Studies on the interaction between nanodiamond and human hemoglobin by surface tension measurement and spectroscopy methods

In this study, a novel method to probe molecular interactions and binding of human hemoglobin (Hb) with nanodiamond (ND) was introduced based on the surface tension measurement. This method complements conventional techniques, which are basically done by zeta potential and dynamic light scattering (DLS) measurements, near and far circular dichroism (CD) spectroscopy, intrinsic and extrinsic fluorescence spectroscopy. Addition of ND to Hb solution increased the surface tension value of Hb–ND complex relative to those of Hb and ND molecules. The zeta potential values reveled that Hb and ND provide identical charge distribution at pH 7.5. DLS measurements demonstrated that Hb, ND, and ND–Hb complex have hydrodynamic radiuses of 98.37 ± 4.57, 122.07 ± 7.88 nm and 62.27 ± 3.70 at pH of 7.5 respectively. Far and near UV-CD results indicated the loss of α-helix structure and conformational changes of Hb, respectively. Intrinsic fluorescence data demonstrated that the fluorescence quenching of Hb by ND was the result of the static quenching. The hydrophobic interaction plays a pivotal role in the interaction of ND with Hb. Fluorescence intensity changes over time revealed conformational change of Hb continues after the mixing of the components (Hb–ND) till 15 min, which is indicative of the denaturation of the Hb relative to the protein control. Extrinsic fluorescence data showed a considerable enhancement of the ANS fluorescence intensity of Hb–ND system relative to the Hb till 60 nM of ND, likely persuaded by greater exposure of nonpolar residues of Hb hydrophobic pocket. The remarkable decrease in T_m value of Hb in Hb–ND complex exhibits interaction of Hb with ND conducts to conformational changes of Hb. This study offers consequential discrimination into the interaction of ND with proteins, which may be of significance for further appeal of these nanoparticles in biotechnology prosecution.     

Can micro-imaging based analysis methods quantify structural integrity of rat vertebrae with and without metastatic involvement?

This study compares the ability of μCT image-based registration, 2D structural rigidity analyses and multimodal continuum-level finite element (FE) modeling in evaluating the mechanical stability of healthy, osteolytic, and mixed osteolytic/osteoblastic metastatically involved rat vertebrae. μMR and μCT images (loaded and unloaded) were acquired of lumbar spinal motion segments from 15rnu/rnu rats (five per group). Strains were calculated based on image registration of the loaded and unloaded μCT images and via analysis of FE models created from the μCT and μMR data. Predicted yield load was also calculated through 2D structural rigidity analysis of the axial unloaded μCT slices. Measures from the three techniques were compared to experimental yield loads. The ability of these methods to predict experimental yield loads were evaluated and image registration and FE calculated strains were directly compared. Quantitatively for all samples, only limited weak correlations were found between the image-based measures and experimental yield load. In comparison to the experimental yield load, we observed a trend toward a weak negative correlation with median strain calculated using the image-based strain measurement algorithm (r=-0.405, p=0.067), weak significant correlations (p<0.05) with FE based median and 10th percentile strain values (r=-0.454, -0.637, respectively), and a trend toward a weak significant correlation with FE based mean strain (r=-0.366, p=0.09). Individual group analyses, however, yielded more and stronger correlations with experimental results. Considering the image-based strain measurement algorithm we observed moderate significant correlations with experimental yield load (p<0.05) in the osteolytic group for mean and median strain values (r=-0.840, -0.832, respectively), and in the healthy group for median strain values (r=-0.809). Considering the rigidity-based predicted yield load, we observed a strong significant correlation with the experimental yield load in the mixed osteolytic/osteoblastic group (r=0.946) and trend toward a moderate correlation with the experimental yield load in the osteolytic group (r=0.788). Qualitatively, strain patterns in the vertebral bodies generated using image registration and FEA were well matched, yet quantitatively a significant correlation was found only between mean strains in the healthy group (r=0.934). Large structural differences in metastatic vertebrae and the complexity of motion segment loading may have led to varied modes of failure. Improvements in load characterization, material properties assignments and resolution are necessary to yield a more generalized ability for image-based registration, structural rigidity and FE methods to accurately represent stability in healthy and pathologic scenarios.     

