Chronic social defeat alters brain vascular-associated cell gene expression patterns leading to vascular dysfunction and immune system activation.

Brain vascular integrity is critical for brain health, and its disruption is implicated in many brain pathologies, including psychiatric disorders. Brain-vascular barriers are a complex cellular landscape composed of endothelial, glial, mural, and immune cells. Yet currently, little is known about these brain vascular-associated cells (BVACs) in health and disease. Previously, we demonstrated that 14 days of chronic social defeat (CSD), a mouse paradigm that produces anxiety and depressive-like behaviors, causes cerebrovascular damage in the form of scattered microbleeds. Here, we developed a technique to isolate barrier-related cells from the mouse brain and subjected the isolated cells to single-cell RNA sequencing. Using this isolation technique, we found an enrichment in BVAC populations, including distinct subsets of endothelial and microglial cells. In CSD compared to non-stress, home-cage control, differential gene expression patterns disclosed biological pathways involving vascular dysfunction, vascular healing, and immune system activation. Overall, our work demonstrates a unique technique to study BVAC populations from fresh brain tissue and suggests that neurovascular dysfunction is a key driver of psychosocial stress-induced brain pathology.

Chromatin remodeling complex NURF regulates thymocyte maturation.

The maturation of T cells requires signaling from both cytokine and T-cell receptors to gene targets in chromatin, but how chromatin architecture influences this process is largely unknown. Here we show that thymocyte maturation post-positive selection is dependent on the nucleosome remodeling factor (NURF). Depletion of Bptf (bromodomain PHD finger transcription factor), the largest NURF subunit, in conditional mouse mutants results in developmental arrest beyond the CD4(+) CD8(int) stage without affecting cellular proliferation, cellular apoptosis, or coreceptor gene expression. In the Bptf mutant, specific subsets of genes important for thymocyte development show aberrant expression. We also observed defects in DNase I-hypersensitive chromatin structures at Egr1, a prototypical Bptf-dependent gene that is required for efficient thymocyte development. Moreover, chromatin binding of the sequence-specific factor Srf (serum response factor) to Egr1 regulatory sites is dependent on Bptf function. Physical interactions between NURF and Srf suggest a model in which Srf recruits NURF to facilitate transcription factor binding at Bptf-dependent genes. These findings provide evidence for causal connections between NURF, transcription factor occupancy, and gene regulation during thymocyte development.

Topoisomerase-modulated genome-wide DNA supercoiling domains colocalize with nuclear compartments and regulate human gene expression.

DNA supercoiling is a biophysical feature of the double helix with a pivotal role in biological processes. However, understanding of DNA supercoiling in the chromatin remains limited. Here, we developed azide-trimethylpsoralen sequencing (ATMP-seq), a DNA supercoiling assay offering quantitative accuracy while minimizing genomic bias and background noise. Using ATMP-seq, we directly visualized transcription-dependent negative and positive twin-supercoiled domains around genes and mapped kilobase-resolution DNA supercoiling throughout the human genome. Remarkably, we discovered megabase-scale supercoiling domains (SDs) across all chromosomes that are modulated mainly by topoisomerases I and IIß. Transcription activities, but not the consequent supercoiling accumulation in the local region, contribute to SD formation, indicating the long-range propagation of transcription-generated supercoiling. Genome-wide SDs colocalize with A/B compartments in both human and Drosophila cells but are distinct from topologically associating domains (TADs), with negative supercoiling accumulation at TAD boundaries. Furthermore, genome-wide DNA supercoiling varies between cell states and types and regulates human gene expression, underscoring the importance of supercoiling dynamics in chromatin regulation and function.

Proteomic analysis of stage I endometrial cancer tissue: identification of proteins associated with oxidative processes and inflammation.

OBJECTIVE: The present study aimed to identify differentially expressed proteins employing a high resolution mass spectrometry (MS)-based proteomic analysis of endometrial cancer cells harvested using laser microdissection. METHODS: A differential MS-based proteomic analysis was conducted from discrete epithelial cell populations gathered by laser microdissection from 91 pathologically reviewed stage I endometrial cancer tissue samples (79 endometrioid and 12 serous) and 10 samples of normal endometrium from postmenopausal women. Hierarchical cluster analysis of protein abundance levels derived from a spectral count analysis revealed a number of proteins whose expression levels were common as well as unique to both histologic types. An independent set of endometrial cancer specimens from 394 patients were used to externally validate the differential expression of select proteins. RESULTS: 209 differentially expressed proteins were identified in a comparison of stage I endometrial cancers and normal post-menopausal endometrium controls (Q<0.005). A number of differentially abundant proteins in stage I endometrial cancer were identified and independently validated by western blot and tissue microarray analyses. Multiple proteins identified with elevated abundance in stage I endometrial cancer are functionally associated with inflammation (annexins) and oxidative processes (peroxiredoxins). PRDX1 and ANXA2 were both confirmed as being overexpressed in stage I cancer compared to normal endometrium by independent TMA (Q=0.008 and Q=0.00002 respectively). CONCLUSIONS: These data provide the basis for further investigation of previously unrecognized novel pathways involved in early stage endometrial carcinogenesis and provide possible targets for prevention strategies that are inclusive of both endometrioid and serous histologic subtypes.

