Breast adipose tissue-derived extracellular vesicles from obese women alter tumor cell metabolism.

Breast adipose tissue is an important contributor to the obesity-breast cancer link. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we find that long-term education of breast cancer cells with EVs obtained from breast adipose tissue of women who are overweight or obese (O-EVs) results in increased proliferation. RNA-seq analysis of O-EV-educated cells demonstrates increased expression of genes involved in oxidative phosphorylation, such as ATP synthase and NADH: ubiquinone oxidoreductase. O-EVs increase respiratory complex protein expression, mitochondrial density, and mitochondrial respiration in tumor cells. The mitochondrial complex I inhibitor metformin reverses O-EV-induced cell proliferation. Several miRNAs-miR-155-5p, miR-10a-3p, and miR-30a-3p-which promote mitochondrial respiration and proliferation, are enriched in O-EVs relative to EVs from lean women. O-EV-induced proliferation and mitochondrial activity are associated with stimulation of the Akt/mTOR/P70S6K pathway, and are reversed upon silencing of P70S6K. This study reveals a new facet of the obesity-breast cancer link with human breast adipose tissue-derived EVs causing metabolic reprogramming of breast cancer cells.

Sex-steroid hormones and risk of postmenopausal estrogen receptor-positive breast cancer: a case-cohort analysis.

PURPOSE: Sex-steroid hormones are associated with postmenopausal breast cancer but potential confounding from other biological pathways is rarely considered. We estimated risk ratios for sex-steroid hormone biomarkers in relation to postmenopausal estrogen receptor (ER)-positive breast cancer, while accounting for biomarkers from insulin/insulin-like growth factor-signaling and inflammatory pathways. METHODS: This analysis included 1208 women from a case-cohort study of postmenopausal breast cancer within the Melbourne Collaborative Cohort Study. Weighted Poisson regression with a robust variance estimator was used to estimate risk ratios (RRs) and 95% confidence intervals (CIs) of postmenopausal ER-positive breast cancer, per doubling plasma concentration of progesterone, estrogens, androgens, and sex-hormone binding globulin (SHBG). Analyses included sociodemographic and lifestyle confounders, and other biomarkers identified as potential confounders. RESULTS: Increased risks of postmenopausal ER-positive breast cancer were observed per doubling plasma concentration of progesterone (RR: 1.22, 95% CI 1.03 to 1.44), androstenedione (RR 1.20, 95% CI 0.99 to 1.45), dehydroepiandrosterone (RR: 1.15, 95% CI 1.00 to 1.34), total testosterone (RR: 1.11, 95% CI 0.96 to 1.29), free testosterone (RR: 1.12, 95% CI 0.98 to 1.28), estrone (RR 1.21, 95% CI 0.99 to 1.48), total estradiol (RR 1.19, 95% CI 1.02 to 1.39) and free estradiol (RR 1.22, 95% CI 1.05 to 1.41). A possible decreased risk was observed for SHBG (RR 0.83, 95% CI 0.66 to 1.05). CONCLUSION: Progesterone, estrogens and androgens likely increase postmenopausal ER-positive breast cancer risk, whereas SHBG may decrease risk. These findings strengthen the causal evidence surrounding the sex-hormone-driven nature of postmenopausal breast cancer.

Proliferation Patterns of MCF-7 Breast Cancer Cells in Lipoaspirate Conditioned Media.

INTRODUCTION: Autologous fat grafting (AFG) is a common technique used to enhance aesthetic outcomes in postmastectomy breast reconstruction patients. Adipokines are hormones secreted by adipose tissue that play a critical role in regulating metabolic processes and the immune system. However, dysregulated adipokine secretion and signaling can contribute to the development and progression of cancer by promoting angiogenesis, altering the immune response, and inducing the epithelial mesenchymal transition. We aimed to assess how breast cancer cells behave in conditioned media derived from fat grafting lipoaspirates and gain a better understanding of the potential interactions that may occur within the tumor microenvironment. METHODS: Patients who were undergoing AFG as a part of breast reconstruction at NY-Presbyterian/Weill Cornell Medical Center between March 2021 and July 2023 were consented and enrolled in the study. This study was approved by the Weill Cornell Medicine Institutional Review Board (#20-10022850-14). Conditioned media is created using 20% of patient lipoaspirate secretome and 80% starving media. The growth of MCF-7, a human ER/PR+ breast cancer cell line, in conditioned media is assessed using CyQUANT. RESULTS: The breast cancer cells incubated in conditioned media displayed similar growth trends as those in complete media, which is enriched for cell growth (P > 0.05). MCF-7 cell behavior in conditioned media differed significantly from their proliferation patterns when serum starved in 100% starving media (P < 0.05). DISCUSSION: Our results suggest that there may be inherent factors within the lipoaspirate that may promote MCF-7 proliferation. One potential implication is that AFG used for breast reconstruction should be delayed until local-regional disease control has been established. In addition, based on the in vitro proliferation patterns of breast cancer cells in conditioned media, the safety profile of AFG may be enhanced if the procedure is performed after attaining negative margins and the completion breast cancer treatment.

