The Evolution of Collagen Fiber Orientation in Engineered Cardiovascular Tissues Visualized by Diffusion Tensor Imaging

The collagen architecture is the major determinant of the function and mechanical behavior of cardiovascular tissues. In order to engineer a functional and load-bearing cardiovascular tissue with a structure that mimics the native tissue to meet in vivo mechanical demands, a complete understanding of the collagen orientation mechanism is required. Several methods have been used to visualize collagen architecture in tissue-engineered (TE) constructs, but they either have a limited imaging depth or have a complicated set up. In this study, Diffusion Tensor Imaging (DTI) is explored as a fast and reliable method to visualize collagen arrangement, and Confocal Laser Scanning Microscopy (CLSM) was used as a validation technique. Uniaxially constrained TE strips were cultured for 2 days, 10 days, 3 and 6 weeks to investigate the evolution of the collagen orientation with time. Moreover, a comparison of the collagen orientation in high and low aspect ratio (length/width) TE constructs was made with both methods. Both methods showed similar fiber orientation in TE constructs. Collagen fibers in the high aspect ratio samples were mostly aligned in the constrained direction, while the collagen fibers in low aspect ratio strips were mainly oriented in the oblique direction. The orientation changed to the oblique direction by extending culture time and could also be visualized. DTI captured the collagen orientation differences between low and high aspect ratio samples and with time. Therefore, it can be used as a fast, non-destructive and reliable tool to study the evolution of the collagen orientation in TE constructs.     

Portable,Battery-Operated,Low-Cost,Bright Field and Fluorescence Microscope

This study describes the design and evaluation of a portable bright-field and fluorescence microscope that can be manufactured for $240 USD. The microscope uses a battery-operated LED-based flashlight as the light source and achieves a resolution of 0.8 µm at 1000× magnification in fluorescence mode. We tested the diagnostic capability of this new instrument to identify infections caused by the human pathogen, Mycobacterium tuberculosis. Sixty-four direct, decontaminated, and serially diluted smears were prepared from sputa obtained from 19 patients suspected to have M. tuberculosis infection. Slides were stained with auramine orange and evaluated as being positive or negative for M. tuberculosis with both the new portable fluorescence microscope and a laboratory grade fluorescence microscope. Concordant results were obtained in 98.4% of cases. This highly portable, low cost, fluorescence microscope may be a useful diagnostic tool to expand the availability of M. tuberculosis testing at the point-of-care in low resource settings.     

Correction to: Effect of Chromium Nanoparticles on Physiological Stress Induced by Exogenous Dexamethasone in Japanese Quails

Biological Trace Element Research - Chromium (Cr), as an essential trace element, plays a critical role in carbohydrate, protein, and lipid metabolism in animals. It has been suggested that the...     

Study on genetic diversity of Magnaporthe grisea using PCR and determination of the mating type alleles distribution in Mazandaran province, Iran

The population structure of Magnaporthe grisea, the causal agent of the rice blast, was analyzed in Mazandaran province, using DNA fingerprinting based on RAPD-PCR by means of three primers including "I", "D" and "H". Total DNA of 47 isolates was extracted and amplified according to a specific PCR program. As a result, variable length fragments were generated. Each isolate was subjected to DNA fingerprinting and clonal lineages were determined. Phenetic analysis differentiated three distinct fingerprint lineages. In order to study on fertility status and distribution of the mating type idiomorphs (alleles), 72 monoconidial isolates from Mazandaran province were paired with four standard fertile hermaphrodite isolates. The mating type of 36 isolates was determined as Mat 1-1. The others (36 isolates) did not form any perithecia in pairing with standard isolates     

Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion

1. Download : Download full-size image

     

Purification of modified mycobacterial A60 antigen by affinity chromatography and its use for rapid diagnostic tuberculosis infection

Tuberculosis has been declared a global emergency. The mainstay for its control is the rapid and accurate identification of infected individual. Antibodies to A60, one of the macromolecular antigen complexes of mycobacteria were commonly used in the rapid detection of Mycobacterium tuberculosis. The aim of this study was to prepare specific antibodies against A60 for detection of tuberculosis infection. Specific polyclonal antibodies against A60, (A60-Ab) were prepared in rabbits using 2 boosted injections of the antigen (A60). The antibodies were purified and treated with normal oral flora to remove any non-specific and cross-reactive antibodies. These antibodies were conjugated to CNBr-activated Sepharose 4B and used to isolate subunits of A60 with more specificity for M. tuberculosis. A new affinity column was designed to prepare modified (purified) A60 antigen. Purified A60 antigen (PA60-Ag) was used to develop antibody production by Immunoaffinity chromatography. 113 patients with a confirmed diagnosis of pulmonary TB at Pasteur Institute were selected for the study. The specificity of the results was analyzed with TB-rapid test by using PA60-antibodies. TB-rapid test revealed that normal oral flora-absorbed antibodies could lead to more specific results than that of the non-absorbed antibodies. The developed, modified A60 antibodies, (PA60-Ab)-rapid test showed higher sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and overall efficiency (93.0%, 86.0%, 90.0%, 91.0%, and 90.0% respectively) for the detection of the Mycobacterium antigen. Moreover, PA60-Ag showed only two protein bands of molecular weight 45 and 66 kDa in SDS-PAGE while untreated A60 showed multiple bands. Thus, our study helped in the purification of a novel and well characterized A60 antigen and good diagnostic potential for detecting tuberculosis infection.     

