Genetika+: precision medicine solutions for mental health.

Genetika+ is developing a precision medicine tool to optimize the treatment of depression by helping physicians find the best drug therapy for their patients. The tool builds on traditional pharmacogenetics, introducing a 'brain-in-a-dish' screening platform for each patient that will overcome the challenge of limited pharmacodynamic knowledge of pharmacogenetics (PGx). In addition to PGx, our platform integrates patient data with innovative blood-derived patient neurons to test all categories of antidepressants and predict the best drug for each patient. This offers patients optimal drug treatment, allowing a faster response, fewer side effects and lower dosing.

Depression patient-derived cortical neurons reveal potential biomarkers for antidepressant response.

Major depressive disorder is highly prevalent worldwide and has been affecting an increasing number of people each year. Current first line antidepressants show merely 37% remission, and physicians are forced to use a trial-and-error approach when choosing a single antidepressant out of dozens of available medications. We sought to identify a method of testing that would provide patient-specific information on whether a patient will respond to a medication using in vitro modeling. Patient-derived lymphoblastoid cell lines from the Sequenced Treatment Alternatives to Relieve Depression study were used to rapidly generate cortical neurons and screen them for bupropion effects, for which the donor patients showed remission or non-remission. We provide evidence for biomarkers specific for bupropion response, including synaptic connectivity and morphology changes as well as specific gene expression alterations. These biomarkers support the concept of personalized antidepressant treatment based on in vitro platforms and could be utilized as predictors to patient response in the clinic.

Associating H2O2-and NO-related changes in the proteome of Mycobacterium smegmatis with enhanced survival in macrophage.

Mycobacterium manages to evade the host cell immune system, partially owing to its ability to survive redox stress after macrophage engulfment. Exposure to redox stress has been linked to later replication, persistence, and latent infection. In this work, mass spectrometry was used to elucidate the cell-wide changes that occur in response to sublethal doses of hydrogen peroxide and nitric oxide over time, with Mycobacterium smegmatis being used as a model organism. A total of 3135 proteins were confidently assigned, of which 1713, 1674, and 1713 were identified under NO, H2O2, and control conditions, respectively. Both treatment conditions resulted in changes of protein expression from the DosR regulon as well as those related to lipid metabolism. Complementary to the changes in the proteome, sublethal exposure to NO and H2O2 improved the survival of the bacteria after macrophage infection. Our data indicate that pre-exposure to sublethal doses of these redox stressors causes an alteration in the expression of proteins related to lipid metabolism, suggesting a link between altered lipid metabolism and enhanced survival in macrophages.

Global proteome and phosphoproteome dynamics indicate novel mechanisms of vitamin C induced dormancy in Mycobacterium smegmatis.

Vitamin C has been found to affect mycobacteria in multiple ways, including increasing susceptibility to antimicrobial drugs, inducing dormancy, and having a bactericidal effect. However, the regulatory events mediating vitamin C related adaptations remain largely elusive. Ser/Thr/Tyr protein phosphorylation plays an important regulatory role in mycobacteria, contributing to environmental adaptation, including dormancy and drug resistance. This study utilised the model organism, Mycobacterium smegmatis, and TiO2 phosphopeptide enrichment combined with mass spectrometry-based proteomics methods to elucidate the mycobacterial signalling and regulatory response to sub-lethal concentrations of vitamin C. After initial validation of peptide spectra, 224 non-redundant phosphosites in 154 proteins were retained with high confidence. Data analysis revealed that 30 peptides were differentially phosphorylated with Vitamin C treatment, including novel phosphosites found on both PknG and GarA. Of these significant proteins, we validated 11 by parallel reaction monitoring of high-confidence phosphopeptides. Interestingly, 17/30 phosphopeptides were annotated as part of transmembrane proteins, suggesting that it is likely vitamin C triggers typical signal transduction events in which the protein periplasmic domain perceives environmental signals and the cytoplasmic domain is then phosphorylated. Finally, the diverse nature of phosphorylated proteins involved in signalling, transport, and carbohydrate biosynthesis indicates the extent of such regulatory phosphorylation events. BIOLOGICAL SIGNIFICANCE: Our findings provide new mechanistic insight into a coordinated network of signalling and regulatory responses to sub-lethal vitamin C in Mycobacterium smegmatis and provide evidence that vitamin C is able to act as a novel extracellular signalling molecule. Vitamin C treatment caused changes in both the proteome and phosphoproteome associated with response to oxidative stress, a shift in metabolic regulation and progression toward dormancy, as well as phospho-dependent activation of specific secretory pathways and activation of specific two component and Ser/Thr/Tyr protein kinase activities. This study confirms the potential of vitamin C as convenient means to study aspects of mycobacterial dormancy, including those regulated at post-translational level.

Mass Spectrometry Offers Insight into the Role of Ser/Thr/Tyr Phosphorylation in the Mycobacteria.

Phosphorylation is a post translational modification which can rapidly regulate biochemical pathways by altering protein function, and has been associated with pathogenicity in bacteria. Once engulfed by host macrophages, pathogenic bacteria are exposed to harsh conditions and must respond rapidly in order to survive. The causative agent of TB, Mycobacterium tuberculosis, is unusual amongst the bacteria because it can survive within the host macrophage for decades in a latent state, demonstrating a remarkable capacity to successfully evade the host immune response. This ability may be mediated in part by regulatory mechanisms such as ser/thr/tyr phosphorylation. Mass spectrometry-based proteomics has afforded us the capacity to identify hundreds of phosphorylation sites in the bacterial proteome, allowing for comparative phosphoproteomic studies in the mycobacteria. There remains an urgent need to validate the reported phosphosites, and to elucidate their biological function in the context of pathogenicity. However, given the sheer number of putative phosphorylation events in the mycobacterial proteome, and the technical difficulty of assigning biological function to a phosphorylation event, it will not be trivial to do so. There are currently six published phosphoproteomic investigations of a member of mycobacteria. Here, we combine the datasets from these studies in order to identify commonly detected phosphopeptides and phosphosites in order to present high confidence candidates for further validation. By applying modern mass spectrometry-based techniques to improve our understanding of phosphorylation and other PTMs in pathogenic bacteria, we may identify candidates for therapeutic intervention.