Molecularly imprinted cryogels for human interferon‐alpha purification from human gingival fibroblast culture

Interferons are important proteins for the immune system because of their antiviral, anti‐proliferating and immunomodulatory activities. Therapeutic value of these proteins against certain types of tumors caused interest and investigations aimed to obtain highly purified interferons. Molecular imprinting is an efficient method for purification with high selectivity, specificity and good reproducibility. In this study, we utilized advantages of molecular imprinting technique for the purification of interferon from human gingival fibroblast culture. For this purpose, interferon α‐2b imprinted poly(hydroxyethyl methacrylate) cryogel (hIFN‐α‐MIP) was prepared. Optimum adsorption conditions were determined, and maximum adsorption capacity of hIFN‐α‐MIP cryogel was found as 254.8 × 10⁴ IU/g from aqueous solution. All interferon measurements are expressed as International Unit (IU), which is a unit measurement used to quantify biologically active substances like interferon based on their biological activity or effect. Selectivity experiments were performed using competitive proteins and repeated adsorption–desorption studies showed that the adsorption capacity maintained almost at a constant value after ten cycles. For the purification of interferon from human gingival fibroblast culture, fast protein liquid chromatography was used and the specific activity of the purified interferon α‐2b on HeLa cell line was found between the values 3.45 × 10⁸ IU/mg and 3.75 × 10⁸ IU/mg. The results are promising, and the molecular imprinting technique is effective for the purification of interferon α‐2b. Copyright © 2013 John Wiley & Sons, Ltd.     

Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice

Bioluminescence recording of Ca(2+) signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca(2+) in various cell compartments. In addition, they would also serve to monitor Ca(2+) in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R(0)) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca(2+) oscillations in single HeLa cells expressing tdTA. Ca(2+) rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca(2+) activity of HeLa cells injected subcutaneously into mice, and Ca(2+) signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca(2+) sensor reported to date and is, therefore, a promising probe to study Ca(2+) dynamics in whole organisms or tissues expressing the transgene.     

Association of IFN-gamma and IL-10 gene variants with the risk of extrapulmonary tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is a chronic infectious disease. Interferon-gamma (IFN-γ) is an important cytokine imparting resistance to mycobacterial diseases. It is believed that IFN-γ and Interleukin-10 (IL-10) play divergent roles in the host immune system against MTB infection. IL-10 is an important inhibitory cytokine and helps balancing the inflammatory and immune responses. IL-10 is involved in down regulation of Th1 cytokines, MHC class II antigen and co-stimulatory molecular expression on macrophages, while IFN-γ results in macrophage activation allowing them to exert the microbicidal role. The objectives were to find out the association of IL-10 (?1082 A/G) and IFN-γ (+874 A/T) single nucleotide polymorphisms (SNPs) with extrapulmonary tuberculosis in ethnic Kashmiri population. A total of 100 extrapulmonary tuberculosis cases and 102 healthy controls were analyzed for IL-10 (?1082 A/G) and IFN- γ (+874 A/T) SNPs using Allele-Specific PCR. We found a significant association of IFN-γ + 874 ‘TT’ genotype with extrapulmonary tuberculosis (p = 0.006) and in case of IL-10 (?1082 A/G) we found a significant association with extrapulmonary tuberculosis under recessive model (GG vs GA + AA) (p = 0.03) in Kashmiri population. IL-10 (?1082 A/G) and IFN-γ (+874 A/T) have a significant association with extrapulmonary tuberculosis in ethnic Kashmiri population.     

Comparison of Univariate and Multivariate Models of 13C SSNMR and XRPD Techniques for Quantification of Nimodipine Polymorphs

The focus of the present investigation was to explore the use of solid-state nuclear magnetic resonance (¹³C ssNMR) and X-ray powder diffraction (XRPD) for quantification of nimodipine polymorphs (form I and form II) crystallized in a cosolvent formulation. The cosolvent formulation composed of polyethylene glycol 400, glycerin, water, and 2.5% drug, and was stored at 5°C for the drug crystallization. The ¹³C ssNMR and XRPD data of the sample matrices containing varying percentages of nimodipine form I and form II were collected. Univariate and multivariate models were developed using the data. Least square method was used for the univariate model generation. Partial least square and principle component regressions were used for the multivariate models development. The univariate models of the ¹³C ssNMR were better than the XRPD as indicated by statistical parameters such as correlation coefficient, R², root mean square error, and standard error. On the other hand, the XRPD multivariate models were better than the ¹³C ssNMR as indicated by precision and accuracy parameters. Similar values were predicted by the univariate and multivariate models for independent samples. In conclusion, the univariate and multivariate models of ¹³C ssNMR and XRPD can be used to quantitate nimodipine polymorphs.KEY WORDS: nimodipine polymorphs, X-ray powder diffraction, solid-state nuclear magnetic resonance, univariate, multivariate     