The Neuromuscular Junction: Measuring Synapse Size,Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.     

Sequence of Age-Associated Changes to the Mouse Neuromuscular Junction and the Protective Effects of Voluntary Exercise

Loss of connections between motor neurons and skeletal muscle fibers contribute to motor impairment in old age, but the sequence of age-associated changes that precede loss of the neuromuscular synapse remains uncertain. Here we determine changes in the size of neuromuscular synapses within the tibialis anterior muscle across the life span of C57BL/6J mice. Immunofluorescence, confocal microscopy and morphometry were used to measure the area occupied by nerve terminal synaptophysin staining and postsynaptic acetylcholine receptors at motor endplates of 2, 14, 19, 22, 25 and 28month old mice. The key findings were: 1) At middle age (14-months) endplate acetylcholine receptors occupied 238±11 µm² and nerve terminal synaptophysin 168±14 µm² (mean ± SEM). 2) Between 14-months and 19-months (onset of old age) the area occupied by postsynaptic acetylcholine receptors declined 30%. At many endplates the large acetylcholine receptor plaque became fragmented into multiple smaller acetylcholine receptor clusters. 3) Between 19- and 25-months, the fraction of endplate acetylcholine receptors covered by synaptophysin fell 21%. By 28-months, half of the endplates imaged retained ≤50 µm² area of synaptophysin staining. 4) Within aged muscles, the degree to which an endplate remained covered by synaptophysin did not depend upon the total area of acetylcholine receptors, nor upon the number of discrete receptor clusters. 5) Voluntary wheel-running exercise, beginning late in middle-age, prevented much of the age-associated loss of nerve terminal synaptophysin. In summary, a decline in the area of endplate acetylcholine receptor clusters at the onset of old age was followed by loss of nerve terminal synaptophysin from the endplate. Voluntary running exercise, begun late in middle age, substantially inhibited the loss of nerve terminal from aging motor endplates.     

Study on morphology,pathogenicity and genetic diversity of Wilsonomyces carpophilus isolates,the causal agent of shot hole of stone fruit trees based on RAPD-PCR in Iran

Shot hole disease is one of the most important diseases of stone fruit trees in Iran. The disease is wide spread among orchards of Prunus spp. During spring and summer of 2007, 80 monoconidial isolates of the pathogen were recovered from infected leaves, fruits and twigs of different Prunus spp. in West Azerbaijan, Tehran, Ghazvin and Razavi Khorasan provinces of Iran and were studied taxonomically. Based on morphological and physiological characteristics and growth optimal temperature, all isolates were identified as Wilsonomyces carpophilus. Seedlings of stone fruits (apricot, almond, peach, nectarine, plum, sweet cherry and sour cherry) were used for pathogenicity tests. All seedlings were susceptible to the fungal isolates and showed disease symptoms on twigs, leaves, buds and petioles. Genetic diversity of 28 selected fungal isolates was investigated based on DNA fingerprinting by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), using four random primers. Based on cluster analysis of the PCR results from the four primers, 10 fingerprinting groups (clonal lineages) and 27 haplotypes were identified. Clonal lineages “C”, “D” and “E”, each with six haplotypes formed the biggest clonal lineages, but other clonal lineages (“B”, “F”, “G”, “H”, “I” and “J”) included only one isolate. No correlation was detected among clonal lineages with the location of selected isolates and their host species. A correlation was found between the substrate (fruit, twig or leaf) and clonal lineages, particularly in “C” clonal lineage. The results showed that the fungus population had high genetic diversity which is distributed among the different areas of Iran.     

Enhancing secondary embryogenesis in Brassica napus by selecting hypocotyl-derived embryos and using plant-derived smoke extract in culture medium

Induction of secondary embryogenesis on transformed androgenic microspore-derived embryos (MDEs) is a convenient approach to avoid chimerism and hemizygosis for the introduced transgene. In this work, we improved two aspects related to secondary embryogenesis in rapeseed (Brassica napus L. cv. Topas) MDEs: the identification of the best source of secondary embryos in the germinated MDEs and the increase in the production of secondary embryos (SEs). We performed a ploidy analysis of the different organs of MDEs-derived plantlets by flow cytometry. Our results showed that 60?% of the MDEs-derived plantlets were mixoploid, with 60?% of them having different ploidies for different organs. We concluded that hypocotyl-derived SEs present in general higher levels of genome duplication, which makes them a source of SEs better than cotyledons in terms of genetic stability and avoidance of hemizygosis. In order to increase production of SEs, we used plant-derived aqueous smoke extracts. The aim was to verify whether these extracts have a positive effect on secondary embryogenesis and if so, to identify the most efficient conditions of use. We tested smoke extracts at different incubation times, concentrations and methods of application to MDEs. The use of smoke extract, either prior to or during germination of MDEs, markedly enhances secondary embryogenesis. The best results were obtained with the use of smoke extract as a pretreatment, incubating MDEs for no longer than 15?min with a 1:250 extract concentration.