Photonic Hydrogel Beads for Controlled Release of Risedronate

pH-sensitive photonic composite hydrogel beads composed of sodium alginate and risedronate sodium (SA/RIS) was prepared crosslinked by Ca2+ owing to the ionic gelation of SA. The structure and surface morphology of the composite hydrogel beads were characterized by SEM. pH-sensitivity of these composite hydrogels beads and the release behaviors of drug from them were investigated. The results showed that the composite hydrogel beads had good pH-sensitivity. The drug loading and encapsulation efficiency were 27.7% and 92% for RIS, respectively. The cumulative release ratios of RIS from the composite hydrogel beads were 2.47% in pH 2.1 solution and 83 % in pH 6.8 solutions within 24 h, respectively. However, the cumulative release ratio of RIS in pH 7.4 solution reached 91% within 7 h. It is proposed that the novel photonic SA/RIS composite hydrogel bead could possess the potential of an increased intestinal absorption and fewer adverse effects of RIS. The pH and salt response of photonic hydrogel bead, as well as the encapsulation of macromolecules, are promising for applications in biomedicine and biotechnology.

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro-to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine. (C) 2014 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.

Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite, Plasmodium vivax, and characterization of its properties

Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni2+-NTA resin giving a yield of 25-30 mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4 x 10(8) min(-1) M-1, 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors.

Matrix-Assisted Refolding, Purification and Activity Assessment Using a `Form Invariant' Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed `form invariant' assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.

Humanized antibody neutralizing 2009 pandemic H1N1 virus

The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of influenza virus neutralizing monoclonal antibodies (MAbs) for treatment purposes is a viable alternative. We previously reported the isolation of a high affinity, potently neutralizing murine MAb MA2077 against 2009 pandemic H1N1 virus. We describe here the humanization of MA2077 and its expression in a mammalian cell line. Six complementarity-determining regions (CDRs) of MA2077 were grafted onto the human germline variable regions; along with six and eight back mutations in the framework of heavy and light chains, respectively, pertaining to the vernier zone and interchain packing residues to promote favorable CDR conformation and facilitate antigen binding. The full length humanized antibody, 2077Hu2, expressed in CHO-K1 cells, showed high affinity to hemagglutinin protein (K-D = 0.75 +/- 0.32 nM) and potent neutralization of pandemic H1N1 virus (IC50 = 0.17 mu g/mL), with marginally higher IC50 as compared to MA2077 (0.08 mu g/mL). In addition, 2077Hu2 also retained the epitope specificity for the ``Sa'' antigenic site on pandemic HA. To the best of our knowledge, this is the first report of a humanized neutralizing antibody against pandemic H1N1 virus.

Retinoic Acid and Iron Metabolism: A Step Towards Design of a Novel Antitubercular Drug

The scenario of tuberculosis has gone deadly due to its high prevalence and emergence of widespread drug resistance. It is now high time to develop novel antimycobacterial strategies and to understand novel mechanisms of existing antimycobacterial compounds so that we are equipped with newer tuberculosis controlling molecules in the days to come. Iron has proven to be essential for pathogenesis of tuberculosis and retinoic acid is known to influence the iron metabolism pathway. Retenoic acid is also known to exhibit antitubercular effect in in vivo system. Therefore there is every possibility that retinoic acid by affecting the iron metabolism pathway exhibits its antimycobacterial effect. These aspects are reviewed in the present manuscript for understanding the antimycobacterial role of retinoic acid in the context of iron metabolism and other immunological aspects.

Expression of Bioactive Callithrix jacchus Follicle-Stimulating Hormone in Pichia pastoris

Callithrix jacchus (common marmoset) is a New World primate monkey, used as an animal model in biomedical research. Marmoset-specific follicle-stimulating hormone (FSH) preparation is required to improve superovulation protocols and to develop homologous FSH monitoring assays in these monkeys. In this study, we document the large-scale expression of recombinant marmoset FSH in methylotropic yeast, Pichia pastoris. The recombinant preparation was found to be immunologically active in Western blotting and radioimmunoassay. The preparation displayed receptor binding ability in radioreceptor assay. Based on the receptor binding ability, the yield of fermentation was estimated to be 7.2 mg/L. FSH-induced cAMP assay and estradiol assay revealed that the recombinant hormone is able to induce signal transduction. Both immunological and in vitro biological activity of marmoset FSH was found to be comparable to purified human pituitary FSH, which served as reference hormone for these assays. Thus, the study suggests that a Pichia expression system can be used for large-scale expression of bioactive recombinant marmoset FSH.

The influence of reduced oxygen availability on gene expression in laboratory (H37Rv) and clinical strains (S7 and S10) of Mycobacterium tuberculosis

Mycobacterium tuberculosis has the ability to persist within the host in a dormant stage. One important condition believed to contribute to dormancy is reduced access to oxygen known as hypoxia. However, the response of M. tuberculosis to such hypoxia condition is not fully characterized. Virtually all dormant models against tuberculosis tested in animals used laboratory strain H37Rv or Erdman strain. But major outbreaks of tuberculosis (TB) occur with the strains that have widely different genotypes and phenotypes compared to H37Rv. In this study, we used a custom oligonucleotide microarray to determine the overall transcriptional response of laboratory strain (H37Rv) and most prevalent clinical strains (S7 and S10) of M. tuberculosis from South India to hypoxia. Analysis of microarray results revealed that a total of 1161 genes were differentially regulated (>= 1.5 fold change) in H37Rv, among them 659 genes upregulated and 502 genes down regulated. Microarray data of clinical isolates showed that a total of 790 genes were differentially regulated in S7 among which 453 genes were upregulated and 337 down regulated. Interestingly, numerous genes were also differentially regulated in S10 (total 2805 genes) of which 1463 genes upregulated and 1342 genes down regulated during reduced oxygen condition (Wayne's model). One hundred and thirty-four genes were found common and upregulated among all three strains (H37Rv, S7, and S10) and can be targeted for drug/vaccine development against TB. (C) 2015 Published by Elsevier B.V.