Association between Estimated Cardiorespiratory Fitness and Abnormal Glucose Risk: A Cohort Study.

BACKGROUND: Cardiorespiratory fitness (CRF) is a predictor of chronic disease that is impractical to routinely measure in primary care settings. We used a new estimated cardiorespiratory fitness (eCRF) algorithm that uses information routinely documented in electronic health care records to predict abnormal blood glucose incidence. METHODS: Participants were adults (17.8% female) 20-81 years old at baseline from the Aerobics Center Longitudinal Study between 1979 and 2006. eCRF was based on sex, age, body mass index, resting heart rate, resting blood pressure, and smoking status. CRF was measured by maximal treadmill testing. Cox proportional hazards regression models were established using eCRF and CRF as independent variables predicting the abnormal blood glucose incidence while adjusting for covariates (age, sex, exam year, waist girth, heavy drinking, smoking, and family history of diabetes mellitus and lipids). RESULTS: Of 8602 participants at risk at baseline, 3580 (41.6%) developed abnormal blood glucose during an average of 4.9 years follow-up. The average eCRF of 12.03 ± 1.75 METs was equivalent to the CRF of 12.15 ± 2.40 METs within the 10% equivalence limit. In fully adjusted models, the estimated risks were the same (HRs = 0.96), eCRF (95% CIs = 0.93-0.99), and CRF (95% CI of 0.94-0.98). Each 1-MET increase was associated with a 4% reduced risk. CONCLUSIONS: Higher eCRF is associated with a lower risk of abnormal glucose. eCRF can be a vital sign used for research and prevention.

Prevalence of Cardiovascular Diseases Among Breast Cancer Survivors: Findings From the NHANES 2003-2018.

PURPOSE: To examine the prevalence of cardiovascular diseases (CVD) among breast cancer (BC) survivors. DESIGN: Cross-sectional observational study using the data from the National Health and Nutrition Examination Survey (NHANES) 2003-2018. SETTING: United States (US). SUBJECTS: A nationally representative sample of US women with a history of BC. MEASURES: Self-reported CVD status (i.e., coronary artery disease (CAD), heart failure, and stroke) and time of the CVD diagnosis were used to categorize BC survivors into three groups: No CVD, preexisting CVD, and post-acquired CVD after BC diagnosis. ANALYSIS: The prevalence of CVD among BC survivors were estimated by demographic characteristics. Complex sampling design of the NHANES was accounted to estimate the population-level prevalence. RESULTS: A total of 658 BC survivors were identified, representing 3.01% (≈3.4 million) of the US women aged ≥18 years old. Of those, ≈6% (≈.2 million) had preexisting CVD and ≈11% (≈.4 million) had at least one CVD diagnosed after BC diagnosis, with an average time elapsed ranging from ≈5 years for heart failure to ≈9 years for CAD and stroke. The prevalence of CVD among BC survivors differed by demographic characteristics including age, education, marital status, menopausal, and physical activity levels. CONCLUSION: Our findings suggest that BC survivors are at risk of suffering from CVD and public health strategies for the long-term management of CVD risk factors in this vulnerable population group is recommended.

Terrorism and the internet: How dangerous is online radicalization?

This work is concerned with the extent and magnitude of threat related to online radicalization in the context of terrorist acts and related offending. Online influences have been depicted as major drivers for the propagation and adoption of extremist ideologies, which often contain an element of collective grievance, and subsequent acts of violence. This is most pronounced in the discussion of so-called lone actor terrorism, but extends to all forms of extremist offending, and beyond. The present work situates online radicalization leading to terrorist acts within the wider context of grievance-based beliefs and attitudes. Further, it addresses current positions and debates surrounding the relevance and mechanisms of online radicalization in terrorist offending. Recent evidence from quantitative studies is reviewed to estimate prevalence of online radicalization and the level of threat that results from it. This is followed by a discussion of plausible, but opposing, interpretations of the estimates presented. While online radicalization does occur, with and without reference to offline processes, the resulting threat is not overly high. This assessment, however, refers to the present only and is unlikely to hold for the future, given the general growth and acceleration of online activity among terrorist actors.

