TSWIFT, a novel method for iterative staining of embedded and mounted human brain sections.

Comprehensive characterization of protein networks in mounted brain tissue represents a major challenge in brain and neurodegenerative disease research. In this study, we develop a simple staining method, called TSWIFT, to iteratively stain pre-mounted formalin fixed, paraffin embedded (FFPE) brain sections, thus enabling high-dimensional sample phenotyping. We show that TSWIFT conserves tissue architecture and allows for relabeling a single mounted FFPE sample more than 10 times, even after prolonged storage at 4 °C. Our results establish TSWIFT as an efficient method to obtain integrated high-dimensional knowledge of cellular proteomes by analyzing mounted FFPE human brain tissue.

TSWIFT, a novel method for iterative staining of embedded and mounted human brain sections.

Comprehensive characterization of protein networks in mounted brain tissue represents a major challenge in brain and neurodegenerative disease research. In this study, we develop a simple staining method, called TSWIFT, to iteratively stain pre-mounted formalin fixed, paraffin embedded (FFPE) brain sections, thus enabling high-dimensional sample phenotyping. We show that TSWIFT conserves tissue architecture and allows for relabeling a single mounted FFPE sample more than 10 times, even after prolonged storage at 4 °C. Using TSWIFT, we profile the abundance and localization of the HSP70 family chaperones HSC70 (HSPA8) and BiP (HSPA5) in mounted human brain tissue. Our results establish TSWIFT as an efficient method to obtain integrated high-dimensional knowledge of cellular proteomes by analyzing mounted FFPE human brain tissue.

Deletion of mFICD AMPylase alters cytokine secretion and affects visual short-term learning in vivo.

Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This posttranslational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mouse FICD (mFICD) AMPylase knockout mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well tolerated in unstressed mice. We also show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM, an antibody secreted early during infections, and the proinflammatory cytokine IL-1ß, without affecting the unfolded protein response. Finally, we demonstrate that visual nonspatial short-term learning is stronger in old mFICD-/- mice than in wild-type controls while other measures of cognition, memory, and learning are unaffected. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo.

Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code.

Cells must be able to cope with the challenge of folding newly synthesized proteins and refolding those that have become misfolded in the context of a crowded cytosol. One such coping mechanism that has appeared during evolution is the expression of well-conserved molecular chaperones, such as those that are part of the heat shock protein 70 (Hsp70) family of proteins that bind and fold a large proportion of the proteome. Although Hsp70 family chaperones have been extensively examined for the last 50 years, most studies have focused on regulation of Hsp70 activities by altered transcription, co-chaperone "helper" proteins, and ATP binding and hydrolysis. The rise of modern proteomics has uncovered a vast array of post-translational modifications (PTMs) on Hsp70 family proteins that include phosphorylation, acetylation, ubiquitination, AMPylation, and ADP-ribosylation. Similarly to the pattern of histone modifications, the histone code, this complex pattern of chaperone PTMs is now known as the "chaperone code." In this review, we discuss the history of the Hsp70 chaperone code, its currently understood regulation and functions, and thoughts on what the future of research into the chaperone code may entail.

Lactoferrin CpG Island Hypermethylation and Decoupling of mRNA and Protein Expression in the Early Stages of Prostate Carcinogenesis.

Lactoferrin (LTF) is an iron-binding protein canonically known for its innate and adaptive immune functions. LTF may also act as a tumor suppressor with antiproliferative action. LTF is inactivated genetically or epigenetically in various cancers, and a CpG island spanning the transcriptional start site of LTF is hypermethylated in prostate cancer cell lines. We, therefore, hypothesized that LTF expression is silenced via CpG island hypermethylation in the early stages of prostate tumorigenesis carcinogenesis. Targeted methylation analysis was performed using a combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes, and laser-capture microdissection followed by bisulfite sequencing on DNA isolated from prostate tissue samples, including both primary and metastatic disease. LTF mRNA in situ hybridization and LTF protein immunohistochemistry were also performed. We report that the LTF CpG island is frequently and densely methylated in high-grade prostatic intraepithelial neoplasia, primary prostate carcinoma, and metastases. We further report a decoupling of lactoferrin mRNA and protein expression, including in lesions where LTF mRNA has presumably been silenced via CpG island methylation. We conclude that LTF mRNA expression is silenced in prostate tumorigenesis via hypermethylation, supporting a role for LTF as a prostate cancer tumor suppressor gene. Likewise, the frequency at which the LTF CpG island is methylated across samples suggests it is an important and conserved step in prostate cancer initiation.

The microbiome in prostate inflammation and prostate cancer.

BACKGROUND: The human microbiome may influence prostate cancer initiation and/or progression through both direct and indirect interactions. To date, the majority of studies have focused on direct interactions including the influence of prostate infections on prostate cancer risk and, more recently, on the composition of the urinary microbiome in relation to prostate cancer. Less well understood are indirect interactions of the microbiome with prostate cancer, such as the influence of the gastrointestinal or oral microbiota on pro- or anti-carcinogenic xenobiotic metabolism, and treatment response. METHODS: We review the literature to date on direct and indirect interactions of the microbiome with prostate inflammation and prostate cancer. RESULTS: Emerging studies indicate that the microbiome can influence prostate inflammation in relation to benign prostate conditions such as prostatitis/chronic pelvic pain syndrome and benign prostatic hyperplasia, as well as in prostate cancer. We provide evidence that the human microbiome present at multiple anatomic sites (urinary tract, gastrointestinal tract, oral cavity, etc.) may play an important role in prostate health and disease. CONCLUSIONS: In health, the microbiome encourages homeostasis and helps educate the immune system. In dysbiosis, a systemic inflammatory state may be induced, predisposing remote anatomical sites to disease, including cancer. The microbiome's ability to affect systemic hormone levels may also be important, particularly in a disease such as prostate cancer that is dually affected by estrogen and androgen levels. Due to the complexity of the potential interconnectedness between prostate cancer and the microbiome, it is vital to further explore and understand the relationships that are involved.