Assessing Long-Term Volume Retention in Breast Fat Grafting: A Comparative Study of Lipoaspirate Processing Techniques.

INTRODUCTION: Autologous fat grafting is a method of improving aesthetic outcomes after both breast reconstruction and aesthetic surgery through volume enhancement and tissue contouring. Long-lasting effects are linked to greater patient satisfaction and more optimal augmentation results. Harvesting, processing, and injection techniques may all affect the longevity of deformity filling. Our objective is to evaluate the effect of lipoaspirate processing modality on longitudinal volume retention after surgery. METHODS: A prospective, single-institution, randomized control trial placed consented postmastectomy fat grafting patients into 1 of 3 treatment arms (active filtration, low-pressure decantation, and standard decantation) in a 1:1:1 ratio. A preoperative 3-dimensional scan of the upper torso was taken as baseline. At the 3-month postoperative visit, another 3D scan was taken. Audodesk Meshmixer was used to evaluate the volume change. RESULTS: The volume of fat injected during the initial procedure did not differ significantly between the treatment arms (P > 0.05). Both active filtration and low-pressure decantation resulted in higher percentage volume retention than traditional decantation (P < 0.05). Active filtration and low-pressure decantation exhibited comparable degrees of fat maintenance at 3 months (P > 0.05). DISCUSSION: Compared with using traditional decantation as the lipoaspirate purification technique, active filtration and low-pressure decantation may have led to higher levels of cell viability by way of reduced cellular debris and other inflammatory components that may contribute to tissue resorption and necrosis. Further immunohistochemistry studies are needed to examine whether active filtration and low-pressure decantation lead to lipoaspirates with more concentrated viable adipocytes, progenitor cells, and factors for angiogenesis.

Targeting obesity-related dysfunction in hormonally driven cancers.

Obesity is a risk factor for at least 13 different types of cancer, many of which are hormonally driven, and is associated with increased cancer incidence and morbidity. Adult obesity rates are steadily increasing and a subsequent increase in cancer burden is anticipated. Obesity-related dysfunction can contribute to cancer pathogenesis and treatment resistance through various mechanisms, including those mediated by insulin, leptin, adipokine, and aromatase signalling pathways, particularly in women. Furthermore, adiposity-related changes can influence tumour vascularity and inflammation in the tumour microenvironment, which can support tumour development and growth. Trials investigating non-pharmacological approaches to target the mechanisms driving obesity-mediated cancer pathogenesis are emerging and are necessary to better appreciate the interplay between malignancy, adiposity, diet and exercise. Diet, exercise and bariatric surgery are potential strategies to reverse the cancer-promoting effects of obesity; trials of these interventions should be conducted in a scientifically rigorous manner with dose escalation and appropriate selection of tumour phenotypes and have cancer-related clinical and mechanistic endpoints. We are only beginning to understand the mechanisms by which obesity effects cell signalling and systemic factors that contribute to oncogenesis. As the rates of obesity and cancer increase, we must promote the development of non-pharmacological lifestyle trials for the treatment and prevention of malignancy.

Creation and characterization of an immortalized canine myoblast cell line: Myok9.