Imaging of Tumor-Associated Vascular Prostate-Specific Membrane Antigen in Woodchuck Model of Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Radiolabeled short peptide ligands targeting prostate-specific membrane antigen (PSMA) were developed initially for imaging and treatment of prostate cancers. While many nonprostate solid tumors including hepatocellular carcinoma (HCC) express little PSMA, their neovasculature expresses a high level of PSMA, which is avid for Gallium-68-labeled PSMA-targeting radio-ligand (68Ga-PSMA-11) for positron emission tomography (PET). However, the lack of a spontaneous animal model of tumor-associated vascular PSMA overexpression has hindered the development and assessment of PSMA-targeting radioligands for imaging and therapy of the nonprostatic cancers. We identified detectable indigenous PSMA expression on tumor neovascular endothelia in a naturally occurring woodchuck model of HCC. METHODS: Molecular docking was performed with 3 bait PSMA ligands and compared between human and woodchuck PSMA. Initially, PET images were acquired dynamically after intravenously injecting 37 MBq (1.0 mCi) of 68Ga-PSMA-11 into woodchuck models of HCC. Subsequently, 10-minute static PET scans were conducted for other animals 1-hour after injection due to HCC and liver background uptake stabilization at 30-45 minutes after injection. Liver tissue samples were harvested after imaging, fresh-frozen for quantitative reverse transcription polymerase chain reaction and western blot for validation, or fixed for histology for correlation. RESULTS: Our preclinical studies confirmed the initial clinical findings of 68Ga-PSMA-11 uptake in HCC. The agents (ligands and antibodies) developed against human PSMA were found to be reactive against the woodchuck PSMA. CONCLUSION: This animal model offers a unique opportunity for investigating the biogenesis of tumor-associated vascular PSMA, its functional role(s), and potentials for future treatment strategies targeting tumor vascular PSMA using already developed PSMA-targeting agents.

Physical activity, sedentary behaviors and all-cause mortality in patients with heart failure: Findings from the NHANES 2007-2014.

BACKGROUND: Limited data are available examining the effects of both moderate- and vigorous-intensity physical activity (MVPA) and sedentary behavior (SB) on longevity among patients with heart failure (HF). This study examined the associations of MVPA and SB with all-cause mortality in HF patients using a nationally representative survey data. METHODS: National Health and Nutrition Examination Survey data (2007-2014) were used. 711 adults with self-reported congestive HF, linked to 2015 mortality data were analyzed. Self-reported MVPA and SB minutes were used to create the three MVPA [No-MVPA, insufficient (I-MVPA; <150 min/wk), and sufficient (S-MVPA; ≥150 min/wk)] and two SB (<8 and ≥8 hrs/d) groups. Cox proportional hazard models were constructed to test the associations of MVPA and SB with all-cause mortality. RESULTS: 119 deaths occurred over an average of 4.9 years of follow-up. Lower MVPA and higher SB were independently associated with poor survival (P < .001). Joint and stratified analyses showed that the protective effect of MVPA was most pronounced among patients with SB<8 hrs/d. There was no difference in the mortality risk by SB levels within I-MVPA and S-MVPA groups; however, in the No-MVPA group, those with SB≥8 hrs/d had a greater risk of mortality compared to those with <8 hrs/d (Hazard ratio = 1.60). CONCLUSION: In this HF cohort, MVPA and SB were independently and jointly associated with all-cause mortality. The beneficial effect of MVPA is attenuated by excessive SB; however, engaging in some amount of MVPA may provide a protective effect and attenuates the detrimental effects associated with excessive SB.

Effects of Replacing Sedentary Time With Physical Activity on Mortality Among Patients With Heart Failure: National Health and Nutrition Examination Survey Follow-Up Study.

