Histopathological correlations of bulk tissue polarimetric images: Case study

Polarimetric imaging and image analysis have gained increased interest in soft tissue analysis at the cellular level. However, polarimetric imaging has widely been tested on thin tissue sections to provide reliable information correlated with histopathological findings. Polarimetric bulk tissue analysis always offered an overall assessment of various tissue optical properties for diagnosis. In this study, the histopathological correlation of bulk tissue polarimetry images for soft tissues is discussed. The first-hand information on the use of bulk tissue Mueller polarimetry and image analysis as an alternative to tissue histopathology is presented for surgically extracted colon and breast tissues.     

High-performance liquid chromatographic determination of 4,4′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone in plasma

An analytical method has been developed for the determination of 4,4′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone (I, trade name A-007) in plasma. Plasma samples are primed with the internal standard, 2,2′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone (II), deproteinized with acetonitrile, centrifuged and filtered prior to assay. The components are then separated on a reversed-phase column with retention times of 4.4 and 6.0 min for I and II, respectively. Ultraviolet detection at 365 nm was employed and little interference with the analyte or the internal standard was noted from other plasma components. This method has been applied to the plasma of rats and monkeys dosed for pharmacokinetic and toxicity studies.     

Isolation of riboflavin carrier proteins from pregnant human and umbilical cord serum: Similarities with chicken egg riboflavin carrier protein

A riboflavin carrier protein has been purified from human pregnancy and umbilical cord sera by affinity chromatography and fast protein liquid chromatography. This protein has a similar molecular weight to the chicken egg riboflavin carrier protein and shares other physicochemical properties, such as pI and riboflavin binding characteristics, with the avian counterpart. A high degree of immunological cross-reactivity is observed between the human and avian riboflavin binding proteins indicating the extensive conservation of this protein throughout evolution.     

Interaction of fishes with pathogenic micro-organisms and application of phages for their control: a review

The present condition of aquacultural industry is influenced by the economical losses due to various aquacultural diseases causing fish mortality. The present review provides benchmark information related to application of bacteriophages in aquacultural industries over available traditional treatment procedures like antibiotics and chemotherapy. The traditional methods are mostly less advantageous due to development of resistance, non-specific targeting of bacteria including intestinal microflora, etc. In short here we discuss the interaction between fish, bacteria and their phages in order to have an alternate treatment method for the pathogens responsible for aquacultural diseases.     

Biochemical characterization and novel inhibitor identification of Mycobacterium tuberculosis Endonuclease VIII 2 (Rv3297)

Nei2 (Rv3297) is a DNA Base Excision Repair (BER) glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5’/3’ fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8 μM-92.7 μM against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR) experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74 nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER.     

Sardine oil loaded vanillic acid grafted chitosan microparticles,a new functional food ingredient: attenuates myocardial oxidative stress and apoptosis in cardiomyoblast cell lines (H9c2)

Fish oil has been widely recognized as an excellent dietary source of polyunsaturated n-3 fatty acids such as EPA and DHA. However, it can undergo oxidation easily resulting in the formation of toxic off flavor compounds such as hydroperoxides. These compounds adversely affect the nutritional quality and may induce several stress reactions in body. To solve this problem, a new antioxidant bio-material, vanillic acid-grafted chitosan (Va-g-Ch), was synthesized and used as a wall material for microencapsulation of fish oil. The sardine oil loaded Va-g-Ch microparticles could be a potential functional food ingredient considering the numerous health benefits of fish oil, chitosan, and vanillic acid. The current study aimed to investigate the possible protective effect of sardine oil-loaded Va-g-Ch microparticles against doxorubicin-induced cardiotoxicity and the underlying mechanisms. In vitro cytotoxicity evaluation was conducted using H9c2 cardiomyocytes. MTT assay revealed that effective cytoprotective effect was induced by a sample concentration of 12.5 μg/mL. Results of apoptosis by double fluorescent staining with acridine orange/ethidium bromide and caspase-3 evaluation by ELISA substantiated the above findings. Further, flow cytometric determination of membrane potential, relative expression of NF-κB by PCR, and ROS determination using DCFH-DA also confirmed the protective effect of encapsulated sardine oil against doxorubicin-induced cardiotoxicity. NF-κB expression was down-regulated nearly by 50% on cells treated with encapsulated sardine oil. Altogether, the results revealed that sardine oil-loaded Va-g-Ch microparticles demonstrated potential cell protection against doxorubicin-induced oxidative stress     

A Stoichiometry Driven Universal Spatial Organization of Backbones of Folded Proteins: Are there Chargaff's Rules for Protein Folding?

Abstract

Protein folding is at least a six decade old problem, since the times of Pauling and Anfinsen. However, rules of protein folding remain elusive till date. In this work, rigorous analyses of several thousand crystal structures of folded proteins reveal a surprisingly simple unifying principle of backbone organization in protein folding. We find that protein folding is a direct consequence of a narrow band of stoichiometric occurrences of amino-acids in primary sequences, regardless of the size and the fold of a protein. We observe that “preferential interactions” between amino-acids do not drive protein folding, contrary to all prevalent views. We dedicate our discovery to the seminal contribution of Chargaff which was one of the major keys to elucidation of the stoichiometry-driven spatially organized double helical structure of DNA.     

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs ¹ (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver ².SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface ³. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs ⁴. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs ^5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms ⁷.With the exception of one report ⁸, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor ⁹ , CD31, and the presence of cytoplasmic fenestration ¹. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium ^10,11.In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.