Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.     

Effect of In Ovo Feeding of the Vitamin B12 on Hatchability,Performance and Blood Constitutes in Broiler Chicken

International Journal of Peptide Research and Therapeutics - The aim of the current study was to determine effect of in ovo feeding of vitamin B12 on hatchability, growth performance and blood...     

Effect of Drought Stress on Certain Morphological and Physiological Characteristics of a Resistant and a Sensitive Canola Cultivar

Water stress is one of the main abiotic factors that reduces plant growth, mainly due to high evaporative demand and low water availability. In order to evaluate the effects of drought stress on certain morphological and physiological characteristics of two canola cultivars, we conducted a factorial experiment based on a completely randomized design. The findings show that drought stress exacerbations result in the plant's response to stress due to increased canola resistance caused by changes in plant pigments, proline, catalase, ascorbate peroxidase, peroxidase, superoxide dismutase and malondialdehyde, glucose, galactose, rhamnose and xylose. These in turn ultimately influence the morphological characteristics of canola. Drought stress reduces the concentration of carotenoids, chlorophyll a, chlorophyll b, total chlorophylls; however, glucose, galactose, rhamnose, xylose, proline, catalase, ascorbate peroxidase, peroxidase, superoxide dismutase, malondialdehyde (in leaves and roots) and the chlorophyll a and b ratios were increased. Reduction of plant height, stem height, root length, fresh and dry weight of canola treated with 300 g/l PEG compared to non‐treatment were 0.264, 0.236, 0.394, 0.183 and 0.395, respectively. From the two canola cultivars, the morphological characteristics of the NIMA increased compared to the Ks7 cultivar. Interaction effects of cultivar and drought stress showed that NIMA cultivar without treatment had the highest number of morphological characteristics such as carotenoid concentration, chlorophyll a, chlorophyll b, total chlorophylls a and b, whereas the cultivar with 300 g/l PEG (drought stress) had the highest amount of proline, malondialdehyde, soluble sugars and enzymes in leaves and roots. Increasing activity of oxidative enzymes and soluble sugars in canola under drought stress could be a sign of their relative tolerance to drought stress.     

Effects of Zinc Supplement on Plasma Homocysteine Level in End-Stage Renal Disease Patients: a Double-Blind Randomized Clinical Trial

Increased homocysteine (hCys) level is an independent risk factor for cardiovascular complications in end-stage renal disease (ESRD) patients. The aim of this study was to evaluate effect of zinc (Zn) supplement on serum hCys level in ESRD patients. One hundred ESRD patients with Zn deficiency were enrolled in this double-blind randomized clinical trial. They were randomly subdivided into two groups and supplemented with Zn (Zn group) or placebo (control group) for 6 weeks. Fasting plasma hCys and Zn levels were measured before and at 43rd days after the start of the study. Serum Zn levels increased significantly (p?<?0.0001), in Zn-treated group in comparison to placebo-treated group. In the Zn-treated group, serum hCys levels reduced significantly (p?<?0.0001), compared to placebo group (p?>?0.05). There was a significant (p?<?0.0001) reduction of mean percentage of hCys in Zn-treated group compared to the placebo group. Our study showed that Zn supplementation decreases serum hCys levels in ESRD patients with Zn deficiency.     

A sensitive fluorescent nanosensor for chloramphenicol based on molecularly imprinted polymer‐capped CdTe quantum dots

A novel fluorescent nanosensor using molecularly imprinted silica nanospheres embedded CdTe quantum dots (CdTe@SiO₂@MIP) was developed for detection and quantification of chloramphenicol (CAP). The imprinted sensor was prepared by synthesis of molecularly imprinting polymer (MIP) on the hydrophilic CdTe quantum dots via reverse microemulsion method using small amounts of solvents. The resulting CdTe@SiO₂@MIP nanoparticles were characterized by fluorescence, UV–vis absorption and FT‐IR spectroscopy and transmission electron microscopy. They preserved 48% of fluorescence quantum yield of the parent quantum dots. CAP remarkably quenched the fluorescence of prepared CdTe@SiO₂@MIP, probably via electron transfer mechanism. Under the optimal conditions, the relative fluorescence intensity of CdTe@SiO₂@MIP decreased with increasing CAP by a Stern–Volmer type equation in the concentration range of 40–500 µg L^–1. The corresponding detection limit was 5.0 µg L^–1. The intra‐day and inter‐day values for the precision of the proposed method were all <4%. The developed sensor had a good selectivity and was applied to determine CAP in spiked human and bovine serum and milk samples with satisfactory results. Copyright © 2015 John Wiley & Sons, Ltd.     

DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi

This paper presents a DNA extraction method suitable for fresh, herbarium-stored, lichenized and other fungal specimens. The method is fast and reliable, comparatively inexpensive and is suitable for obtaining PCR amplification quality DNA from large numbers of samples in a short time. The method has been tested with over 300 samples ofAscochyta, Phyllosticta, Ramalina, Parmelia andPhysconia. Amplifiable fungal DNA was extracted from pure cultures, leaf lesions, whole thalli and dissected only-fungal sections of lichenized fungi. In addition, the method has proved suitable for use with herbarium specimens of both lichenized and non-lichenized fungi, stored as dried pure cultures or dried infected plant material.     

