Browsing by Author "Bandara, Aloka B."

Now showing 1 - 11 of 11
  • Thumbnail Image
    The Application of a Nanomaterial Optical Fiber Biosensor Assay for Identification of Brucella Nomenspecies
    McCutcheon, Kelly; Bandara, Aloka B.; Zuo, Ziwei; Heflin, James R.; Inzana, Thomas J. (MDPI, 2019-05-21)
    Bacteria in the genus Brucella are the cause of brucellosis in humans and many domestic and wild animals. A rapid and culture-free detection assay to detect Brucella in clinical samples would be highly valuable. Nanomaterial optical fiber biosensors (NOFS) are capable of recognizing DNA hybridization events or other analyte interactions with high specificity and sensitivity. Therefore, a NOFS assay was developed to detect Brucella DNA from cultures and in tissue samples from infected mice. An ionic self-assembled multilayer (ISAM) film was coupled to a long-period grating optical fiber, and a nucleotide probe complementary to the Brucella IS711 region and modified with biotin was bound to the ISAM by covalent conjugation. When the ISAM/probe duplex was exposed to lysate containing ≥100 killed cells of Brucella, or liver or spleen tissue extracts from Brucella-infected mice, substantial attenuation of light transmission occurred, whereas exposure of the complexed fiber to non-Brucella gram-negative bacteria or control tissue samples resulted in negligible attenuation of light transmission. Oligonucleotide probes specific for B. abortus, B. melitensis, and B. suis could also be used to detect and differentiate these three nomenspecies. In summary, the NOFS biosensor assay detected three nomenspecies of Brucella without the use of polymerase chain reaction within 30 min and could specifically detect low numbers of this bacterium in clinical samples.
  • Thumbnail Image
    Colonization efficiency of multidrug-resistant Neisseria gonorrhoeae in a female mouse model
    Kikiowo, Babatomiwa; Bandara, Aloka B.; Abutaleb, Nader S.; Seleem, Mohamed N. (Oxford University Press, 2023-10-18)
    The rapid occurrence of gonococcal resistance to all classes of antibiotics could lead to untreatable gonorrhea. Thus, development of novel anti-Neisseria gonorrhoeae drugs is urgently needed. Neisseria gonorrhoeae FA1090 is the most used in gonococcal infection mouse models because of its natural resistance to streptomycin. Streptomycin inhibits the urogenital commensal flora that permits gonococcal colonization. However, this strain is drug-susceptible and cannot be used to investigate the efficacy of novel agents against multidrug-resistant N. gonorrhoeae. Hence, to test the in vivo efficacy of new therapeutics against N. gonorrhoeae resistant to the frontline antibiotics, azithromycin, or ceftriaxone, we constructed streptomycin-resistant mutants of N. gonorrhoeae CDC-181 (azithromycin-resistant) and WHO-X (ceftriaxone-resistant). We identified the inoculum size needed to successfully colonize mice. Both mutants, CDC-181-rpsLA128G and WHO-X-rpsLA128G, colonized the genital tract of mice for 14 days with 100% colonization observed for at least 7 days. CDC-181-rpsLA128G demonstrated better colonization of the murine genital tract compared to WHO-X-rpsLA128G. Lower inoculum of WHO-X-rpsLA128G (105 and 106 CFU) colonized mice better than higher inoculum. Overall, our results indicate that CDC-181-rpsLA128G and WHO-X-rpsLA128G can colonize the lower genital tract of mice and are suitable to be used in mouse models to investigate the efficacy of antigonococcal agents.
  • Thumbnail Image
    Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog
    Bandara, Aloka B.; Drake, Joshua C.; Brown, David A. (2021-06-12)
    Background Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Results Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Conclusions Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.
  • Thumbnail Image
    Development of a live, attenuated, recombinant vaccine for Brucellosis
    (United States Patent and Trademark Office, 2008-04-29)
    A recombinant, attenuated strain of Brucella suis or Brucella melitensis with a deficiency in carboxyl-terminal protease activity or tail-specific protease activity can be used as a vaccine for the prevention or treatment of Brucellosis. Prior exposure to the Brucella species is identified by detecting a genetic sequence for carboxyl-terminal (i.e. tail-specific) protease activity in a biological sample.
