Contribution of T cell subsets to the pathophysiology of Pneumocystis-related immunorestitution disease

Immune-mediated lung injury is an important component of Pneumocystis pneumonia (PcP)-related immunorestitution disease (IRD). However, the individual contribution of CD4(+) and CD8(+) T cells to the pathophysiology of IRD remains undetermined. Therefore, IRD was modeled in severe combined immunodeficient mice, and specific T cell depletion was used to determine how T cell subsets interact to affect the nature and severity of disease. CD4(+) cells were more abundant than CD8(+) cells during the acute stage of IRD that coincided with impaired pulmonary physiology and organism clearance. Conversely, CD8(+) cells were more abundant during the resolution phase following P. carinii clearance. Depletion of CD4(+) T cells protected mice from the acute pathophysiology of IRD. However, these mice could not clear the infection and developed severe PcP at later time points when a pathological CD8(+) T cell response was observed. In contrast, mice depleted of CD8(+) T cells efficiently cleared the infection but developed more severe disease, an increased frequency of IFN-gamma-producing CD4(+) cells, and a prolonged CD4(+) T cell response than mice with both CD4(+) and CD8(+) cells. These data suggest that CD4(+) T cells mediate the acute respiratory disease associated with IRD. In contrast, CD8(+) T cells contributed to neither lung injury nor organism clearance when CD4(+) cells were present, but instead served to modulate CD4 function. In the absence of CD4(+) cells, CD8(+) T cells produced a nonprotective, pathological immune response. These data suggest that the interplay of CD4(+) and CD8(+) T cells affects the ultimate outcome of PcP-related IRD.     

Kinetic modeling of a bi-enzymatic system for efficient conversion of lactose to lactobionic acid

A model has been developed to describe the interaction between two enzymes and an intermediary redox mediator. In this bi-enzymatic process, the enzyme cellobiose dehydrogenase oxidizes lactose at the C-1 position of the reducing sugar moiety to lactobionolactone, which spontaneously hydrolyzes to lactobionic acid. 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt is used as electron acceptor and is continuously regenerated by laccase. Oxygen is the terminal electron acceptor and is fully reduced to water by laccase, a copper-containing oxidase. Oxygen is added to the system by means of bubble-free oxygenation. Using the model, the productivity of the process is investigated by simultaneous solution of the rate equations for varying enzyme quantities and redox mediator concentrations, solved with the aid of a numerical solution. The isocharts developed in this work provide an easy-to-use graphical tool to determine optimal process conditions. The model allows the optimization of the employed activities of the two enzymes and the redox mediator concentration for a given overall oxygen mass transfer coefficient by using the isocharts. Model predictions are well in agreement with the experimental data.     

Smoothened antagonists for hair inhibition

A series of aminomethylpyrazoles were prepared and evaluated using cell-based Smoothened β-lactamase reporter assay and Smoothened binding assay. Potent Smoothened antagonists 10k and 10l were found to inhibit hair growth in vivo in the C3H/HeN mouse hair growth model. The more selective compound 10l was tested negative in the 3T3 NRU assay, indicating a low risk for causing photo-irritation and was efficacious using the C3H/HeN mouse hair growth model although it was slightly less efficacious than that of the reference compound eflornithine (7).     

Synthesis and bio-evaluation of alkylaminoaryl phenyl cyclopropyl methanones as antitubercular and antimalarial agents

A series of 4-alkylaminoaryl phenyl cyclopropyl methanones (6a–6u and 8a–8c) were synthesized from 4-fluorochalcones (3a and 3b) by cyclopropanation of double bond followed by nucleophilic substitution of F with different amines. The compounds were screened for their antitubercular and antimalarial activities against Mycobacterium tuberculosis H37Rv and Plasmodium falciparum 3D7 strains in vitro respectively. Several compounds (6a, 6d–6h, 6p, 6q and 8a–8c) exhibited good in vitro antitubercular activities with MIC values 3.12–12.5 μg/mL and preferentially inhibited the growth of P. falciparum in vitro (4a, 4c, 6a–6d, 6f, 6s, 8a and 8c) with IC₅₀ as low as 0.080 and 0.035 μg/mL and SI values 4975 and 6948, respectively. Molecular docking studies and in vitro evaluation against FAS-II enzymes using reporter gene assays were carried out to elucidate the mode of action of these molecules. Two compounds 4a and 6g showed significant inhibition at 25 μM concentration of the compound.     

