Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells.

The pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin 1ß (IL-1ß) are expressed simultaneously and have tumor-promoting roles in breast cancer. In parallel, mesenchymal stem cells (MSCs) undergo conversion at the tumor site to cancer-associated fibroblasts (CAFs), which are generally connected to enhanced tumor progression. Here, we determined the impact of consistent inflammatory stimulation on stromal cell plasticity. MSCs that were persistently stimulated by TNFα + IL-1ß (generally 14-18 days) gained a CAF-like morphology, accompanied by prominent changes in gene expression, including in stroma/fibroblast-related genes. These CAF-like cells expressed elevated levels of vimentin and fibroblast activation protein (FAP) and demonstrated significantly increased abilities to contract collagen gels. Moreover, they gained the phenotype of inflammatory CAFs, as indicated by the reduced expression of α smooth muscle actin (αSMA), increased proliferation, and elevated expression of inflammatory genes and proteins, primarily inflammatory chemokines. These inflammatory CAFs released factors that enhanced tumor cell dispersion, scattering, and migration; the inflammatory CAF-derived factors elevated cancer cell migration by stimulating the chemokine receptors CCR2, CCR5, and CXCR1/2 and Ras-activating receptors, expressed by the cancer cells. Together, these novel findings demonstrate that chronic inflammation can induce MSC-to-CAF conversion, leading to the generation of tumor-promoting inflammatory CAFs.

Putative Blood Somatic Mutations in Post-Traumatic Stress Disorder-Symptomatic Soldiers: High Impact of Cytoskeletal and Inflammatory Proteins.

BACKGROUND: We recently discovered autism/intellectual disability somatic mutations in postmortem brains, presenting higher frequency in Alzheimer's disease subjects, compared with the controls. We further revealed high impact cytoskeletal gene mutations, coupled with potential cytoskeleton-targeted repair mechanisms. OBJECTIVE: The current study was aimed at further discerning if somatic mutations in brain diseases are presented only in the most affected tissue (the brain), or if blood samples phenocopy the brain, toward potential diagnostics. METHODS: Variant calling analyses on an RNA-seq database including peripheral blood samples from 85 soldiers (58 controls and 27 with symptoms of post-traumatic stress disorder, PTSD) was performed. RESULTS: High (e.g., protein truncating) as well as moderate impact (e.g., single amino acid change) germline and putative somatic mutations in thousands of genes were found. Further crossing the mutated genes with autism, intellectual disability, cytoskeleton, inflammation, and DNA repair databases, identified the highest number of cytoskeletal-mutated genes (187 high and 442 moderate impact). Most of the mutated genes were shared and only when crossed with the inflammation database, more putative high impact mutated genes specific to the PTSD-symptom cohorts versus the controls (14 versus 13) were revealed, highlighting tumor necrosis factor specifically in the PTSD-symptom cohorts. CONCLUSION: With microtubules and neuro-immune interactions playing essential roles in brain neuroprotection and Alzheimer-related neurodegeneration, the current mutation discoveries contribute to mechanistic understanding of PTSD and brain protection, as well as provide future diagnostics toward personalized military deployment strategies and drug design.

Discovery of autism/intellectual disability somatic mutations in Alzheimer's brains: mutated ADNP cytoskeletal impairments and repair as a case study.

With Alzheimer's disease (AD) exhibiting reduced ability of neural stem cell renewal, we hypothesized that de novo mutations controlling embryonic development, in the form of brain somatic mutations instigate the disease. A leading gene presenting heterozygous dominant de novo autism-intellectual disabilities (ID) causing mutations is activity-dependent neuroprotective protein (ADNP), with intact ADNP protecting against AD-tauopathy. We discovered a genomic autism ADNP mutation (c.2188C>T) in postmortem AD olfactory bulbs and hippocampi. RNA-Seq of olfactory bulbs also identified a novel ADNP hotspot mutation, c.2187_2188insA. Altogether, 665 mutations in 596 genes with 441 mutations in AD patients (389 genes, 38% AD-exclusive mutations) and 104 genes presenting disease-causing mutations (OMIM) were discovered. OMIM AD mutated genes converged on cytoskeletal mechanisms, autism and ID causing mutations (about 40% each). The number and average frequencies of AD-related mutations per subject were higher in AD subjects compared to controls. RNA-seq datamining (hippocampus, dorsolateral prefrontal cortex, fusiform gyrus and superior frontal gyrus-583 subjects) yielded similar results. Overlapping all tested brain areas identified unique and shared mutations, with ADNP singled out as a gene associated with autism/ID/AD and presenting several unique aging/AD mutations. The large fusiform gyrus library (117 subjects) with high sequencing coverage correlated the c.2187_2188insA ADNP mutation frequency to Braak stage (tauopathy) and showed more ADNP mutations in AD specimens. In cell cultures, the ADNP-derived snippet NAP inhibited mutated-ADNP-microtubule (MT) toxicity and enhanced Tau-MT association. We propose a paradigm-shifting concept in the perception of AD whereby accumulating mosaic somatic mutations promote brain pathology.

