Prospective Artificial Intelligence to Dissect the Dengue Immune Response and Discover Therapeutics.

Dengue virus (DENV) poses a serious threat to global health as the causative agent of dengue fever. The virus is endemic in more than 128 countries resulting in approximately 390 million infection cases each year. Currently, there is no approved therapeutic for treatment nor a fully efficacious vaccine. The development of therapeutics is confounded and hampered by the complexity of the immune response to DENV, in particular to sequential infection with different DENV serotypes (DENV1-5). Researchers have shown that the DENV envelope (E) antigen is primarily responsible for the interaction and subsequent invasion of host cells for all serotypes and can elicit neutralizing antibodies in humans. The advent of high-throughput sequencing and the rapid advancements in computational analysis of complex data, has provided tools for the deconvolution of the DENV immune response. Several types of complex statistical analyses, machine learning models and complex visualizations can be applied to begin answering questions about the B- and T-cell immune responses to multiple infections, antibody-dependent enhancement, identification of novel therapeutics and advance vaccine research.

Traditional and Digital Biomarkers: Two Worlds Apart?

The identification and application of biomarkers in the clinical and medical fields has an enormous impact on society. The increase of digital devices and the rise in popularity of health-related mobile apps has produced a new trove of biomarkers in large, diverse, and complex data. However, the unclear definition of digital biomarkers, population groups, and their intersection with traditional biomarkers hinders their discovery and validation. We have identified current issues in the field of digital biomarkers and put forth suggestions to address them during the DayOne Workshop with participants from academia and industry. We have found similarities and differences between traditional and digital biomarkers in order to synchronize semantics, define unique features, review current regulatory procedures, and describe novel applications that enable precision medicine.

Identification of Mycobacterium avium subsp. hominissuis secreted proteins using an in vitro system mimicking the phagosomal environment.

BACKGROUND: Mycobacterium avium subsp. hominissuis is a common intracellular pathogen that infects patients with HIV/AIDS and cause lung infection in patients with underlying lung pathology. M.avium preferably infects macrophages and uses diverse mechanisms to alter phagosome maturation. Once in the macrophage, the pathogen can alter the host cellular defenses by secreting proteins into the cytosol of host cells, but despite considerable research, only a few secreted effector proteins have been identified. We hypothesized that the environmental cues inside the phagosome can trigger bacterial protein secretion. To identify M. avium secretome within the phagosome, we utilized a previously established in vitro system that mimics the metal ion concentrations and pH of the M. avium phagosome. RESULTS: M. avium was exposed to phagosome metal concentrations for different time points and exported proteins were profiled and analyzed against bacterial proteins secreted in the culture medium. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 46 were unique to bacteria incubated in the metal mixture. Ten of potential effectors were selected and secretion of these proteins was monitored within M. avium infected mononuclear phagocytic cells using the beta-lactamase FRET-based reporter system. In addition, pull-down assay was performed for secreted calmodulin-like protein MAV_1356 protein to evaluate for eukaryotic target. All examined M. avium proteins were secreted into the macrophage cytosol, and gene expression analysis suggested that the metal environment likely stimulates secretion of pre-made proteins. Further investigation of bacterial secreted MAV_1356 protein, lead to the observation that the MAV_1356 interacts with the host proteins Annexin A1 and Protein S100-A8. CONCLUSIONS: We established an in vitro system for the study if proteins secreted intracellularly, and revealed that the metal mixture mimicking the concentration of metals in the phagosome environment, triggers protein secretion.

Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics.

Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.