  • Thumbnail Image
    Formation of the Francisella tularensis Biofilm is Affected by Cell Surface Glycosylation, Growth Medium, and a Glucan Exopolysaccharide
    Champion, Anna E.; Catanzaro, Kelly C. Freudenberger; Bandara, Aloka B.; Inzana, Thomas J. (Springer Nature, 2019-08-22)
    Biofilms are matrix-associated communities that enable bacteria to colonise environments unsuitable for free-living bacteria. The facultative intracellular pathogen Francisella tularensis can persist in water, amoebae, and arthropods, as well as within mammalian macrophages. F. tularensis Types A and B form poor biofilms, but F. tularensis mutants lacking lipopolysaccharide O-antigen, O-antigen capsule, and capsule-like complex formed up to 15-fold more biofilm than fully glycosylated cells. The Type B live vaccine strain was also 50% less capable of initiating surface attachment than mutants deficient in O-antigen and capsule-like complex. However, the growth medium of all strains tested also influenced the formation of biofilm, which contained a novel exopolysaccharide consisting of an amylose-like glucan. In addition, the surface polysaccharide composition of the bacterium affected the protein: DNA: polysaccharide composition of the biofilm matrix. In contrast, F. novicida attached to surfaces more efficiently and made a more robust biofilm than Type A or B strains, but loss of O-antigen or capsule-like complex did not significantly affect F. novicida biofilm formation. These results indicated that suppression of surface polysaccharides may promote biofilm formation by F. tularensis Types A and B. Whether biofilm formation enhances survival of F. tularensis in aquatic or other environmental niches has yet to be determined.
  • Thumbnail Image
    Further Characterization of the Capsule-Like Complex (CLC) Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane Vesicles
    Champion, Anna E.; Bandara, Aloka B.; Mohapatra, Nrusingh; Fulton, Kelly M.; Twine, Susan M.; Inzana, Thomas J. (Frontiers, 2018-06-15)
    Francisella tularensis is the etiologic agent of tularemia, and subspecies tularensis (type A) is the most virulent subspecies. The live vaccine strain (LVS) of subspecies holarctica produces a capsule-like complex (CLC) that consists of a large variety of glycoproteins. Expression of the CLC is greatly enhanced when the bacteria are subcultured in and grown on chemically defined medium. Deletion of two genes responsible for CLC glycosylation in LVS results in an attenuated mutant that is protective against respiratory tularemia in a mouse model. We sought to further characterize the CLC composition and to determine if a type A CLC glycosylationmutant would be attenuated inmice. The CLCs isolated from LVS extracted with 0.5% phenol or 1M urea were similar, as determined by gel electrophoresis and Western blotting, but the CLC extracted with urea was more water-soluble. The CLC extracted with either 0.5% phenol or 1M urea from type A strains was also similar to the CLC of LVS in antigenic properties, electrophoretic profile, and by transmission electron microscopy (TEM). The solubility of the CLC could be further enhanced by fractionation with Triton X-114 followed by N-Lauroylsarcosine detergents; the largest (>250 kDa) molecular size component appeared to be an aggregate of smaller components. Outer membrane vesicles/tubules (OMV/T) isolated by differential centrifugation and micro-filtration appeared similar to the CLC by TEM, and many of the proteins present in the OMV/T were also identified in soluble and insoluble fractions of the CLC. Further investigation is warranted to assess the relationship between OMV/T and the CLC. The CLC conjugated to keyhole limpet hemocyanin or flagellin was highly protective against high-dose LVS intradermal challenge and partially protective against intranasal challenge. A protective response was associated with a significant rise in cytokines IL-12, IL-10, and IFN-γ. However, a type A CLC glycosylation mutant remained virulent in BALB/cmice, and immunization with the CLC did not protectmice against high dose respiratory challenge with type A strain SCHU S4.
  • Thumbnail Image
    Isolation and Mutagenesis of a Capsule-Like Complex (CLC) from Francisella tularensis, and Contribution of the CLC to F. tularensis Virulence in Mice
    Bandara, Aloka B.; Champion, Anna E.; Wang, X.; Berg, G.; Apicella, Michael A.; McLendon, M.; Azadi, P.; Snyder, D. S.; Inzana, Thomas J. (PLOS, 2011-04-22)
    Background: Francisella tularensis is a category-A select agent and is responsible for tularemia in humans and animals. The surface components of F. tularensis that contribute to virulence are not well characterized. An electron-dense capsule has been postulated to be present around F. tularensis based primarily on electron microscopy, but this specific antigen has not been isolated or characterized. Methods and Findings: A capsule-like complex (CLC) was effectively extracted from the cell surface of an F. tularensis live vaccine strain (LVS) lacking O-antigen with 0.5% phenol after 10 passages in defined medium broth and growth on defined medium agar for 5 days at 32uC in 7% CO2. The large molecular size CLC was extracted by enzyme digestion, ethanol precipitation, and ultracentrifugation, and consisted of glucose, galactose, mannose, and Proteinase K-resistant protein. Quantitative reverse transcriptase PCR showed that expression of genes in a putative polysaccharide locus in the LVS genome (FTL_1432 through FTL_1421) was upregulated when CLC expression was enhanced. Open reading frames FTL_1423 and FLT_1422, which have homology to genes encoding for glycosyl transferases, were deleted by allelic exchange, and the resulting mutant after passage in broth (LVSD1423/1422_P10) lacked most or all of the CLC, as determined by electron microscopy, and CLC isolation and analysis. Complementation of LVSD1423/1422 and subsequent passage in broth restored CLC expression. LVSD1423/1422_P10 was attenuated in BALB/c mice inoculated intranasally (IN) and intraperitoneally with greater than 80 times and 270 times the LVS LD50, respectively. Following immunization, mice challenged IN with over 700 times the LD50 of LVS remained healthy and asymptomatic. Conclusions: Our results indicated that the CLC may be a glycoprotein, FTL_1422 and -FTL_1423 were involved in CLC biosynthesis, the CLC contributed to the virulence of F. tularensis LVS, and a CLC-deficient mutant of LVS can protect mice against challenge with the parent strain.