Base Induced Condensation of Malononitrile with Erlenmeyer Azlactones: An Unexpected Synthesis of Multi‐Substituted Δ2‐Pyrrolines and Their Cytotoxicity

An efficient, metal free approach to synthesize multi‐substituted Δ²‐pyrroline derivatives by mild base catalyzed cyclocondensation of malononitrile with Erlenmeyer azlactones via 1,2 addition was developed. The modularity of this reaction was used to assemble a range of poly‐substituted pyrrolines. Further, synthesized products were screened for cytotoxic properties on different cancer cell lines such as A549 (Human lung adenocarcinoma cells), HeLa (Human cervical adenocarcinoma cells), Jurkat (Human chronic myeloid leukemia cells) and K562 (Human leukemic T cell Lymphoblast cells). Among the synthesized library of compounds, 6f and 6q displayed potent cytotoxic activity.     

An in vivo Transient Expression System Can Be Applied for Rapid and Effective Selection of Artificial MicroRNA Constructs for Plant Stable Genetic Transformation

The utility of artificial microRNAs (amiRNAs) to induce loss of gene function has been reported for many plant species, but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable. In this study, expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation. A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments. Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs. This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.     

Antibacterial activity of herbal extracts against five plant pathogenic bacteria

The present study was designed to evaluate in vitro antibacterial activity of herbal extracts against five plant pathogenic bacteria (viz. Xanthomonas campestris, Xanthomonas axonopodis pv. punicae, Erwinia spp., Pseudomonas syringae and Xanthomonas citri). Herbal extracts of leaves and rinds of Garcinia indica, rhizomes of Curcuma aromatica, roots of Glyccyrrhiza glabra, leaves of Nyctanthes arbor-tristis and seeds of Vernonia anthelmintica were used for screening. Screening was done using agar well diffusion method. Relatively potent extracts were shortlisted from this study and were further studied to find out their minimum bactericidal concentration (MBC). From the studies, it was observed that extracts of C. aromatica, G. indica and G. glabra have shown lowest MBC values among other tested plant extracts. This study indicates the potential of these potent plant extracts in the management of diseases caused by plant pathogenic bacteria.     

Isolation and characterization of a novel <Emphasis Type="Italic">α</Emphasis>-amylase from a metagenomic library of Western Ghats of Kerala,India

In the present study, metagenomic library of Western Ghats soil sample was constructed in a fosmid vector (pCC1FOS) and screened for biocatalytic properties. The clones showed amylolytic activity on Luria-Bertani starch agar plates and one of them was studied in detail. The enzyme exhibited stability at elevated temperature with 60°C being the optimal temperature. The enzyme retained more than 30% activity after 60 min incubation at 80°C. It also showed more than 70% activity retention in 1.5 M NaCl solution. The pH optimum of the enzyme was at pH = 5.0. The enzyme possesses good activity in the presence of chelating and strong reducing agents with activity enhancements or retention being observed at 5 mM β-mercaptoethanol, dithiothreitol and N-bromosuccinimide. However, almost complete loss of activity was observed with 5 mM EDTA, while activity enhancement was observed upon incubation with Ca²⁺ suggesting it to be a Ca²⁺-dependent α-amylase, which was further confirmed by a thin-layer chromatography (TLC). The TLC run revealed that digestion pattern was similar to commercial α-amylase. The 16S rRNA gene sequence (GenBank accession number HQ680979) BLAST showed 95% similarities with Exiguobacterium sp. AFB-11 and AFB 18, with query sequence coverage of 99%.