Isolation and characterization of Streptomyces bacteriophages and Streptomyces strains encoding biosynthetic arsenals.

The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcare's most potent antibiotics are becoming obsolete. Approximately two-thirds of the world's antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 104 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.

Reaching into the toolbox: Stem cell models to study neuropsychiatric disorders.

Recent advances in genetics, molecular biology, and stem cell biology have accelerated our understanding of neuropsychiatric disorders, like autism spectrum disorder (ASD), major depressive disorder (MDD), bipolar disorder (BD), and schizophrenia (SZ). This progress highlights the incredible complexity of both the human brain and mental illnesses from the biochemical to the cellular level. Contributing to the complexity of neuropsychiatric disorders are their polygenic nature, cellular and brain region interconnectivity, and dysregulation of human-specific neurodevelopmental processes. Here, we discuss available tools, including CRISPR-Cas9, and the applications of these tools to develop cell-based two-dimensional (2D) models and 3D brain organoid models that better represent and unravel the intricacies of neuropsychiatric disorder pathophysiology.

Altered Neuronal Support and Inflammatory Response in Bipolar Disorder Patient-Derived Astrocytes.

Bipolar disorder (BD) is characterized by cyclical mood shifts. Studies indicate that BD patients have a peripheral pro-inflammatory state and alterations in glial populations in the brain. We utilized an in vitro model to study inflammation-related phenotypes of astrocytes derived from induced pluripotent stem cells (iPSCs) generated from BD patients and healthy controls. BD astrocytes showed changes in transcriptome and induced a reduction in neuronal activity when co-cultured with neurons. IL-1ß-stimulated BD astrocytes displayed a unique inflammatory gene expression signature and increased secretion of IL-6. Conditioned medium from stimulated BD astrocytes reduced neuronal activity, and this effect was partially blocked by IL-6 inactivating antibody. Our results suggest that BD astrocytes are functionally less supportive of neuronal excitability and this effect is partially mediated by IL-6. We confirmed higher IL-6 in blood in a distinct cohort of BD patients, highlighting the potential role of astrocyte-mediated inflammatory signaling in BD neuropathology.

Deficient LEF1 expression is associated with lithium resistance and hyperexcitability in neurons derived from bipolar disorder patients.

Bipolar disorder (BD) is a psychiatric condition characterized by depressive and manic episodes that affect 2% of the world population. The first-line long-term treatment for mood stabilization is lithium (Li). Induced pluripotent stem cell modeling of BD using hippocampal dentate gyrus-like neurons derived from Li-responsive (LR) and Li-non-responsive (NR) patients previously showed neuronal hyperexcitability. Li treatment reversed hyperexcitability only on the LR neurons. In this study we searched for specific targets of Li resistance in NR neurons and found that the activity of Wnt/ß-catenin signaling pathway was severely affected, with a significant decrease in expression of LEF1. Li targets the Wnt/ß-catenin signaling pathway by inhibiting GSK-3ß and releasing ß-catenin that forms a nuclear complex with TCF/LEF1, activating the Wnt/ß-catenin transcription program. Therefore, we propose that downregulation of LEF1 may account for Li resistance in NR neurons. Our results show that valproic acid (VPA), a drug used to treat NR patients that also acts downstream of GSK-3ß, upregulated LEF1 and Wnt/ß-catenin gene targets, increased transcriptional activity of complex ß-catenin/TCF/LEF1, and reduced excitability in NR neurons. In addition, decreasing LEF1 expression in control neurons using shLEF1 caused hyperexcitability, confirming that the impact of VPA on excitability in NR neurons was connected to changes in LEF1 and in the Wnt/ß-catenin pathway. Our results suggest that LEF1 may be a useful target for the discovery of new drugs for BD treatment.