Androgen action in prostate function and disease.

Benign prostatic hyperplasia (BPH) is an enlargement of the prostate gland that is frequently found in aging men. Androgens are essential for the development and differentiated function of the prostate, as well as for proliferation and survival of prostatic cells. In man, dog and rodent, there are age-related decreases in serum testosterone. Despite the lower serum testosterone levels, benign prostatic hyperplasia increases with age in men and dogs, while age-dependent prostatic hyperplasia develops in the dorsal and lateral lobes of the rat prostate. The possible mechanisms that lead to prostate hyperplasia have been extensively studied over many years. It is clear that androgens, estrogens and growth factors contribute to the condition, but the exact etiology remains unknown. Prostate cancer (CaP) represents a significant cause of death among males worldwide. As is the case of BPH, it is clear that androgens (testosterone and dihydrotestosterone) and their metabolites play important roles in the disease, but cause-effect relationships have not been established. Androgen deprivation therapy has been used for decades, primarily in the metastatic stage, to inhibit androgen-dependent prostate cancer cell growth. Androgen deprivation, which can be achieved by targeting hormone biosynthesis or androgen receptor activation, results in symptom amelioration. However, most patients will develop hormone refractory cancer or castration-resistant prostate cancer (CRPC). Prostatic epithelial cells demonstrate enormous plasticity in response to androgen ablation. This characteristic of prostatic epithelial cells may give rise to different populations of cells, some of which may not be dependent on androgen. Consequently, androgen receptor positive and negative cells might co-exist within CRPC. A clear understanding of this possible cellular heterogeneity and plasticity of prostate epithelial cells is necessary to develop an optimal strategy to treat or prevent CRPC.

Bcl-2 protein expression correlates with cell survival and androgen independence in rat prostatic lobes.

Castration of young and old male Brown Norway rats induces apoptosis in the ventral, but not in the dorsal and lateral, lobes of the prostate gland, and apoptosis in old rats is diminished by 50% compared with that in young rats. In this study we examined the lobe-specific and age-dependent expression of Bcl-2 and Bax proteins. Bcl-2 levels in the ventral lobe were 5-fold lower compared with expression in the dorsal and lateral lobes. Bax expression in the ventral lobe was 2- and 20-fold higher than that in the lateral and dorsal lobes, respectively. In all three lobes, Bcl-2 was detected in epithelial cells, but not in stromal cells, whereas Bax protein was localized in both cell types. After castration, Bcl-2 expression in the ventral lobe decreased significantly from the control level after 2-3 d, but increased significantly by 7-10 d. By contrast, Bax expression increased significantly by d 1, gradually decreased by 2-4 d, and was nearly undetectable by 7-10 d postcastration. In the dorsal and lateral lobes, neither Bcl-2 nor Bax expression was significantly altered after castration. In the ventral lobe of old rats after castration, Bcl-2 followed a pattern of expression similar to that observed in young rats. However, Bax levels were 50% lower in old rats compared with those in young rats on d 1 after castration. Therefore, cell death follows the down-regulation of Bcl-2 expression in the ventral lobe of young and old rats. Moreover, the higher relative levels of Bcl-2 expression in the dorsal and lateral lobes of intact animals and in the ventral lobe by 7-10 d after castration serve to protect cells from apoptosis.

Expression of the B-cell receptor component CD79a on immature myeloid cells contributes to their tumor promoting effects.

The role of myeloid derived suppressor cells (MDSCs) in promoting tumorigenesis is well-established, and significant effort is being made to further characterize surface markers on MDSCs both for better diagnosis and as potential targets for therapy. Here we show that the B cell receptor adaptor molecule CD79a is unexpectedly expressed on immature bone marrow myeloid cells, and is upregulated on MDSCs generated in multiple different mouse models of metastatic but not non-metastatic cancer. CD79a on MDSCs is upregulated and activated in response to soluble factors secreted by tumor cells. Activation of CD79a on mouse MDSCs, by crosslinking with a specific antibody, maintained their immature phenotype (CD11b+Gr1+), enhanced their migration, increased their suppressive effect on T cell proliferation, and increased secretion of pro-tumorigenic cytokines such as IL-6 and CCL22. Furthermore, crosslinking CD79a on myeloid cells activated signaling through Syk, BLNK, ERK and STAT3 phosphorylation. In vivo, CD79+ myeloid cells showed enhanced ability to promote primary tumor growth and metastasis. Finally we demonstrate that CD79a is upregulated on circulating myeloid cells from lung cancer patients, and that CD79a+ myeloid cells infiltrate human breast tumors. We propose that CD79a plays a functional role in the tumor promoting effects of myeloid cells, and may represent a novel target for cancer therapy.