The availability of an in vitro canine cell line would reduce the need for dogs for primary in vitro cell culture and reduce overall cost in pre-clinical studies. An immortalized canine muscle cell line, named Myok9, from primary myoblasts of a normal dog has been developed by the authors. Immortalization was performed by SV40 viral transfection of the large T antigen into the primary muscle cells. Proliferation assays, growth curves, quantitative PCR, western blotting, mass spectrometry, and light microscopy were performed to characterize the MyoK9 cell line at different stages of growth and differentiation. The expression of muscle-related genes was determined to assess myogenic origin. Myok9 cells expressed dystrophin and other muscle-specific proteins during differentiation, as detected with mass spectrometry and western blotting. Using the Myok9 cell line, new therapies before moving to pre-clinical studies to enhance the number and speed of analyses and reduce the cost of early experimentation can be tested now. This cell line will be made available to the research community to further evaluate potential therapeutics.

Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle-ear epithelial cells.

Nontypeable Haemophilus influenzae (NTHi), one of the most common acute otitis media (OM) pathogens, is postulated to promote middle-ear epithelial remodeling in the progression of OM from acute to chronic. The goal of this study was to examine early quantitative proteomic secretome effects of NTHi lysate exposure in a human middle-ear epithelial cell (HMEEC) line. NTHi lysates were used to stimulate HMEEC, and conditional quantitative stable isotope labeling with amino acids in cell culture of cell secretions was performed. Mass spectrometry analysis identified 766 proteins across samples. Of interest, several heterogeneous nuclear ribonucleoproteins (hnRNPs) were regulated by NTHi lysate treatment, especially hnRNP A2B1 and hnRNP Q, known to be implicated in microRNA (miRNA) packaging in exosomes. After purification, the presence of exosomes in HMEEC secretions was characterized by dynamic light scattering (<100 nm), transmission electron microscopy, and CD63/heat shock protein 70 positivity. hnRNP A2B1 and hnRNP Q were confirmed to be found in exosomes by Western blot and proteomic analysis. Finally, exosomal miRNA content comprised 110 unique miRNAs, with 5 found to be statistically induced by NTHi lysate (miR-378a-3p + miR-378i, miR-200a-3p, miR-378g, miR30d-5p, and miR-222-3p), all known to target innate immunity genes. This study demonstrates that NTHi lysates promote release of miRNA-laden exosomes from middle-ear epithelium in vitro. -Val, S., Krueger, A., Poley, M., Cohen, A., Brown, K., Panigrahi, A., Preciado, D. Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle-ear epithelial cells.

IL-10 suppresses TNF-α-induced expression of human aromatase gene in mammary adipose tissue.

White adipose tissue inflammation is linked with increased aromatase gene expression and estrogen production, a major risk factor for breast cancer in obese postmenopausal women. TNF-α, a proinflammatory cytokine, is a key driver of aromatase promoter I.4-mediated expression in adipose tissue. In this study, we have shown that IL-10, an anti-inflammatory cytokine, suppressed both TNF-α-stimulated human aromatase reporter-luciferase (hARO-Luc) expression in mouse bone marrow mesenchymal stromal cells and aromatase gene expression in human breast adipose stromal cells (ASCs). IL-10 blocked TNF-α-stimulated ERK1/2 activation in ASCs, suggesting an inhibitory effect through the MAPK signaling pathway. The links among obesity, IL-10, and aromatase were confirmed in ovariectomized (OVX) hARO-Luc mice, where increased adiposity was associated with upregulation of aromatase reporter activity and reduced IL-10 level in the mammary fat pad. OVX mice also exhibited changes in gut microbiota, similar to that in obese women, indicating altered immune function. In summary, our results suggest that increased adiposity, induced by the lack of ovarian hormones, results in enhanced expression of aromatase in mammary adipose tissue, mediated by reduction in local IL-10. These findings may bring new insights into the mechanisms involved in the development of postmenopausal breast cancer, as well as novel approaches for prevention.-Martínez-Chacón, G., Brown, K. A., Docanto, M. M., Kumar, H., Salminen, S., Saarinen, N., Mäkelä, S. IL-10 suppresses TNF-α-induced expression of human aromatase gene in mammary adipose tissue.

AMP-activated protein kinase signaling regulated expression of urea cycle enzymes in response to changes in dietary protein intake.

Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although urea cycle enzyme levels rise in response to a high-protein (HP) diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either HP or low-protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptomic data. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated adenosine monophosphate-activated protein kinase (AMPK) as a possible regulator. Primary human hepatocytes, treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) an activator of AMPK, were used to test whether AMPK regulates expression of urea cycle genes. The abundance of carbamoylphosphate synthetase 1 and ornithine transcarbamylase mRNA increased in hepatocytes treated with AICAR, which supports a role for AMPK signaling in regulation of the urea cycle. Because AMPK is either a target of drugs used to treat type-2 diabetes, these drugs might increase the expression of urea cycle enzymes in patients with partial urea cycle disorders, which could be the basis of a new therapeutic approach.

The benefits of adding metformin to tamoxifen to protect the endometrium-A randomized placebo-controlled trial.

BACKGROUND: We investigated whether metformin prevents tamoxifen-induced endometrial changes and insulin resistance (IR) after a diagnosis of breast cancer. METHODS: This was a single-centre, randomized, double-blind, placebo-controlled, parallel group trial. Postmenopausal women with hormone receptor-positive breast cancer taking tamoxifen were randomly allocated to metformin 850 mg or identical placebo, twice daily, for 52 weeks. Outcome measures included double endometrial thickness (ET) measured by transvaginal ultrasound, fasting insulin, glucose and IR estimated by the homeostasis model of assessment (HOMA-IR). RESULTS: A total of 112 women were screened and 102 randomized. Results are presented as median (range). The 101 women who took at least one dose of medication were aged 56 (43-72) years, with 5(0.5-28) years postmenopause, and had taken tamoxifen for 28.9 (0-367.4) weeks. The baseline ET was 2.9 mm (1.4-21.9) for the placebo group (n = 52) and 2.5 mm (1.3-14.8) for the metformin group (n = 50). At 52 weeks, the median ET was statistically significantly lower for the metformin (n = 36) than for the placebo group (n = 45) (2.3 mm (1.4-7.8) vs 3.0 (1.2-11.3); P = 0.05). 13.3% allocated to placebo had an ET greater than 4 mm vs 5.7% for metformin (P = 0.26). There was no endometrial atypia or cancer. Compared with placebo, metformin resulted in significantly greater baseline-adjusted reductions in weight (P < 0.001), waist circumference (0.03) and HOMA-IR (P < 0.001). CONCLUSIONS: Metformin appears to inhibit tamoxifen-induced endometrial changes and has favourable metabolic effects. Further research into the adjuvant use of metformin after breast cancer and to prevent EH and cancer is warranted.

Characterization of mucoid and serous middle ear effusions from patients with chronic otitis media: implication of different biological mechanisms?

BACKGROUND: Chronic otitis media with effusion (COME) is characterized by persistent middle ear effusions that are in most cases highly viscous, but some patients present with serous fluid. This study aimed at comprehensively characterizing the macromolecular composition of mucoid vs. serous middle ear effusions (MEEs). METHODS: MEEs from patients with COME were analyzed for proteins by mass spectrometry (MS) and western blot techniques, total DNA quantity, bacterial DNA (16S sequencing), and cytokine content. Proteomics datasets were studied in Ingenuity Pathway Analysis (IPA). RESULTS: Mucoid samples showed a global tendency of increased pro-inflammatory mediators. Interleukin-1ß (IL-1ß) and IL-10 were significantly more abundant in serous samples (p < 0.01). Mucoid samples had higher DNA quantity (p = 0.04), more likely to be positive in MUC5B protein (p = 0.008) and higher peptide counts (12,786 vs. 2225), as well as an overall larger number of identified proteins (331 vs. 177), compared to serous. IPA found the mucoid sample dataset to be related to immune cell function and epithelial remodeling, whereas the serous sample dataset showed acute responses and blood-related proteins. Interestingly, serous samples showed more bacterial DNA than mucoid ones, with less bacterial genera variability. CONCLUSION: This study demonstrates divergent immune responses in children with COME by effusion quality.

A 3-Dimensional Biomimetic Platform to Interrogate the Safety of Autologous Fat Transfer in the Setting of Breast Cancer.

