Browsing by Author "Briken, Volker"
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Item Anthracene-Walled Acyclic CB[n] Receptors: in vitro and in vivo Binding Properties toward Drugs of Abuse(Wiley, 2022-03-03) DiMaggio, Delaney; Brockett, Adam T.; Shuster, Michael; Murkli, Steven; Zhai, Canjia; King, David; O'Dowd, Brona; Cheng, Ming; Brady, Kimberly; Briken, Volker; Roesch, Matthew R.; Isaacs, LyleWe report studies of the interaction of six acyclic CB[n]-type receptors toward a panel of drugs of abuse by a combination of isothermal titration calorimetry and 1H NMR spectroscopy. Anthracene walled acyclic CB[n] host (M3) displays highest binding affinity toward methamphetamine (Kd=15 nM) and fentanyl (Kd=4 nM). Host M3 is well tolerated by Hep G2 and HEK 293 cells up to 100 μM according to MTS metabolic and adenylate kinase release assays. An in vivo maximum tolerated dose study with Swiss Webster mice showed no adverse effects at the highest dose studied (44.7 mg kg−1). Host M3 is not mutagenic based on the Ames fluctuation test and does not inhibit the hERG ion channel. In vivo efficacy studies showed that pretreatment of mice with M3 significantly reduces the hyperlocomotion after treatment with methamphetamine, but M3 does not function similarly when administered 30 seconds after methamphetamine.Item Automated Workflow for Advanced Single Cell and Bacterium Tracking in Host-Pathogen Interactions(2024) Augenstreich, Jacques; Poddar, Anushka; Belew, Ashton T.; El-Sayed, Najib; Briken, Volker; Briken, VolkerIn the study of intracellular pathogens like Mycobacterium tuberculosis, time-lapse microscopy is a valuable tool for understanding dynamic cellular processes involved in host cell defense. Quantification of signals at localized compartments within the cell and around bacteria can provide even deeper insight into interactions between bacteria and host cell organelles. However, existing quantitative analysis at a single-bacterial level remains limited and dependent on manual tracking methods. We developed a near-fully automated workflow that performs unbiased, high-throughput cell segmentation and quantitative tracking of both single cells and single bacteria/phagosomes within multi-channel, z-stack, time-lapse confocal microscopy videos. We took advantage of the PyImageJ library to bring Fiji functionality into a Python environment and combined deep-learning-based segmentation from Cellpose with tracking algorithms from Trackmate and visualization within ImageJ. Following both cell and bacteria tracking, our workflow provides a versatile toolkit of functions for measuring relevant signal parameters at the single-cell level (such as velocity or bacterial burden) and at the single-bacteria level (for assessment of phagosome maturation). Ultimately, our workflow’s capabilities in both single-cell and single-bacteria quantification can help decipher the virulence factors of pathogens and pave the way for the development of innovative therapeutic approaches. The customizable nature of the methods extends the applications of the workflow far beyond the field of mycobacteria and presents opportunities for advancement in host-pathogen interaction research in a variety of systems.Item The non-pathogenic mycobacteria M. smegmatis and M. fortuitum induce rapid host cell apoptosis via a caspase-3 and TNF dependent pathway(2010-09-10) Bohsali, Amro; Abdalla, Hana; Velmurugan, Kamalakannan; Briken, VolkerBackground: The HIV pandemic raised the potential for facultative-pathogenic mycobacterial species like, Mycobacterium kansasii, to cause disseminating disease in humans with immune deficiencies. In contrast, nonpathogenic mycobacterial species, like M. smegmatis, are not known to cause disseminating disease even in immunocompromised individuals. We hypothesized that this difference in phenotype could be explained by the strong induction of an innate immune response by the non-pathogenic mycobacterial species. Results: A comparison of two rapid-growing, non-pathogenic species (M. smegmatis and M. fortuitum) with two facultative-pathogenic species (M. kansasii and M. bovis BCG) demonstrated that only the non-pathogenic bacteria induced strong apoptosis in human THP-1 cells and murine bone marrow-derived macrophages (BMDM) and dendritic cells (BMDD). The phospho-myo-inositol modification of lipoarabinomannan (PI-LAM) isolated from nonpathogenic species may be one of the cell wall components responsible for the pro-inflammatory activity of the whole bacteria. Indeed, PI-LAM induces high levels of apoptosis and IL-12 expression compared to the mannosyl modification of LAM isolated from facultative-pathogenic mycobacteria. The apoptosis induced by non-pathogenic M. smegmatis was dependent upon caspase-3 activation and TNF secretion. Consistently, BALB/c BMDM responded by secreting large amounts of TNF upon infection with non-pathogenic but not facultative-pathogenic mycobacteria. Interestingly, C57Bl/6 BMDM do not undergo apoptosis upon infection with non-pathogenic mycobacteria despite the fact that they still induce an increase in TNF secretion. This suggests that the host cell signaling pathways are different between these two mouse genotypes and that TNF is necessary but not sufficient to induce host cell apoptosis. Conclusion: These results demonstrate a much stronger induction of the innate immune response by nonpathogenic versus facultative-pathogenic mycobacteria as measured by host cell apoptosis, IL-12 and TNF cytokine induction. These observations lend support to the hypothesis that the strong induction of the innate immune response is a major reason for the lack of pathogenicity in fast-growing mycobacteria.