Genome-wide transcription analysis of interaction between the human macrophage and Mycobacterium tuberculosis during concurrent drug administration by conventional and novel methods

Targeted drug delivery to alveolar macrophages harboring Mycobacterium tuberculosis (Mtb) holds promise of high efficacy against pulmonary tuberculosis (TB). It was investigated whether inhalable microparticles (MP) can rescue macrophages from ‘alternative’ activation induced by pathogenic Mtb in addition to achieving targeted drug delivery. A genome-wide transcription analysis (Affymetrix HG-U133 Plus 2.0 DNA microarray) of THP-1 cell line derived macrophages was undertaken after exposing them to infection with 10 MOI of MTB H37Rv at 0, 12 and 24 hours post infection. The Molecular markers of macrophage bactericidal activity were assayed in THP-1- and primary human peripheral blood mononuclear cell (PBMC)-derived macrophages, in the presence or absence of soluble anti-tuberculosis drugs, drug-containing MP and blank MP. About 1,500 genes were differentially upregulated and about 500 genes differentially downregulated in response to various modes of treatment. Variations were also observed in the kinetics of gene expression. Cluster analysis indicated activation of several pathways related to innate immune response (cytokines, chemokines, receptors and ligands), apoptosis, cytoskeleton and membrane remodeling, general metabolism and general housekeeping. Some of these results were validated at the functional level, by studying caspase activities, concentrations and time-courses of effector molecules , rates/extents of apoptosis and nitrite oxide induction. Production of cytokines and NO, apoptosis, and bacterial survival were studied as pharmacodynamic outcomes. Cytokine responses of THP-1 derived macrophages were estimated. MP reversed suppression of tumor necrosis factor (TNF) induced by infection, and transiently upregulated γ-interferon (IFN-γ). Drug-free MP surprisingly induced IFN-γ, but not TNF. Primary cells responded to MP, regardless of drug content, by upregulation of NO; but THP-1-derived cells did not respond to blank MP. About 19% of infected cells exposed to MP underwent apoptosis as compared to ~11% cells treated with soluble drugs or blank MP. Cell death induced by blank MP was caspase-independent. Only drug-containing MP induced apoptosis through caspase-8 and caspase-9. Bacterial survival after different treatments varied between individuals. In the best case, while untreated infection resulted in survival of 900±141 colony forming units (CFU), treatment with soluble drugs, drug-containing MP and blank MP respectively, reduced CFU counts to 8.5± 0.7, 3±1.4 and 102±138.6. The results suggest a role of the drug delivery system in macrophage activation as a component of therapeutic strategy against TB

Effects of Multi-Enzymes on Growth Performance, and Effects of Multi-Enzymes and Probiotics on Nutrient Utilization in Broilers Fed Different Level of Fibers.

M.S. Thesis. University of Hawaiʻi at Mānoa 2018

Filogenetska analiza izolata pasjeg parvovirusa izdvojenih u Mathuri u Indiji.

Canine parvovirus (CPV) is an important pathogen causing acute hemorrhagic gastroenteritis in dogs and myocarditis in pups. The present study deals with an analysis of partial nucleotide sequences of the VP1/VP2 gene of CPV isolates from Mathura, India to establish their phylogenetic relationship with other CPV isolates. Out of 100 samples from dogs showing the clinical signs of gastroenteritis viz., vomiting, diarrhea and dysentery, 63 were found positive for CPV-2 by polymerase chain reaction (PCR). Among the 63 positive samples, eight samples were processed further for nucleotide sequencing. Phylogenetic analysis revealed that the CPV variants were not only closely related among themselves but also showed minimum divergence from their ancestors, such as MEV, indicating very little divergence since their origin. From the study, it may be concluded that canine parvovirus-2 variants may represent a potential threat to canine populations. Thus more efforts is required to increase epidemiological monitoring and surveillance, along with the measures necessary to control this disease in the canine population, and to assess the efficacy of the current vaccines.Pasji parvovirus važan je uzročnik hemoragijskog gastroenteritisa u odraslih pasa i miokarditisa u štenadi. U ovom su istraživanju djelomično analizirane nukleotidne sekvencije gena VP1/VP2 izolata pasjeg parvovirusa iz Mathure u Indiji sa svrhom da se njihova filogenetska svojstva usporede s drugim izolatima toga virusa. Od 100 uzoraka izdvojenih iz pasa s gastroenteritisom odnosno s povraćanjem i proljevom, 63 su bila pozitivna na pasji parvovirus 2 upotrebom lančane reakcije polimerazom. Od toga je osam uzoraka uzeto za određivanje njihova nukleotidnog slijeda. Filogenetska analiza je pokazala da varijante pasjeg parvovirusa nisu bile samo međusobno usko srodne, već su s neznatnim skretanjem bile srodne i sa svojim predcima, kao što je virus enteritisa američke vidrice, što upućuje na njihove neznatne razlike od njihova nastanka. Može se zaključiti da varijante pasjeg parvovirusa 2 predstavljaju moguću prijetnju za populaciju pasa. Potrebno je uložiti više napora u smjeru epizootioloških istraživanja i donošenja mjera nadzora te kontrole ove zarazne bolesti zajedno s naporima za procjenu učinkovitosti postojećih cjepiva

Filogenetska analiza izolata pasjeg parvovirusa izdvojenih u Mathuri u Indiji.