INTRODUCTION: Obesity is a known risk factor for the development and prognosis of breast cancer. Adipocytes have been identified as a source of exogenous lipids in other cancer types and may similarly provide energy to fuel malignant survival and growth in breast cancer. This relationship is of particular relevance to plastic surgery, because many reconstructions after oncologic mastectomy achieve optimal aesthetics and durability using adjunctive autologous fat transfer (AFT). Despite the increasing ubiquity and promise of AFT, many unanswered questions remain, including safety in the setting of breast cancer. Clinical studies to examine this question are underway, but an in vitro system is critical to elucidate the complex interplay between the cells that normally reside at the surgical recipient site. To study these interactions and characterize possible lipid transfer between adipocytes to breast cancer cells, we designed a 3-dimensional in vitro model using primary patient-derived tissues. METHODS: Breast adipose tissue was acquired from patients undergoing breast reduction surgery. The tissue was enzymatically digested and sorted to retrieve adipocytes and adipose stromal cells. Polydimethylsiloxane wells were filled with type I collagen-encapsulated adipocytes labeled with the fluorescent lipid dye boron dipyrromethene, as well as unlabeled adipose stromal cells. A monolayer of red fluorescently labeled MDA-MB-231 and MDA-MB-468 breast cancer cells was seeded on the surface of the construct. Lipid transfer at the interface between adipocytes and breast cancer cells was analyzed. RESULTS: Confocal microscopy revealed a dense culture of native adipocytes containing fluorescent lipid droplets in the 3-dimensional collagen culture platform. RFP-positive breast cancer cells were found in close proximity to lipid-laden adipocytes. Lipid transfer from adipocytes to breast cancer cells was observed by the presence of boron dipyrromethene-positive lipid droplets within RFP-labeled breast cancer cells. CONCLUSION: We have established a 3-dimensional model to study complex breast cancer-adipose tissue interactions. Direct transfer of fluorescently labeled lipids from adipocytes to breast cancer cells may indicate aberrant metabolism to fuel malignant growth and adaptive survival. Our novel platform can untangle the complex interplay within the breast cancer tumor microenvironment for high-throughput analysis and better elucidate the safety of AFT in postoncologic mastectomy.

Hsp90 and PKM2 Drive the Expression of Aromatase in Li-Fraumeni Syndrome Breast Adipose Stromal Cells.

Li-Fraumeni syndrome (LFS) patients harbor germ line mutations in the TP53 gene and are at increased risk of hormone receptor-positive breast cancers. Recently, elevated levels of aromatase, the rate-limiting enzyme for estrogen biosynthesis, were found in the breast tissue of LFS patients. Although p53 down-regulates aromatase expression, the underlying mechanisms are incompletely understood. In the present study, we found that LFS stromal cells expressed higher levels of Hsp90 ATPase activity and aromatase compared with wild-type stromal cells. Inhibition of Hsp90 ATPase suppressed aromatase expression. Silencing Aha1 (activator of Hsp90 ATPase 1), a co-chaperone of Hsp90 required for its ATPase activity, led to both inhibition of Hsp90 ATPase activity and reduced aromatase expression. In comparison with wild-type stromal cells, increased levels of the Hsp90 client proteins, HIF-1α, and PKM2 were found in LFS stromal cells. A complex comprised of HIF-1α and PKM2 was recruited to the aromatase promoter II in LFS stromal cells. Silencing either HIF-1α or PKM2 suppressed aromatase expression in LFS stromal cells. CP-31398, a p53 rescue compound, suppressed levels of Aha1, Hsp90 ATPase activity, levels of PKM2 and HIF-1α, and aromatase expression in LFS stromal cells. Consistent with these in vitro findings, levels of Hsp90 ATPase activity, Aha1, HIF-1α, PKM2, and aromatase were increased in the mammary glands of p53 null versus wild-type mice. PKM2 and HIF-1α were shown to co-localize in the nucleus of stromal cells of LFS breast tissue. Taken together, our results show that the Aha1-Hsp90-PKM2/HIF-1α axis mediates the induction of aromatase in LFS.

Neurofilament light polypeptide gene N98S mutation in mice leads to neurofilament network abnormalities and a Charcot-Marie-Tooth Type 2E phenotype.