A sedentary lifestyle is prevalent among patients with heart failure (HF) and is associated with poor prognosis and survival, possibly owing to the displacement of health-enhancing behaviors, such as physical activity (PA). However, there is limited evidence examining the displacement effects of reducing duration of sedentary time (ST) on clinical outcomes in patients with HF. The current study examined the theoretical effects of relocating ST with PA on all-cause and cardiovascular disease (CVD)-specific mortality risks in patients with HF. We analyzed 265 patients with HF who participated in the National Health and Nutrition Examination Survey from 2003 to 2006. Cox proportional hazards regression model was fitted to estimate mortality risks based on objectively measured ST well as time spent in light-intensity PA (LPA) and moderate- and vigorous-intensity PA (MVPA). The theoretical changes in the hazard ratio (HR) by replacing ST with LPA or MVPA were examined using isotemporal substitutional modeling. On average, patients with HF spent 70% of waking hours per day in ST (9.01 hours), followed by LPA (29%; 3.75 hours) and MVPA (1%; 0.13 hours). Ten-minute substitution of ST with LPA was associated with significantly lower all-cause and CVD-specific mortality risks (hazard ratio [HR]=0.93 for both). The mortality risks progressively decreased as more ST was relocated to LPA. The relocation effects of ST with MVPA were not statistically significant, possibly because of limited MVPA accrued in this clinical population. The current study provides empirical evidence about the potential health benefits of replacing a modest amount of ST with LPA among patients with HF.

Peak oxygen consumption achieved at the end of cardiac rehabilitation predicts long-term survival in patients with coronary heart disease.

AIMS: Cardiac rehabilitation (CR) improves survival in patients with coronary heart disease (CHD), which is largely mediated by the improvements in cardiorespiratory fitness (CRF) defined as peak oxygen consumption (VO2). Therefore, measuring CRF is essential to predict long-term outcomes in this population. It is unclear, however, whether peak VO2 achieved at the end of CR (END-peak VO2) predicts survival or whether the changes of CRF achieved during CR provide a greater prognostic value. To determine whether END-peak VO2 independently predicts long-term survival in patients with CHD undergoing CR. We also aimed at identifying cut-offs for END-peak VO2 that could be used in clinical practice. METHODS AND RESULTS: Retrospective analysis of 853 patients with CHD referred to CR who completed a maximal cardiopulmonary exercise test. Survival analysis was performed to examine the risk of all-cause mortality (average follow-up years: 6.65) based on peak VO2. The Contal and O'Quigley's method was used to determine the optimal cut-off of END-peak VO2 based on the log-rank statistic. END-peak VO2 was inversely associated with mortality risk [hazard ratio (HR) = 0.84; 95% confidence interval (CI) = 0.78-0.90], independent of changes in peak VO2 adjusted for the baseline peak VO2. The estimated cut-off of END-peak VO2 at ≥17.6 mL/kg/min best predicted the survival with high predictive accuracy and patients with END-peak VO2 under the cut-off had a greater risk of mortality (HR = 2.93; 95% CI = 1.81-4.74). CONCLUSIONS: In patient with CHD undergoing CR, END-peak VO2 is an independent predictor for long-term survival. Studies utilizing higher intensity CR programmes, with and without pharmacologic strategies, to increase peak VO2 to a greater degree in those achieving a suboptimal END-peak VO2, are urgently needed.

Physical Activity Levels of 1053 Omani 4th Grade Children: The Importance of Gender and Sport Team Participation in Achieving 60 Minutes of Daily Moderate-to-Vigorous Physical Activity.

Sufficient daily physical activity is associated with many positive mental, physical, and societal benefits in children. Unfortunately, most children worldwide do not achieve recommended levels of daily physical activity (PA), and a majority of evidence is from Western countries and based on subjective measures. This study examined the prevalence and correlates of objectively measured PA levels among Omani children in 2017 (pre-pandemic). A two-stage cluster sampling was used to recruit the 4th grade children across five regions of Oman. A final analytic sample included 1053 children (504 boys, 549 girls) with a mean age of 9.21 years old. PA was objectively measured using a wrist-worn Polar Active Watch during three consecutive school days. Screen-based sedentary behaviors and other PA-related behaviors were subjectively measured. On average, boys were less sedentary and more active, with a greater likelihood of meeting current recommendations when compared with girls. The self-reported time spent in screen-based sedentary behaviors was relatively low for both boys and girls and was not associated with PA; however, sports team participation was associated with a greater likelihood of meeting the current recommendation. The present study provides empirical data on objectively measured PA in Omani children. The gender disparities concerning daily PA, including sports team participation, should receive further attention.

Acetic Acid Supplementation: Effect on Resting and Exercise Energy Expenditure and Substrate Utilization.