PRMT1 inhibition induces differentiation of colon cancer cells.

Differentiation therapy has been recently revisited as a prospective approach in cancer therapy by targeting the aberrant growth, and repairing the differentiation and cell death programs of cancer cells. However, differentiation therapy of solid tumors is a challenging issue and progress in this field is limited. We performed High Throughput Screening (HTS) using a novel dual multiplex assay to discover compounds, which induce differentiation of human colon cancer cells. Here we show that the protein arginine methyl transferase (PRMT) type 1 inhibitor, MS023, is a potent inducer of colon cancer cell differentiation with a large therapeutic window. Differentiation changes in the highly aggressive human colon cancer cell line (HT-29) were proved by proteomic and genomic approaches. Growth of HT-29 xenograft in nude mice was significantly delayed upon MS023 treatment and immunohistochemistry of tumor indicated differentiation changes. These findings may lead to development of clinically effective anti-cancer drugs based on the mechanism of cancer cell differentiation.

RNA sequencing of bipolar disorder lymphoblastoid cell lines implicates the neurotrophic factor HRP-3 in lithium's clinical efficacy.

Objectives: Lithium remains the oldest and most effective treatment for mood stabilisation in bipolar disorder (BD), even though at least half of patients are only partially responsive or do not respond. This study aimed to identify biomarkers associated with lithium response in BD, based on comparing RNA sequencing information derived from lymphoblastoid cell lines (LCLs) of lithium-responsive (LR) versus lithium non-responsive (LNR) BD patients, to assess gene expression variations that might bear on treatment outcome. Methods: RNA sequencing was carried out on 24 LCLs from female BD patients (12 LR and 12 LNR) followed by qPCR validation in two additional independent cohorts (41 and 17 BD patients, respectively). Results: Fifty-six genes showed nominal differential expression comparing LR and LNR (FC ≥ |1.3|, P ≤ 0.01). The differential expression of HDGFRP3 and ID2 was validated by qPCR in the independent cohorts. Conclusions: We observed higher expression levels of HDGFRP3 and ID2 in BD patients who favourably respond to lithium. Both of these genes are involved in neurogenesis, and HDGFRP3 has been suggested to be a neurotrophic factor. Additional studies in larger BD cohorts are needed to confirm the potential of HDGFRP3 and ID2 expression levels in blood cells as tentative favourable lithium response biomarkers.

Quantitative analysis of population-scale family trees with millions of relatives.

Family trees have vast applications in fields as diverse as genetics, anthropology, and economics. However, the collection of extended family trees is tedious and usually relies on resources with limited geographical scope and complex data usage restrictions. We collected 86 million profiles from publicly available online data shared by genealogy enthusiasts. After extensive cleaning and validation, we obtained population-scale family trees, including a single pedigree of 13 million individuals. We leveraged the data to partition the genetic architecture of human longevity and to provide insights into the geographical dispersion of families. We also report a simple digital procedure to overlay other data sets with our resource.

Revealing the inventory of type III effectors in Pantoea agglomerans gall-forming pathovars using draft genome sequences and a machine-learning approach.