  • Thumbnail Image
    Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy
    Drake, Joshua C.; Wilson, Rebecca J.; Laker, Rhianna C.; Guan, Yuntian; Spaulding, Hannah R.; Nichenko, Anna S.; Shen, Wenqing; Shang, Huayu; Dorn, Maya; Huang, Kian; Zhang, Mei; Bandara, Aloka B.; Brisendine, Matthew H.; Kashatus, Jennifer A.; Sharma, Poonam R.; Young, Alexander; Gautam, Jitendra; Cao, Ruofan; Wallrabe, Horst; Chang, Paul A.; Wong, Michael; Desjardins, Eric M.; Hawley, Simon A.; Christ, George J.; Kashatus, David F.; Miller, Clint L.; Wolf, Matthew J.; Periasamy, Ammasi; Steinberg, Gregory R.; Hardie, D. Grahame; Yan, Zhen (National Academy of Sciences, 2021-09-14)
    Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5′ AMP-activated protein kinase (AMPKα1/ α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.
  • Thumbnail Image
    Modifying Fatty Acid Composition of Bovine Milk by Abomasal Infusion or Dietary Supplementation of Seed Oils or Fish Oil
    Bandara, Aloka B. (Virginia Tech, 1997-12-17)
    The potential for enhancing oleic acid (cis-18:1) and linoleic acid (18:2) content and lowering medium chain fatty acid (MCFA) content of bovine milk was investigated by abomasal infusion or dietary supplementation of oils. In experiment 1, olive oil, sesame oil, sunflower oil, or fish oil was abomasally infused (155 to 219 g/d) into Jersey cows during the last 6 d of each of four 14-d periods. In experiment 2, canola oil, olive oil, high-oleic sunflower oil, or distilled water (control) was abomasally infused (342 to 371 g/d) into three Holsteins and three Jerseys during the last 5 d of each of four 10-d periods. The intestinal digestibility and concentration of cis-18:1 and 18:2 in milk were proportional to flow of these fatty acids to the duodenum. Also, greater concentration of cis-18:1 in milk was associated with lowered yield of MCFA. During olive oil or sesame oil infusion in experiment 1, for each 100 g of cis-18:1 infused into the abomasum, milk cis-18:1 yield was increased by an average of 47 g, and MCFA yield was reduced by 42 g. The yield of 18:2 in milk was increased by approximately 46 g for each 100 g of infused 18:2 during olive oil or sesame oil infusion. Milk produced during sesame oil infusion, however, had an off-flavor when evaluated by a taste panel. In experiment 2, each 100 g of cis-18:1 infused daily increased milk cis-18:1 yield in Holsteins and Jerseys by 41 and 39 g/d, respectively, whereas recovery of infused 18:2 was 34 g/d for Jerseys and 42 g/d for Holsteins. In experiment 3, 22 Jersey cows were fed a basal diet, or the basal diet supplemented with 3.5% high-oleic canola oil, 3.5% soybean oil, or 1.75% high-oleic canola oil plus 1.75% soybean oil for 5 wk. Dietary canola oil supplementation increased conjugated linoleic acid (CLA) percentage in milk to a moderate level without raising trans-18:1 percentage, whereas feeding either supplement containing soybean oil raised both CLA and trans-18:1 percentages. Concentrations of trans-18:1 and CLA in milk apparently reflected the extent of unsaturated fatty acid biohydrogenation in the rumen. Dietary supplementation with canola oil increased yield of cis-18:1 in milk by 21 g for each 100 g of supplemental cis-18:1 intake. Yield of 18:2 in milk was raised by 3 g for each 100 g of supplemental 18:2 intake by cows fed soybean oil. Using abomasal infusion as an indicator of the maximum potential for apparent recovery of cis-18:1 in milk (39 to 49%), cis-18:1 recovery in response to supplemental cis-18:1 in the diet was approximately half of the potential response due to partial biohydrogenation in the rumen. The apparent recovery of dietary 18:2 in milk was reduced to only one-tenth of the potential yield (31 to 47%) indicated by abomasal infusion of seed oils. Results indicated that the fatty acid profile of bovine milk was altered in a manner that would be beneficial to human health when cows were fed supplemental oleic acid, but further research should focus on safe and economical methods to protect dietary unsaturated fatty acids from biohydrogenation.