Inhibition of cAMP-dependent protein kinase under conditions occurring in the cardiac dyad during a Ca2+ transient

The space between the t-tubule invagination and the sarcoplasmic reticulum (SR) membrane, the dyad, in ventricular myocytes has been predicted to experience very high [Ca2+] for short periods of time during a Ca2+ transient. The dyadic space accommodates many protein kinases responsible for the regulation of Ca2+ handling proteins of the cell. We show in vitro that cAMP-dependent protein kinase (PKA) is inhibited by high [Ca2+] through a shift in the ratio of CaATP/MgATP toward CaATP. We further generate a three-dimensional mathematical model of Ca2+ and ATP diffusion within dyad. We use this model to predict the extent to which PKA would be inhibited by an increased CaATP/MgATP ratio during a Ca2+ transient in the dyad in vivo. Our results suggest that under normal physiological conditions a myocyte paced at 1 Hz would experience up to 55% inhibition of PKA within the cardiac dyad, with inhibition averaging 5% throughout the transient, an effect which becomes more pronounced as the myocyte contractile frequency increases (at 7 Hz, PKA inhibition averages 28% across the dyad throughout the duration of a Ca2+ transient).     

Ca2+ regulation in the near-membrane microenvironment in smooth muscle cells

The microenvironment between the plasma membrane and the near-membrane sarcoplasmic reticulum (SR) may play an important role in Ca(2+) regulation in smooth muscle cells. We used a three-dimensional mathematical model of Ca(2+) diffusion and regulation and experimental measurements of SR Ca(2+) uptake and the distribution of the SR in isolated smooth muscle cells to predict the extent that the near-membrane SR could load Ca(2+) after the opening of single plasma membrane Ca(2+) channels. We also modeled the effect of SR uptake on 1), single-channel Ca(2+) transients in the near-membrane space; 2), the association of Ca(2+) with Ca(2+) buffers in this space; and 3), the amount of Ca(2+) reaching the central cytoplasm of the cell. Our results indicate that, although single-channel Ca(2+) transients could increase SR Ca(2+) to a certain extent, SR Ca(2+) uptake is not rapid enough to greatly affect the magnitude of these transients or their spread to the central cytoplasm unless the Ca(2+) uptake rate of the peripheral SR is an order-of-magnitude higher than the mean rate derived from our experiments. Immunofluorescence imaging, however, did not reveal obvious differences in the density of SR Ca(2+) pumps or phospholamban between the peripheral and central SR in smooth muscle cells.     

Effects of very rapid versus vapor phase freezing on human sperm parameters

The aim of the present study was to compare the effects of two freezing methods, vapor phase and very rapid freezing, with and without cryoprotectant on semen parameters in men with normal semen criteria. Cryopreservation was done on semen samples from 31 men by two methods of vapor phase freezing and very rapid freezing, with and without Test Yolk buffered glycerol (TYBG) as cryoprotectant. The motility, viability, acrosome and DNA integrity were evaluated on fresh and post-thaw samples. Post-thaw sperm progressive motility was significantly higher in the presence of TYBG in the vapor phase cryopreservation (%6.30 ± 3.74) compared with the very rapid freezing method (%2.2 ± 1.97 and %4.00 ± 2.42 in the presence and absence of TYBG, respectively). There was no significant difference in viability, acrosome status and DNA integrity between two methods in presence or absence of TYBG. The very rapid freezing method in the absence of TYBG showed better sperm motility but viability, acrosome and DNA integrity were similar to the presence of TYBG. The results show that cryopreservation of human spermatozoa together with seminal plasma by using vapor phase method is better than very rapid freezing method to preserve sperm progressive motility; however very rapid freezing method is quick and simple and do not require special cryoprotectant. It can be used for cryopreservation of small number of spermatozoa in IVF centers.     

An atlas of CNV maps in cattle,goat and sheep

Copy number variation(CNV) is the most prevalent type of genetic structural variation that has been recognized as an important source of phenotypic variation in humans, animals and plants. However, the mechanisms underlying the evolution of CNVs and their function in natural or artificial selection remain unknown. Here, we generated CNV region(CNVR) datasets which were diverged or shared among cattle, goat, and sheep, including 886 individuals from 171 diverse populations. Using 9 environmental factors for genome-wide association study(GWAS), we identified a series of candidate CNVRs, including genes relating to immunity, tick resistance, multi-drug resistance, and muscle development. The number of CNVRs shared between species is significantly higher than expected(P0.00001), and these CNVRs may be more persist than the single nucleotide polymorphisms(SNPs) shared between species. We also identified genomic regions under long-term balancing selection and uncovered the potential diversity of the selected CNVRs close to the important functional genes. This study provides the evidence that balancing selection might be more common in mammals than previously considered, and might play an important role in the daily activities of these ruminant species.     