Charcot-Marie-Tooth disease (CMT) is the most commonly inherited neurological disorder with a prevalence of 1 in 2500 people worldwide. Patients suffer from degeneration of the peripheral nerves that control sensory information of the foot/leg and hand/arm. Multiple mutations in the neurofilament light polypeptide gene, NEFL, cause CMT2E. Previous studies in transfected cells showed that expression of disease-associated neurofilament light chain variants results in abnormal intermediate filament networks associated with defects in axonal transport. We have now generated knock-in mice with two different point mutations in Nefl: P8R that has been reported in multiple families with variable age of onset and N98S that has been described as an early-onset, sporadic mutation in multiple individuals. Nefl(P8R/+) and Nefl(P8R/P8R) mice were indistinguishable from Nefl(+/+) in terms of behavioral phenotype. In contrast, Nefl(N98S/+) mice had a noticeable tremor, and most animals showed a hindlimb clasping phenotype. Immunohistochemical analysis revealed multiple inclusions in the cell bodies and proximal axons of spinal cord neurons, disorganized processes in the cerebellum and abnormal processes in the cerebral cortex and pons. Abnormal processes were observed as early as post-natal day 7. Electron microscopic analysis of sciatic nerves showed a reduction in the number of neurofilaments, an increase in the number of microtubules and a decrease in the axonal diameters. The Nefl(N98S/+) mice provide an excellent model to study the pathogenesis of CMT2E and should prove useful for testing potential therapies.

The presence and impact of estrogen metabolism on the biology of triple-negative breast cancer.

PURPOSE: While triple-negative breast cancer (TNBC) is negative for estrogen receptor alpha, a substantial proportion of carcinomas express estrogen receptor beta (ERß); consequently, estrogen actions and metabolism may be relevant in this cancer subtype. METHODS: A cohort of 81 TNBC patients from Tohoku University Hospital, Japan were characterised with regard to the expression of estrogen receptor beta and enzymes known to modulate levels of estrogens in breast and other tissues (Aromatase, 17-beta- Hydroxysteroid dehydrogenases 1, 2 and 6). This was done at the protein level by means of immunohistochemistry. As this cohort has been previously characterised for androgens, this also allows for comparison between the expressions of estrogen-related proteins and of androgen-related proteins. Preliminary mechanistic studies in cell culture were also undertaken. RESULTS: 17ßHSD2 was detected in the highest number of cases followed by 17ßHSD1, 17ßHSD6 and aromatase. When comparing the expression of ERß with that of the enzymes, it was positively correlated with the expression of 17ßHSD6 (p < 0.05) and trended towards correlation with dual expression of 17ßHSD1 and 2 (p < 0.07). 17ßHSD1 was associated with significantly reduced tumour volume (p = 0.0025), while ERß was associated with a trend towards reduced lymphovascular invasion, (p < 0.061). Interestingly, in survival analysis, 17ßHSD6 expression was the only one of these five factors that influenced survival, with positive samples being associated with longer disease-free survival compared to those that were negative for 17ßHSD6 (p < 0.05). In assessing associations with expression of proteins in the androgenic pathway, expression of aromatase appeared to be associated with androgenic pathways in TNBC patients (p < 0.05). Due to this association and the potential relevance to androgen-directed therapies in TNBC, we evaluated this interaction in vitro. We observed androgen-dependent upregulation of aromatase and ERß in a subset of AR expressing TNBC cell lines (MDA-MB-453, SUM-185-PE and MFM-223). CONCLUSION: Overall this study suggests the presence of, and a potential protective effect of estrogens in TNBC.

Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology.

BACKGROUND: Otitis media (OM) is characterized by acute infection progressing to chronic middle ear effusion (MEE). Extracellular secretion of microRNAs (miRNAs) in exosomes is a newly discovered mechanism for cells exerting distant cell genetic regulation. Whether MEE contains exosomes with specific miRNAs is unknown. This study aimed to purify and characterize the exosomal and miRNA content of MEE. METHOD: MEEs were subjected to Exoquick exosomal purification and EXOCET exosomal quantification. Extracted vesicles were analyzed by dynamic light scattering (DLS), transmission electron microscopy (TEM), and immunoblotting of HSP-70. NanoString hybridization was performed to profile miRNAs. Exosomal protein content was profiled by Liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: EXOCET assays showed presence of exosomes (0-0.5 × 107/ml) in MEEs. DLS confirmed exosomal size between 10 and 200 nm. Western blot analysis showed presence of HSP-70. Twenty-nine miRNAs were found to be unique to MEEs. The most abundant miRNA was miR-223, a miRNA typically secreted by neutrophils. Proteomics demonstrated typical neutrophil markers as well as common innate immune molecules. CONCLUSION: To our knowledge, this the first report demonstrating the presence of exosomes transporting miRNAs in MEEs. These findings open a broad and novel area of research in OM pathophysiology as driven by miRNA cell communication.