  • Thumbnail Image
    Neuromuscular Dysfunction Precedes Cognitive Impairment in a Mouse Model of Alzheimer's Disease
    Brisendine, Matthew H.; Nichenko, Anna S.; Bandara, Aloka B.; Willoughby, Orion S.; Amiri, Niloufar; Weingrad, Zach; Specht, Kalyn S.; Bond, Jacob M.; Addington, Adele; Jones III, Ronald G.; Murach, Kevin A.; Poelzing, Steven; Craige, Siobhan M.; Grange, Robert W.; Drake, Joshua C. (Oxford University Press, 2023-12-04)
    Alzheimer's disease (AD) develops along a continuum that spans years prior to diagnosis. Decreased muscle function and mitochondrial respiration occur years earlier in those that develop AD; however, it is unknown what causes these peripheral phenotypes in a disease of the brain. Exercise promotes muscle, mitochondria, and cognitive health and is proposed to be a potential therapeutic for AD, but no study has investigated how skeletal muscle adapts to exercise training in an AD-like context. Utilizing 5xFAD mice, an AD model that develops ad-like pathology and cognitive impairments around 6 mo of age, we examined in vivo neuromuscular function and exercise adapations (mitochondrial respiration and RNA sequencing) before the manifestation of overt cognitive impairment. We found 5xFAD mice develop neuromuscular dysfunction beginning as early as 4 mo of age, characterized by impaired nerve-stimulated muscle torque production and compound nerve action potential of the sciatic nerve. Furthermore, skeletal muscle in 5xFAD mice had altered, sex-dependent, adaptive responses (mitochondrial respiration and gene expression) to exercise training in the absence of overt cognitive impairment. Changes in peripheral systems, specifically neural communication to skeletal muscle, may be harbingers for AD and have implications for lifestyle interventions, like exercise, in AD.
  • Thumbnail Image
    Photonic Biosensor Assays to Detect and Distinguish Subspecies of Francisella tularensis
    Cooper, Kristie L.; Bandara, Aloka B.; Wang, Yunmiao; Wang, Anbo; Inzana, Thomas J. (MDPI, 2011-03-07)
    The application of photonic biosensor assays to diagnose the category-A select agent Francisella tularensis was investigated. Both interferometric and long period fiber grating sensing structures were successfully demonstrated; both these sensors are capable of detecting the optical changes induced by either immunological binding or DNA hybridization. Detection was made possible by the attachment of DNA probes or immunoglobulins (IgG) directly to the fiber surface via layer-by-layer electrostatic self-assembly. An optical fiber biosensor was tested using a standard transmission mode long period fiber grating of length 15 mm and period 260 µm, and coated with the IgG fraction of antiserum to F. tularensis. The IgG was deposited onto the optical fiber surface in a nanostructured film, and the resulting refractive index change was measured using spectroscopic ellipsometry. The presence of F. tularensis was detected from the decrease of peak wavelength caused by binding of specific antigen. Detection and differentiation of F. tularensis subspecies tularensis (type A strain TI0902) and subspecies holarctica (type B strain LVS) was further accomplished using a single-mode multi-cavity fiber Fabry-Perot interferometric sensor. These sensors were prepared by depositing seven polymer bilayers onto the fiber tip followed by attaching one of two DNA probes: (a) a 101-bp probe from the yhhW gene unique to type-A strains, or (b) a 117-bp probe of the lpnA gene, common to both type-A and type-B strains. The yhhW probe was reactive with the type-A, but not the type-B strain. Probe lpnA was reactive with both type-A and type-B strains. Nanogram quantities of the target DNA could be detected, highlighting the sensitivity of this method for DNA detection without the use of PCR. The DNA probe reacted with 100% homologous target DNA, but did not react with sequences containing 2-bp mismatches, indicating the high specificity of the assay. These assays will fill an important void that exists for rapid, culture-free, and field-compatible diagnosis of F. tularensis.