A Hu sheep genome with the first ovine Y chromosome reveal introgression history after sheep domestication

The Y chromosome plays key roles in male fertility and reflects the evolutionary history of paternal lineages. Here, we present a de novo genome assembly of the Hu sheep with the first draft assembly of ovine Y chromosome(o MSY), using nanopore sequencing and Hi-C technologies. The o MSY that we generated spans 10.6 Mb from which 775 Y-SNPs were identified by applying a large panel of whole genome sequences from worldwide sheep and wild Iranian mouflons. Three major paternal lineages(HY1a, HY1b and HY2) were defined across domestic sheep, of which HY2 was newly detected. Surprisingly, HY2 forms a monophyletic clade with the Iranian mouflons and is highly divergent from both HY1a and HY1b. Demographic analysis of Y chromosomes, mitochondrial and nuclear genomes confirmed that HY2 and the maternal counterpart of lineage C represented a distinct wild mouflon population in Iran that diverge from the direct ancestor of domestic sheep, the wild mouflons in Southeastern Anatolia. Our results suggest that wild Iranian mouflons had introgressed into domestic sheep and thereby introduced this Iranian mouflon specific lineage carrying HY2 to both East Asian and Africa sheep populations.     

The effect of adrenocorticotropic hormone on alpha‐2‐macroglobulin in osteoblasts derived from human mesenchymal stem cells

Nowadays, alpha‐2‐macroglobulin (A2M) gene has allocated escalating interest among several genes involved in the pathogenesis of avascular necrosis of the femoral head (ANFH). This molecule could interact with several osteogenic‐related proteins. It was reported that adrenocorticotropic hormone (ACTH) affects bones through its receptor located on osteoblasts, suggesting it as a potential target in ANFH treatment. In this study, the effect of ACTH on A2M expression was investigated in osteoblasts as well as during the differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. In this study, MSCs derived from bone marrow were isolated and purified using Ficoll gradient and several passaging. MSCs were characterized by induction with osteogenic and adipogenic medium followed by Oil Red O, Alizarin Red and alkaline phosphatase staining. Besides, MSCs were exposed to various concentrations of ACTH to evaluate the cell variability by MTT assay. MSCs and differentiated osteoblasts were treated with 10^?8 molar ACTH for 16 and 26 days, respectively. Then, the total RNA was extracted and A2M expression was quantified by real‐time qPCR. The protein expression levels of osteoblast markers including alkaline phosphatase (ALPL) and bone gamma‐carboxyglutamate protein (BGLAP) were also measured. The results showed that A2M expression in cells treated with ACTH was up‐regulated significantly compared to the control group. Similarly, the expression of osteoblast gene markers including ALPL and BGLAP was significantly increased. ACTH, as an osteoblastic differentiation enhancer, up‐regulates A2M, which promotes osteoblastic differentiation probably through TGF‐β induction.     

Design and Synthesis of Novel Cytotoxic Indole‐Thiosemicarbazone Derivatives: Biological Evaluation and Docking Study

In this work, two novel series of indole‐thiosemicarbazone derivatives were designed, synthesized, and evaluated for their cytotoxic activity against MCF‐7, A‐549, and Hep‐G2 cell lines in comparison to etoposide and colchicine as the reference drugs. Generally, the synthesized compounds showed better cytotoxicity towards A‐549 and Hep‐G2 than MCF‐7. Among them, (2E)‐2‐{[2‐(4‐chlorophenyl)‐1H‐indol‐3‐yl]methylidene}‐N‐(4‐methoxyphenyl)hydrazinecarbothioamide ( 8l ) was found to be the most potent compound against A‐549 and Hep‐G2, at least three times more potent than etoposide. The morphological analysis by the acridine orange/ethidium bromide double staining test and flow cytometry analysis indicated that compound 8l induced apoptosis in A‐549 cells. Moreover, molecular docking methodology was exploited to elucidate the details of molecular interactions of the studied compounds with putative targets.