Canine parvovirus (CPV) is an important pathogen causing acute hemorrhagic gastroenteritis in dogs and myocarditis in pups. The present study deals with an analysis of partial nucleotide sequences of the VP1/VP2 gene of CPV isolates from Mathura, India to establish their phylogenetic relationship with other CPV isolates. Out of 100 samples from dogs showing the clinical signs of gastroenteritis viz., vomiting, diarrhea and dysentery, 63 were found positive for CPV-2 by polymerase chain reaction (PCR). Among the 63 positive samples, eight samples were processed further for nucleotide sequencing. Phylogenetic analysis revealed that the CPV variants were not only closely related among themselves but also showed minimum divergence from their ancestors, such as MEV, indicating very little divergence since their origin. From the study, it may be concluded that canine parvovirus-2 variants may represent a potential threat to canine populations. Thus more efforts is required to increase epidemiological monitoring and surveillance, along with the measures necessary to control this disease in the canine population, and to assess the efficacy of the current vaccines.Pasji parvovirus važan je uzročnik hemoragijskog gastroenteritisa u odraslih pasa i miokarditisa u štenadi. U ovom su istraživanju djelomično analizirane nukleotidne sekvencije gena VP1/VP2 izolata pasjeg parvovirusa iz Mathure u Indiji sa svrhom da se njihova filogenetska svojstva usporede s drugim izolatima toga virusa. Od 100 uzoraka izdvojenih iz pasa s gastroenteritisom odnosno s povraćanjem i proljevom, 63 su bila pozitivna na pasji parvovirus 2 upotrebom lančane reakcije polimerazom. Od toga je osam uzoraka uzeto za određivanje njihova nukleotidnog slijeda. Filogenetska analiza je pokazala da varijante pasjeg parvovirusa nisu bile samo međusobno usko srodne, već su s neznatnim skretanjem bile srodne i sa svojim predcima, kao što je virus enteritisa američke vidrice, što upućuje na njihove neznatne razlike od njihova nastanka. Može se zaključiti da varijante pasjeg parvovirusa 2 predstavljaju moguću prijetnju za populaciju pasa. Potrebno je uložiti više napora u smjeru epizootioloških istraživanja i donošenja mjera nadzora te kontrole ove zarazne bolesti zajedno s naporima za procjenu učinkovitosti postojećih cjepiva

Peristaltic Induced Flow of a Particulate Suspension in a Non-Uniform Geometry

The flow induced by sinusoidal peristaltic waves of a particle-fluid suspension in a twodimensional diverging channel under low Reynolds number and long wavelength approximation has been investigated. The analytical expression for the flow characteristics-the flow rate, pressure rise and friction force have been derived. Moreover, we present some results concerning the dependence of these quantities on the geometrical parameters

Confocal Microscopy: a Powerful Tool for Biological Research

Conventional light microscopy allows the observation of living as well as fixed cells and tissues to generate two-dimensional images. The out-of-focus information often obscures the ultrastructural details, especially in thick specimens with overlapping structures. The earliest available light microscopy visualized the objects in hydrated state in two-dimensions during their temporal development. The emergence of electron microscopy (EM) provided superb resolution of ultrastructural details, but it was applicable only for objects in the dehydrated state and thereby potentially introducing handling artifacts. The usefulness of optical methods, however, has been limited by the poor depth discrimination. Often, the fluorescence and reflectance images are severely degraded by the scattered- or emitted-light from tissue structures outside the plane of focus. These limitations have been partially overcome by video image processing and deconvolution. Laser scanning confocal microscopy (LSCM) overcomes the above difficulties and produces improved light microscopic images of fixed as well as living cells and tissues. In terms of resolution of the image, the confocal microscope occupies a position in between the light and electron microscopes. Confocal microscope generates information from a well-defined optical section rather than from the entire specimen, thereby eliminating the out-of-focus glare and increasing the contrast, clarity and detection sensitivity. Optical sectioning is noninvasive and less time consuming compared to reconstruction algorithms to give 3-D images. Optical sectioning is achieved not only in the xy plane (perpendicular to the optical axis of the microscope) but also vertically in the xz or yz plane (parallel to the optical axis). With vertical sectioning, cells are scanned laterally (x or y axis) as well as in depth (z axis). Stacks of optical sections taken at successive focal plane (known as z series) are then reconstructed to generate a 3-D version of the specimen. The 3-D image can be directly visualized where each data point represents the quantity of specific contrast parameter used at a certain point in space. The image processing can be additionally used to enhance the confocal images. It is widely used in the fluorescence mode for different specimens and in bright field reflection mode for objects of different forms

Presentació

an early differentiated state: a preliminary repor