Caveolin-1 null (-/-) mice show dramatic reductions in life span

Caveolae are 50-100 nm flask-shaped invaginations of the plasma membrane found in most cell types. Caveolin-1 is the principal protein component of caveolae membranes in nonmuscle cells. The recent development of Cav-1-deficient mice has allowed investigators to study the in vivo functional role of caveolae in the context of a whole animal model, as these mice lack morphologically detectable caveolae membrane domains. Surprisingly, Cav-1 null mice are both viable and fertile. However, it remains unknown whether loss of caveolin-1 significantly affects the overall life span of these animals. To quantitatively determine whether loss of Cav-1 gene expression confers any survival disadvantages with increasing age, we generated a large cohort of mice (n = 180), consisting of Cav-1 wild-type (+/+) (n = 53), Cav-1 heterozygous (+/-) (n = 70), and Cav-1 knockout (-/-) (n = 57) animals, and monitored their long-term survival over a 2 year period. Here, we show that Cav-1 null (-/-) mice exhibit an approximately 50% reduction in life span, with major declines in viability occurring between 27 and 65 weeks of age. However, Cav-1 heterozygous (+/-) mice did not show any changes in long-term survival, indicating that loss of both Cav-1 alleles is required to mediate a reduction in life span. Mechanistically, these dramatic reductions in life span appear to be secondary to a combination of pulmonary fibrosis, pulmonary hypertension, and cardiac hypertrophy in Cav-1 null mice. Taken together, our results provide the first demonstration that loss of Cav-1 gene expression and caveolae organelles dramatically affects the long-term survival of an organism. In addition, aged Cav-1 null mice may provide a new animal model to study the pathogenesis and treatment of progressive hypertrophic cardiomyopathy and sudden cardiac death syndrome.     

Significance of inducible nitric oxide synthase in acute myocarditis caused by Trypanosoma cruzi (Tulahuen strain)

Chagas' disease, caused by Trypanosoma cruzi, is associated with myocarditis and expression of myocardial cytokines and inducible nitric oxide synthase (NOS2). To assess the functional significance of NOS2 in murine Chagas' disease, we infected NOS2 knockout (NOS2(-/-)) and C57BL/6x129sv (wild type) mice with the Tulahuen strain of T. cruzi. Serial transthoracic echocardiography was performed to assess the progression of left and right ventricular dysfunction in infected mice. Uninfected wild type and NOS2(-/-) mice served as controls. At day 10 post-infection (p.i.), infected wild type mice had larger left ventricular end-diastolic diameter (2.52+/-0.14-vs-2.11+/-0.06 mm, P<0.02) and right ventricle (0.6+/-0.2-vs-0 visual grade, P<0.02) as compared with uninfected wild type mice. At day 19 p.i., compared with uninfected controls, infected wild type mice had larger left ventricular end-diastolic diameter (3.30+/-0.29-vs-2.11+/-0.07 mm), left ventricular end-systolic diameter (1.86+/-0.29-vs-0.88+/-0.05 mm), right ventricle (1.6+/-0.2-vs-0 visual grade), lower heart rate (413+/-27-vs-557+/-25 beats per min), left ventricular relative wall thickness (0.44+/-0.05-vs-0.64+/-0.03) and fractional shortening (45+/-4-vs-57+/-2%) [P<0.05 for all]. In contrast, no differences in left ventricular end-diastolic diameter or fractional shortening were noted among infected and uninfected NOS2(-/-) mice at day 19 p.i. Compared with uninfected controls, infected NOS2(-/-) mice had significantly lower heart rate (272+/-23-vs-512+/-31 beats per min, P<0.01) and larger right ventricle (0.6+/-0.2-vs-0, P<0.05 visual grade). The magnitude of right ventricular dilation in NOS2(-/-) mice was less than that observed in infected wild type mice. At necropsy, the heart weight was greater (129+/-16-vs-109+/-7 mg, P=0.02) and myocardial inflammation more severe in infected wild type compared with infected NOS2(-/-) mice. Myocardial interleukin (IL)-1beta, IL-6, tumour necrosis factor-alpha, and interferon-gamma were induced in all infected mice. These data indicate that nitric oxide derived from NOS2 plays an important role in the development and progression of ventricular dilation and systolic dysfunction in acute murine chagasic myocarditis caused by infection with the Tulahuen strain.     

Cardioprotective effects of phosphoramidon on myocardial structure and function in murine Chagas' disease

Chagas' disease is an important cause of cardiomyopathy. Endothelin-1, a vasoactive peptide has been implicated in the pathogenesis of chagasic cardiomyopathy. C57BL/6 x 129sv and CD1 mice were thus, infected with trypomastigotes of Trypanosoma cruzi (Brazil strain) and these infected mice were compared with infected mice treated with phosphoramidon. This compound inhibits endothelin-converting enzyme and neutral endopeptidases and does not affect the growth of the parasite in culture. Phosphoramidon was given in a dose of 10mg/kg for the initial 15 days post-infection None of the C57Bl/6 x 129sv mice died as a result of infection. However, there was marked myocardial inflammation and fibrosis in infected, untreated mice. The hearts of the infected, phosphoramidon-treated mice showed significantly less pathology. Cardiac magnetic resonance imaging of infected mice revealed right ventricular dilation that was less severe in those treated with phosphoramidon. Phosphoramidon-treated CD1 mice survived the acute infection. Transthoracic echocardiography demonstrated left ventricular dilation and reduced percent fractional shortening and relative wall thickness. These alterations were also attenuated as a result of phosphoramidon treatment. These data suggest that endothelin-1 contributes to the pathogenesis of chagasic cardiomyopathy and interventions that inhibit the synthesis of endothelin-1 and/or neutral endopeptidase might have a protective effect on myocardial structure and function in murine Chagas' disease.