Fine needle aspiration biopsy of the spleen in pyrexia of unknown origin

To evaluate the diagnostic utility, value and potential risk of fine needle aspiration biopsy of spleen (sFNAB) in patients with splenomegaly in pyrexia of unknown origin (PUO), a retrospective analysis of medical records and cytological material of 31 patients on whom FNAB was performed between April 1994 and October 1997 was done. The patients were HIV- and presented with PUO. All other relevant investigations were negative. The spleen was either palpable or detected to have space-occupying lesions on ultrasonography (USG). The splenic aspirates showed tuberculosis in 11 patients (35.4%) and inconclusive or reactive changes in nine patients (25.8%). One case out of this group proved to be Kaposi's sarcoma on autopsy. The other diseases encountered were leishmaniasis (n = 3), non-Hodgkin's lymphoma (n = 4), fungal infections (n = 2), Hodgkin's lymphoma (n = 1). The patients who were diagnosed as having tuberculosis had epithelioid cells, giant cells, necrosis and inflammatory cells in various combinations. AFB positivity was 63.6%. The other cases which showed granulomas but no AFB were diagnosed on empirical grounds and all responded to the anti-tuberculosis therapy. No complications were encountered with the procedure. Therefore the authors conclude that sFNAB is rewarding in patients where all other non-invasive modalities of diagnosis have failed.     

Quantitative determination of butorphanol and its metabolites in human plasma by gas chromatography-electron capture negative-ion chemical ionization mass spectrometry

Two separate analytical methods have been developed for the determination of butorphanol and its metabolites in human plasma. One method is specific for butorphanol (I) while the other determines the metabolites, hydroxybutorphanol (II) and norbutorphanol (III). Both procedures incorporate solid-phase extraction, chemical derivatization and separation, and detection using gas chromatography-electron-capture negative-ion chemical ionization mass spectrometry (GC-ECNCI-MS). Both methods use the cyclopropyl analog of I (BC-2605, IV) as the internal standard and the procedures for extraction of the analytes from plasma are identical. However, following extraction, either the pentafluorobenzoyl ester of I or the tris- and bis-trifluoroacetyl esters of II and III, respectively, were prepared. The derivatives were analyzed by GC-ECNCI-MS with selected-ion monitoring of the molecular ions. The standard curves were linear over the concentration ranges of 20–2000, 20–1000 and 50–1000 pg/ml for I, II and III, respectively. All standard curves from the assay validation had r² values of ≥0.994, 0.991 and 0.985 for I, II and III, respectively. For all three compounds, the intra- and inter-assay precisions (CV) and inter-assay accuracy (deviation from nominal) were within 12% for plasma quality control samples. All derivatives were stable in the reconstitution solvent for at least 24 h. The assays are being used for the determination of plasma concentrations of I, II and III in humans following repeated administration of nasal spray.     

Microbial xylanases and their industrial applications: a review

Despite an increased knowledge of microbial xylanolytic systems in the past few years, further studies are required to achieve a complete understanding of the mechanism of xylan degradation by microorganisms and their enzymes. The enzyme system used by microbes for the metabolism of xylan is the most important tool for investigating the use of the second most abundant polysaccharide (xylan) in nature. Recent studies on microbial xylanolytic systems have generally focussed on induction of enzyme production under different conditions, purification, characterization, molecular cloning and expression, and use of enzyme predominantly for pulp bleaching. Rationale approaches to achieve these goals require a detailed knowledge of the regulatory mechanism governing enzyme production. This review will focus on complex xylan structure and the microbial enzyme complex involved in its complete breakdown, studies on xylanase regulation and production and their potential industrial applications, with special reference to biobleaching.     

Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture

Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post‐translational insertion of a sulfur atom into disulfide bonds at the heavy–heavy and heavy–light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run‐to‐run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable. To investigate variables that might impact trisulfide formation, cell culture parameters were varied in bench‐scale bioreactor studies. Trisulfide analysis of the samples from these runs revealed that the trisulfide content in the bond between heavy and light chains varied considerably from <1% to 39%. Optimizing the culture duration and feeding strategy resulted in more consistent trisulfide levels. Cysteine concentration in the feed medium had a direct correlation with the trisulfide level in the product. Systematic studies revealed that cysteine in the feed and the bioreactor media was contributing hydrogen sulfide which reacted with the IgG1 mAb in the supernatant leading to the insertion of sulfur atom and formation of a trisulfide bond. Cysteine feed strategies were developed to control the trisulfide modification in the recombinant monoclonal antibody. Biotechnol. Bioeng. 2012; 109: 2523–2532. © 2012 Wiley Periodicals, Inc.     

Effects of arbuscular mycorrhiza and phosphorus application on artemisinin concentration in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Annual wormwood (Artemisia annua L.) produces an array of complex terpenoids including artemisinin, a compound of current interest in the treatment of drug-resistant malaria. However, this promising antimalarial compound remains expensive and is hardly available on the global scale. Synthesis of artemisinin has not been proved to be feasible commercially. Therefore, increase in yield of naturally occurring artemisinin is an important area of investigation. The effects of inoculation by two arbuscular mycorrhizal (AM) fungi, Glomus macrocarpum and Glomus fasciculatum, either alone or supplemented with P-fertilizer, on artemisinin concentration in A. annua were studied. The concentration of artemisinin was determined by reverse-phase high-performance liquid chromatography with UV detection. The two fungi significantly increased concentration of artemisinin in the herb. Although there was significant increase in concentration of artemisinin in nonmycorrhizal P-fertilized plants as compared to control, the extent of the increase was less compared to mycorrhizal plants grown with or without P-fertilization. This suggests that the increase in artemisinin concentration may not be entirely attributed to enhanced P-nutrition and improved growth. A strong positive linear correlation was observed between glandular trichome density on leaves and artemisinin concentration. Mycorrhizal plants possessed higher foliar glandular trichome (site for artemisinin biosynthesis and sequestration) density compared to nonmycorrhizal plants. Glandular trichome density was not influenced by P-fertilizer application. The study suggests a potential role of AM fungi in improving the concentration of artemisinin in A. annua.     

Higher expression of ferritin protects Chlamydia trachomatis infected HeLa 229 cells from reactive oxygen species mediated cell death

Apoptosis plays an important role in modulating the pathogenesis of a variety of infectious diseases. Chlamydial infection protects cells against different forms of apoptosis: extrinsic, intrinsic, and granzyme B mediated. Redox reactions are central to the life and death decision of cells and pathogens and an intimate relationship exists between oxidative stress and iron metabolism. The link between redox status and ferritin was largely unexplored in chlamydia-infected cells. In the present study, we showed that Chlamydia trachomatis (CT) infection induced FHC protein in HeLa cells. FHC induction by CT-infected cells stably expressing FHC blunted ROS production compared with mock infected cells, and the infected cells were relatively resistant to apoptosis induced by H?O?. We also demonstrated that endogenous FHC overexpression correlates well with the stabilization of the mitochondrial membrane potential in CT-infected cells. Increased expression of FHC is independent of iron supplementation (FAC) and depletion (DFO) in CT-infected cells. These data suggest that FHC up-regulation is an acute response of HeLa cells against CT infection and that FHC exerts anti-apoptotic activity against oxidative stress.     

