Development of Spray Dried Liposomal Dry Powder Inhaler of Dapsone

This investigation was undertaken to evaluate practical feasibility of site specific pulmonary delivery of liposomal encapsulated Dapsone (DS) dry powder inhaler for prolonged drug retention in lungs as an effective alternative in prevention of Pneumocystis carinii pneumonia (PCP) associated with immunocompromised patients. DS encapsulated liposomes were prepared by thin film evaporation technique and resultant liposomal dispersion was passed through high pressure homogenizer. DS nano-liposomes (NLs) were separated by ultra centrifugation and characterized. NLs were dispersed in phosphate buffer saline (PBS) pH 7.4 containing different carriers like lactose, sucrose, and hydrolyzed gelatin, and 15% l-leucine as antiadherent. The resultant dispersion was spray dried and spray dried formulation were characterized to ascertain its performance. In vitro pulmonary deposition was assessed using Andersen Cascade Impactor as per USP. NLs were found to have average size of 137 ± 15 nm, 95.17 ± 3.43% drug entrapment, and zeta potential of 0.8314 ± 0.0827 mV. Hydrolyzed gelatin based formulation was found to have low density, good flowability, particle size of 7.9 ± 1.1 μm, maximum fine particle fraction (FPF) of 75.6 ± 1.6%, mean mass aerodynamic diameter (MMAD) 2.2 ± 0.1 μm, and geometric standard deviation (GSD) 2.3 ± 0.1. Developed formulations were found to have in vitro prolonged drug release up to 16 h, and obeys Higuchi's Controlled Release model. The investigation provides a practical approach for direct delivery of DS encapsulated in NLs for site specific controlled and prolonged release behavior at the site of action and hence, may play a promising role in prevention of PCP.     

Immunoreactivity of five antigens of Mycobacterium tuberculosis in patients attending a public health care facility in an area with high endemicity for TB

The objective of this study was to evaluate people attending a primary health clinic in Rio de Janeiro, Brazil for immunoreactivity to five Mycobacterium tuberculosis antigens, as these antigens are markers of immune response and factors associated with active TB. The serum antibody titers of different categories of patients (defined by microbiological and radiological characteristics and by response to therapy on follow‐up) to 38 kDa, 16 kDa, MPT64, ESAT‐6 and MT10.3 antigens were determined blind with ELISA. Positive tests to each antigen were defined with ROC analysis. OR were calculated for factors associated with humoral response in patients with active TB. A total of 201 patients underwent serological testing. Patients with confirmed active TB responded more frequently to MPT64 (44%), 16 kDa (37.7%) and 38 kDa (36.1%). ESAT‐6 and MT10.3 were also able to distinguish people in TB groups from controls. TB infected subjects responded less frequently to ESAT‐6 and MT10.3 (3.7% and 11%, respectively). Sensitivity and specificity to all antigens combined were 58.4% and 60.7%, respectively. Reactivity to 38 kDa and to MPT64 was more likely among alcohol users OR 2.61 (95%CI;1.05–6.94) and OR 3.27 (95%CI;1.33–8.15), respectively. 16 kDa antigen elicited a more protective response among smokers, OR 0.29 (95%CI; 0.10–0.83). It was concluded that reactivity to all antigens tested represented markers of active disease. ESAT‐6 and MT10.3 could not be identified as markers of TB infection in this community. Sensitivity was higher to all antigens combined, but at a cost of lower specificity. Interestingly, among factors associated with positive immunoreactivity, alcohol use and smoking seem to polarize the humoral response in different directions. This finding deserves further investigation.     

Stimulation of Th1-polarizing cytokines,C-C chemokines,maturation of dendritic cells,and adjuvant function by the peptide binding fragment of heat shock protein 70

The peptide binding C-terminal portion of heat shock protein (HSP)70 (aa 359-610) stimulates human monocytes to produce IL-12, TNF-alpha, NO, and C-C chemokines. The N-terminal, ATPase portion (HSP70(1-358)) failed to stimulate any of these cytokines or chemokines. Both native and the truncated HSP70(359-610) stimulation of chemokine production is mediated by the CD40 costimulatory molecule. Maturation of dendritic cells was induced by stimulation with native HSP70, was not seen with the N-terminal HSP70(1-358), but was enhanced with HSP70(359-610), as demonstrated by up-regulation of CD83, CCR7, CD86, CD80, and HLA class II. In vivo studies in macaques showed that immunization with HSP70(359-610) enhances the production of IL-12 and RANTES. Immunization with peptide-bound HSP70(359-610) in mice induced higher serum IgG2a and IgG3 Abs than the native HSP70-bound peptide. This study suggests that the C-terminal, peptide-binding portion of HSP70 is responsible for stimulating Th1-polarizing cytokines, C-C chemokines, and an adjuvant function.     

Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis

The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. Proteins 2015; 83:389–394. © 2014 Wiley Periodicals, Inc.     

Nano-liposomal dry powder inhaler of tacrolimus: preparation, characterization, and pulmonary pharmacokinetics

The studies were undertaken to evaluate feasibility of pulmonary delivery of liposomaly encapsulated tacrolimus dry powder inhaler for prolonged drug retention in lungs as rescue therapy to prevent refractory rejection of lungs after transplantation. Tacrolimus encapsulated liposomes were prepared by thin film evaporation technique and liposomal dispersion was passed through high pressure homogenizer. Tacrolimus nano-liposomes (NLs) were separated by centrifugation and characterized. NLs were dispersed in phosphate buffer saline (PBS) pH 7.4 containing different additives like lactose, sucrose, and trehalose, and L-leucine as antiadherent. The dispersion was spray dried and spray dried powders were characterized. In vitro and in vivo pulmonary deposition was performed using Andersen Cascade Impactor and intratracheal instillation in rats respectively. NLs were found to have average size of 140 nm, 96% +/- 1.5% drug entrapment, and zeta potential of 1.107 mV. Trehalose based formulation was found to have low density, good flowability, particle size of 9.46 +/- 0.8 microm, maximum fine particle fraction (FPF) of 71.1 +/- 2.5%, mean mass aerodynamic diameter (MMAD) 2.2 +/- 0.1 microm, and geometric standard deviation (GSD) 1.7 +/- 0.2. Developed formulations were found to have in vitro prolonged drug release up to 18 hours, following Higuchi's Controlled Release model. In vivo studies revealed maximal residence of tacrolimus within lungs of 24 hours, suggesting slow clearance from the lungs. The investigation provides a practical approach for direct delivery of tacrolimus encapsulated in NLs for controlled and prolonged retention at the site of action. It may play a promising role as rescue therapy in reducing the risk of acute rejection and chronic rejection.     

Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes

The high-quality rice genome sequence is serving as a reference for comparative genome analysis in crop plants, especially cereals. However, early comparisons with bread wheat showed complex patterns of conserved synteny (gene content) and colinearity (gene order). Here, we show the presence of ancient duplicated segments in the progenitor of wheat, which were first identified in the rice genome. We also show that single-copy (SC) rice genes, those representing unique matches with wheat expressed sequence tag (EST) unigene contigs in the whole rice genome, show more than twice the proportion of genes mapping to syntenic wheat chromosome as compared to the multicopy (MC) or duplicated rice genes. While 58.7% of the 1,244 mapped SC rice genes were located in single syntenic wheat chromosome groups, the remaining 41.3% were distributed randomly to the other six non-syntenic wheat groups. This could only be explained by a background dispersal of genes in the genome through transposition or other unknown mechanism. The breakdown of rice–wheat synteny due to such transpositions was much greater near the wheat centromeres. Furthermore, the SC rice genes revealed a conserved primordial gene order that gives clues to the origin of rice and wheat chromosomes from a common ancestor through polyploidy, aneuploidy, centromeric fusions, and translocations. Apart from the bin-mapped wheat EST contigs, we also compared 56,298 predicted rice genes with 39,813 wheat EST contigs assembled from 409,765 EST sequences and identified 7,241 SC rice gene homologs of wheat. Based on the conserved colinearity of 1,063 mapped SC rice genes across the bins of individual wheat chromosomes, we predicted the wheat bin location of 6,178 unmapped SC rice gene homologs and validated the location of 213 of these in the telomeric bins of 21 wheat chromosomes with 35.4% initial success. This opens up the possibility of directed mapping of a large number of conserved SC rice gene homologs in wheat. Overall, only 46.4% of these SC genes code for proteins with known functional domains; the remaining 53.6% have unknown function, and hence, represent an important, but yet, under explored category of genes. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Global Gene Transcriptome Analysis in Vaccinated Cattle Revealed a Dominant Role of IL-22 for Protection against Bovine Tuberculosis

Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex group of bacteria. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB. Identification of biomarkers in tuberculosis research remains elusive and the goal is to identify host correlates of protection. We hypothesized that by studying global gene expression we could identify in vitro predictors of protection that could help to facilitate vaccine development. Calves were vaccinated with BCG or with a heterologous BCG prime adenovirally vectored subunit boosting protocol. Protective efficacy was determined after M. bovis challenge. RNA was prepared from PPD-stimulated PBMC prepared from vaccinated-protected, vaccinated-unprotected and unvaccinated control cattle prior to M. bovis challenge and global gene expression determined by RNA-seq. 668 genes were differentially expressed in vaccinated-protected cattle compared with vaccinated-unprotected and unvaccinated control cattle. Cytokine-cytokine receptor interaction was the most significant pathway related to this dataset with IL-22 expression identified as the dominant surrogate of protection besides INF-γ. Finally, the expression of these candidate genes identified by RNA-seq was evaluated by RT-qPCR in an independent set of PBMC samples from BCG vaccinated and unvaccinated calves. This experiment confirmed the importance of IL-22 as predictor of vaccine efficacy.     

Multiwalled carbon nanotubes-ceramic electrode modified with substrate-selective imprinted polymer for ultra-trace detection of bovine serum albumin

This study describes the synthesis of a new class of substrate-selective molecularly imprinted polymer. This involved tetraethylene glycol 3-morpholin propionate acrylate (functional monomer) and bovine serum albumin (template) for polymerization in aqueous condition, using "surface grafting-from" approach directly on a vinyl exposed multiwalled carbon nanotubes-ceramic electrode. The analyte recapture at pH 6.8 in aqueous environment simultaneously involved hydrophobically driven hydrogen bonds and ionic interactions between negatively charged bovine serum albumin and positively charged imprinted nanofilm. The selectively encapsulated bovine serum albumin first gets reduced at -0.9V and then oxidized within the cavity, without getting stripped off, to respond a differential pulse voltammetry signal. The limit of detection [0.42ngmL(-1) (3σ, RSD≤1.02%)] obtained was free from any cross-reactivity and matrix complications in aqueous, pharmaceutical, serum, and liquid milk samples. The proposed sensor can be used as a practical sensor for ultra-trace analysis of bovine serum albumin in clinical settings.     

Interaction of the Bacillus thuringiensis delta endotoxins Cry1Ac and Cry3Aa with the gut of the pea aphid, Acyrthosiphon pisum (Harris)

Hemipteran pests including aphids are not particularly susceptible to the effects of insecticidal Cry toxins derived from the bacterium Bacillus thuringiensis. We examined the physiological basis for the relatively low toxicity of Cry1Ac and Cry3Aa against the pea aphid, Acyrthosiphon pisum (Harris). Cry1Ac was efficiently hydrolyzed by aphid stomach membrane associated cysteine proteases (CP) producing a 60 kDa mature toxin, whereas Cry3Aa was incompletely processed and partially degraded. Cry1Ac bound to the aphid gut epithelium but showed low aphid toxicity in bioassays. Feeding of aphids on Cry1Ac in the presence or absence of GalNAc, suggested that Cry1Ac gut binding was glycan mediated. In vitro binding of biotinylated-Cry1Ac to gut BBMVs and competition assays using unlabeled Cry1Ac and GalNAc confirmed binding specificity as well as glycan mediation of Cry1Ac binding. Although Cry3Aa binding to the aphid gut membrane was not detected, Cry3Aa bound 25 and 37 kDa proteins in aphid gut BBMV in ligand blot analysis and competition assays confirmed the binding specificity of Cry3Aa. This, combined with low toxicity in feeding assays, suggests that Cry3Aa does bind the gut epithelium to some extent. This is the first systematic examination of the physiological basis for the low efficacy of Cry toxins against aphids, and analysis of Cry toxin-aphid gut interaction.