A neuroglobin-overexpressing transgenic mouse

Neuroglobin (Ngb) is a recently discovered vertebrate globin expressed primarily in neurons. Ngb expression is induced by hypoxia and ischemia, and Ngb protects neurons from these insults. However, its normal physiological role and the mechanism underlying its neuroprotective action are uncertain. We report production of a transgenic mouse in which Ngb is overexpressed under the control of the chicken beta-actin promoter. This mouse should prove helpful for studying Ngb-mediated effects in vitro and in vivo.     

Vitamin D levels in asymptomatic adults--a population survey in Karachi, Pakistan

Background

It is well established that low levels of 25(OH) Vitamin D (<30 ng/dL) are a common finding world over, affecting over a billion of the global population. Our primary objective was to determine the prevalence of vitamin D deficiency and insufficiency in the asymptomatic adult population of Karachi, Pakistan and the demographic, nutritional and co-morbidity characteristics associated with serum vitamin D levels.

Methods

A cross-sectional population survey was conducted at two spaced out densely populated areas of the city. Serum levels of 25OHVitamin D were measured and GFR as renal function was assessed by using 4 variable MDRD formula.

Results

Our sample of 300 had a median age of 48(interquartile range 38–55) years. The median level of serum vitamin D was 18.8 (IQ range 12.65–24.62) ng/dL. A total of 253 (84.3%) respondents had low levels (<30 ng/dL) of 25OH vitamin D. Serum PTH and vitamin D were negatively correlated (r = −0.176, p = 0.001). The median PTH in the vitamin D sufficiency group was 38.4(IQ range28.0–48.8)pg/mL compared with 44.4(IQ range34.3–56.8) pg/mL in the deficiency group (p = 0.011).The median serum calcium level in the sample was 9.46(IQ range 9.18–9.68) ng/dL. Low serum levels of vitamin D were not associated with hypertension (p = 0.771) or with an elevated spot blood pressure (p = 0.164).In our sample 75(26%) respondents had an eGFR corresponding to stage 2 and stage 3 CKD. There was no significant correlation between levels of vitamin D and eGFR (r = −0.127, p-value = 0.277).Respondents using daily vitamin D supplements had higher 25 OH vitamin D levels (p-value = 0.021).

Conclusion

We observed a high proportion of the asymptomatic adult population having low levels of vitamin D and subclinical deterioration of eGFR. The specific cause(s) for this observed high prevalence of low 25OH vitamin D levels are not clear and need to be investigated further upon.     

Changes in soil associated with continuous growth of some vegetation

Summary and conclusions From a study of the composition of the soil and the subsoil under three grasses,Imperata cylindrica, Pennisetum orientale, Pennisetum polystachyum and three legumesTephrosia candida, Medicago sativa andPueraria hirsuta and of those of natural bare soil in the neighbourhood of each, it could be observed that the soils under vegetation contained more moisture, organic matter, organic nitrogen clay and soluble salts but had lower pH values than the bare soils. The soils under grasses had less moisture, lower pH and lower salinity but higher clay content and exhibited greater aggregation than the soils under legumes. Though the soils under grasses had significantly higher quantities of organic matter than the soils under legumes there was no significant difference in the organic nitrogen contents between them.     

Immobilization of catalase via adsorption onto -histidine grafted functional pHEMA based membrane

Poly(2-hydroxyethylmethacrylate) (pHEMA) based flat sheet membrane was prepared by UV-initiated photopolymerization technique. The membrane was then grafted with -histidine. Catalase immobilization onto the membrane from aqueous solutions containing different amounts of catalase at different pH was investigated in a batch system. The maximum catalase immobilization capacity of the pHEMA–histidine membrane was 86 μg cm⁻². The activity yield was decreased with the increase of the enzyme loading. It was observed that there was a significant change between V_max value of the free catalase and V_max value of the adsorbed catalase on the pHEMA–histidine membrane. The K_m value of the immobilized enzyme was higher 1.5 times than that of the free enzyme. Optimum operational temperature was 5°C higher than that of the free enzyme and was significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed without loss of adsorption capacity or enzyme activity.