Pantoea agglomerans, a widespread epiphytic bacterium, has evolved into a hypersensitive response and pathogenicity (hrp)-dependent and host-specific gall-forming pathogen by the acquisition of a pathogenicity plasmid containing a type III secretion system (T3SS) and its effectors (T3Es). Pantoea agglomerans pv. betae (Pab) elicits galls on beet (Beta vulgaris) and gypsophila (Gypsophila paniculata), whereas P. agglomerans pv. gypsophilae (Pag) incites galls on gypsophila and a hypersensitive response (HR) on beet. Draft genome sequences were generated and employed in combination with a machine-learning approach and a translocation assay into beet roots to identify the pools of T3Es in the two pathovars. The genomes of the sequenced Pab4188 and Pag824-1 strains have a similar size (∼5 MB) and GC content (∼55%). Mutational analysis revealed that, in Pab4188, eight T3Es (HsvB, HsvG, PseB, DspA/E, HopAY1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on beet and gypsophila. In Pag824-1, nine T3Es (HsvG, HsvB, PthG, DspA/E, HopAY1, HopD1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on gypsophila, whereas the PthG effector triggers HR on beet. HsvB, HsvG, PthG and PseB appear to endow pathovar specificities to Pab and Pag, and no homologous T3Es were identified for these proteins in other phytopathogenic bacteria. Conversely, the remaining T3Es contribute to the virulence of both pathovars, and homologous T3Es were found in other phytopathogenic bacteria. Remarkably, HsvG and HsvB, which act as host-specific transcription factors, displayed the largest contribution to disease development.

Insights from the Genome Sequence of Acidovorax citrulli M6, a Group I Strain of the Causal Agent of Bacterial Fruit Blotch of Cucurbits.

Acidovorax citrulli is a seedborne bacterium that causes bacterial fruit blotch of cucurbit plants including watermelon and melon. A. citrulli strains can be divided into two major groups based on DNA fingerprint analyses and biochemical properties. Group I strains have been generally isolated from non-watermelon cucurbits, while group II strains are closely associated with watermelon. In the present study, we report the genome sequence of M6, a group I model A. citrulli strain, isolated from melon. We used comparative genome analysis to investigate differences between the genome of strain M6 and the genome of the group II model strain AAC00-1. The draft genome sequence of A. citrulli M6 harbors 139 contigs, with an overall approximate size of 4.85 Mb. The genome of M6 is ∼500 Kb shorter than that of strain AAC00-1. Comparative analysis revealed that this size difference is mainly explained by eight fragments, ranging from ∼35-120 Kb and distributed throughout the AAC00-1 genome, which are absent in the M6 genome. In agreement with this finding, while AAC00-1 was found to possess 532 open reading frames (ORFs) that are absent in strain M6, only 123 ORFs in M6 were absent in AAC00-1. Most of these M6 ORFs are hypothetical proteins and most of them were also detected in two group I strains that were recently sequenced, tw6 and pslb65. Further analyses by PCR assays and coverage analyses with other A. citrulli strains support the notion that some of these fragments or significant portions of them are discriminative between groups I and II strains of A. citrulli. Moreover, GC content, effective number of codon values and cluster of orthologs' analyses indicate that these fragments were introduced into group II strains by horizontal gene transfer events. Our study reports the genome sequence of a model group I strain of A. citrulli, one of the most important pathogens of cucurbits. It also provides the first comprehensive comparison at the genomic level between the two major groups of strains of this pathogen.

Revealing bacterial targets of growth inhibitors encoded by bacteriophage T7.

Today's arsenal of antibiotics is ineffective against some emerging strains of antibiotic-resistant pathogens. Novel inhibitors of bacterial growth therefore need to be found. The target of such bacterial-growth inhibitors must be identified, and one way to achieve this is by locating mutations that suppress their inhibitory effect. Here, we identified five growth inhibitors encoded by T7 bacteriophage. High-throughput sequencing of genomic DNA of resistant bacterial mutants evolving against three of these inhibitors revealed unique mutations in three specific genes. We found that a nonessential host gene, ppiB, is required for growth inhibition by one bacteriophage inhibitor and another nonessential gene, pcnB, is required for growth inhibition by a different inhibitor. Notably, we found a previously unidentified growth inhibitor, gene product (Gp) 0.6, that interacts with the essential cytoskeleton protein MreB and inhibits its function. We further identified mutations in two distinct regions in the mreB gene that overcome this inhibition. Bacterial two-hybrid assay and accumulation of Gp0.6 only in MreB-expressing bacteria confirmed interaction of MreB and Gp0.6. Expression of Gp0.6 resulted in lemon-shaped bacteria followed by cell lysis, as previously reported for MreB inhibitors. The described approach may be extended for the identification of new growth inhibitors and their targets across bacterial species and in higher organisms.