Detoxification of sugarcane bagasse hydrolysate improves ethanol production by Candida shehatae NCIM 3501

Sugarcane bagasse hydrolysis with 2.5% (v/v) HCl yielded 30.29g/L total reducing sugars along with various fermentation inhibitors such as furans, phenolics and acetic acid. The acid hydrolysate when treated with anion exchange resin brought about maximum reduction in furans (63.4%) and total phenolics (75.8%). Treatment of hydrolysate with activated charcoal caused 38.7% and 57.5% reduction in furans and total phenolics, respectively. Laccase reduced total phenolics (77.5%) without affecting furans and acetic acid content in the hydrolysate. Fermentation of these hydrolysates with Candida shehatae NCIM 3501 showed maximum ethanol yield (0.48g/g) from ion exchange treated hydrolysate, followed by activated charcoal (0.42g/g), laccase (0.37g/g), overliming (0.30g/g) and neutralized hydrolysate (0.22g/g).     

Characterization of Ku70(2)-NLS as bipartite nuclear localization sequence for non-viral gene delivery

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku70(2)-NLS), a nuclear transport active mutant of Ku70(2)-NLS (s1Ku70(2)-NLS) and a nuclear transport deficient mutant of Ku70(2)-NLS (s2Ku70(2)). We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.     

Rhizosphere: its structure,bacterial diversity and significance

Sustainable agricultural practices are the answer to multifaceted problems that have resulted due to prolonged and indiscriminate use of chemical based agronomic tools to improve crop productions for the last many decades. The hunt for suitable ecofriendly options to replace the chemical fertilizers and pesticides has thus been aggravated. Owing to their versatile and unmatchable capacities microbial agents offer an attractive and feasible option to develop the biological tools to replace/supplement the chemicals. Exploring the microorganisms that reside in close proximity to the plant is thus a justified move in the direction to achieve this target. One of the most lucrative options is to look into the rhizosphere. Rhizosphere may be defined as the narrow zone of soil that surrounds and get influenced by the roots of the plants. It is rich in nutrients compared to the bulk soil and hence exhibit intense biological and chemical activities. A wide range of macro and microorganisms including bacteria, fungi, virus, protozoa, algae, nematodes and microarthropods co-exist in rhizosphere and show a variety of interactions between themselves as well as with the plant. Plant friendly bacteria residing in rhizosphere which exert beneficial affect on it are called as plant growth promoting rhizobacteria (PGPR). Here we review the structure and bacterial diversity of the rhizosphere. The major points discussed here are: (1) structure and composition of the rhizosphere (2) range of bacteria found in rhizosphere and their interactions with the plant with a particular emphasis on PGPR (3) mechanisms of plant growth promotion by the PGPR (4) rhizosphere competence.     

Evidence for increased de novo synthesis of NAD in immune-activated RAW264.7 macrophages: a self-protective mechanism?

Chondrogenesis in cartilage development and repair and cartilage degeneration in arthritis can be regulated by mechanical-load-induced physical factors such as tissue deformation, interstitial fluid flow and pressure, and electrical fields or streaming potentials. Previous animal and tissue explant studies have shown that time-varying dynamic tissue loading can increase the synthesis and deposition of matrix molecules in an amplitude-, frequency-, and spatially dependent manner. To provide information on the cell-level physical factors which may stimulate chondrocytes to increase production and export of aggrecan, the main proteoglycan component of the cartilage matrix, we characterized local changes in aggrecan synthesis within cyclically loaded tissue explant disks and compared those changes to values of predicted local physical factors. Aggrecan synthesis following a 23-h compression/radiolabel protocol was measured with a spatial resolution of approximately 0.1 mm across the 1.5-mm radius of explanted disks using a quantitative autoradiography method. A uniform stimulation of aggrecan synthesis was observed at an intermediate frequency of 0.01 Hz, while, at a higher frequency of 0.1 Hz, stimulation was only seen at peripheral radial positions. Profiles of radial solid matrix deformation and interstitial fluid pressure and velocity predicted to be occurring across the radius of the disk during sinusoidal loading were estimated using a composite poroelastic model. Tissue regions experiencing high interstitial fluid velocities corresponded to those displaying increased aggrecan synthesis. These results reinforce the role of load-induced flow of interstitial fluid in the stimulation of aggrecan production during dynamic loading of cartilage.