Functional role of RNA polymerase II and P70 S6 kinase in KCl withdrawal-induced cerebellar granule neuron apoptosis.

KCl withdrawal-induced apoptosis in cerebellar granule neurons is associated with aberrant cell cycle activation, and treatment with cyclin-dependent kinase (Cdk) inhibitors protects cells from undergoing apoptosis. Because the Cdk inhibitor flavopiridol is known to inhibit RNA polymerase II (Pol II)-dependent transcription elongation by inhibiting the positive transcription elongation factor b (P-TEFb, a complex of CDK9 and cyclin T), we examined whether inhibition of RNA Pol II protects neurons from apoptosis. Treatment of neurons with 5, 6-dichloro-1-ß-D-ribobenzimidazole (DRB), an RNA Pol II-dependent transcription elongation inhibitor, and flavopiridol inhibited phosphorylation and activation of Pol II and protected neurons from undergoing apoptosis. In addition to Pol II, neurons subjected to KCl withdrawal showed increased phosphorylation and activation of p70 S6 kinase, which was inhibited by both DRB and flavopiridol. Immunostaining analysis of the neurons deprived of KCl showed increased nuclear levels of phospho-p70 S6 kinase, and neurons protected with DRB and flavopiridol showed accumulation of the kinase into large spliceosome assembly factor-positive speckle domains within the nuclei. The formation of these foci corresponded with cell survival, and removal of the inhibitors resulted in dispersal of the speckles into smaller foci with subsequent apoptosis induction. Because p70 S6 kinase is known to induce translation of mRNAs containing a 5'-terminal oligopyrimidine tract, our data suggest that transcription and translation of this subset of mRNAs may contribute to KCl withdrawal-induced apoptosis in neurons.

Discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to Duchenne muscular dystrophy patients.

It is expected that serum protein biomarkers in Duchenne muscular dystrophy (DMD) will reflect disease pathogenesis, progression and aid future therapy developments. Here, we describe use of quantitative in vivo stable isotope labeling in mammals to accurately compare serum proteomes of wild-type and dystrophin-deficient mdx mice. Biomarkers identified in serum from two independent dystrophin-deficient mouse models (mdx-Δ52 and mdx-23) were concordant with those identified in sera samples of DMD patients. Of the 355 mouse sera proteins, 23 were significantly elevated and 4 significantly lower in mdx relative to wild-type mice (P-value < 0.001). Elevated proteins were mostly of muscle origin: including myofibrillar proteins (titin, myosin light chain 1/3, myomesin 3 and filamin-C), glycolytic enzymes (aldolase, phosphoglycerate mutase 2, beta enolase and glycogen phosphorylase), transport proteins (fatty acid-binding protein, myoglobin and somatic cytochrome-C) and others (creatine kinase M, malate dehydrogenase cytosolic, fibrinogen and parvalbumin). Decreased proteins, mostly of extracellular origin, included adiponectin, lumican, plasminogen and leukemia inhibitory factor receptor. Analysis of sera from 1 week to 7 months old mdx mice revealed age-dependent changes in the level of these biomarkers with most biomarkers acutely elevated at 3 weeks of age. Serum analysis of DMD patients, with ages ranging from 4 to 15 years old, confirmed elevation of 20 of the murine biomarkers in DMD, with similar age-related changes. This study provides a panel of biomarkers that reflect muscle activity and pathogenesis and should prove valuable tool to complement natural history studies and to monitor treatment efficacy in future clinical trials.

Clinical utility of serum biomarkers in Duchenne muscular dystrophy.

Assessments of disease progression and response to therapies in Duchenne muscular dystrophy (DMD) patients remain challenging. Current DMD patient assessments include complex physical tests and invasive procedures such as muscle biopsies, which are not suitable for young children. Defining alternative, less invasive and objective outcome measures to assess disease progression and response to therapy will aid drug development and clinical trials in DMD. In this review we highlight advances in development of non-invasive blood circulating biomarkers as a means to assess disease progression and response to therapies in DMD.