process as a moving-interface problem using an interface-capturing approach

We present a novel numerical model of the fracture-healing process using interface-capturing techniques, a well-known approach from fields like fluid dynamics, to describe tissue growth. One advantage of this method is its direct connection to experimentally observable parameters, including tissue-growth velocities. In our model, osteogenesis, chondrogenesis and revascularisation are triggered by mechanical stimuli via mechano-transduction based on previously established hypothesis of Claes and Heigele. After experimentally verifying the convergence of the numerical method, we compare the predictions of our model with those of the already established Ulm bone-healing model, which serves as a benchmark, and corroborate our results with existing animal experiments. We demonstrate that the new model can predict the history of the interfragmentary movement and forecast a tissue evolution that appears similar to the experimental results. Furthermore, we compare the relative tissue concentration in the healing domain with outcomes of animal experiments. Finally, we discuss the possible application of the model to new fields, where numerical simulations could also prove beneficial.