The purpose of this study was to investigate the influence of acetic acid (apple cider vinegar; ACV) supplementation on resting and exercise energy expenditure and substrate utilization. Using a randomized, double blind, crossover design, 16 healthy subjects were supplemented for 4 d with either ACV (30-ml/d) mixed in 1 L of a non-nutritive lemon-flavored drink or a placebo (PLA). They were then assessed via indirect calorimetry for resting energy expenditure (REE) and substrate utilization. This was immediately followed by the assessment of steady state cycling exercise energy expenditure at 40 W (EEE-40) and 80 W (EEE-80) and substrate utilization. Results: Neither REE nor resting substrate utilization were significantly different between groups (p ≥ .05). During cycling exercise at both 40W and 80W, there were no significant differences observed between groups for energy expenditure (EEE-40: ACV 4.13 ± 0.79, PLA 4.37 ± 0.61 kcal/min; EEE-80: ACV 6.09 ± 0.87, PLA 6.26 ± 0.72 kcal/min) or substrate utilization (40W carbohydrate: ACV 0.72 ± 0.19, PLA 0.76 ± 0.16; fat: ACV 0.15 ± 0.07, PLA 0.16 ± 0.06 g/min), (80W carbohydrate: ACV 1.28 ± 0.32, PLA 1.34 ± 0.35; fat: ACV 0.14 ± 0.10, PLA 0.14 ± 0.10 g/min) (p ≥ .05). Conclusions: Recent findings suggest that chronic acetic acid supplementation is associated with significant reductions in body weight and body fat; however, the findings of the present study suggest that a semi-acute (4 d) acetic acid supplementation does not impact resting or exercise energy expenditure or substrate utilization.

Mesenchymal stromal cell mitochondrial transfer to human induced T-regulatory cells mediates FOXP3 stability.

The key obstacle to clinical application of human inducible regulatory T cells (iTreg) as an adoptive cell therapy in autoimmune disorders is loss of FOXP3 expression in an inflammatory milieu. Here we report human iTreg co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) during short-term ex vivo expansion enhances the stability of iTreg FOXP3 expression and suppressive function in vitro and in vivo, and further that a key mechanism of action is MSC mitochondrial (mt) transfer via tunneling nanotubules (TNT). MSC mt transfer is driven by mitochondrial metabolic function (CD39/CD73 signaling) in proliferating iTreg and promotes iTreg expression of FOXP3 stabilizing factors BACH2 and SENP3. These results elucidate cellular and molecular mechanisms underlying human MSC mt transfer to proliferating cells. MSC mt transfer stabilizes FOXP3 expression in iTregs, thereby enhancing and sustaining their suppressive function in inflammatory conditions in vitro and in vivo.

Liver background uptake of [18F]FLT in PET imaging.

High liver uptake presents a problem for 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) as a radiotracer for imaging cellular proliferation in the liver with positron emission tomography (PET). This investigation re-visited some issues related to the high liver background uptake of [18F]FLT with an animal model of woodchucks. Several enzymes involved in the hepatic catabolism of FLT, thymidine phosphorylase (TP, TYMP), uridine 5'-diphospho-glucuronosyl-transferases (UDP-GTs, short for UGTs), and ß-glucuronidase (GUSB), their homology as well as hepatic expression between the human and the woodchuck was examined. Inhibitors of these enzymes, TP inhibitor (TPI) tipiracil hydrochloride, UGT inhibitor probenecid, ß-glucuronidase inhibitor L-aspartate, were administered to the animals at human equivalent doses either intravenously (i.v.) and orally before the injection of tracer-dose [18F]FLT for PET imaging to examine any changes in liver uptake. Liver tissue samples were harvested from the animals after PET imaging and used to perform polymerase chain reaction (PCR) for TP expression or assays for enzymatic activities of TP and ß-glucuronidase. Non-radiolabeled (cold) FLT was also applied for enzyme saturation. Animals administered with TPI displayed lower radioactivity in the liver in comparison with the baseline scan. The application of probenecid did not change [18F]FLT liver uptake even though it reduced renal uptake. L-aspartate reduced the liver background uptake of [18F]FLT slightly. The application of cold FLT reduced overall uptake of [18F]FLT including the liver background. Therefore, the combined application of cold FLT and [18F]FLT merits further clinical investigation for reducing liver background uptake of [18F]FLT.

Transcriptomic Analysis of Human Mesenchymal Stem Cell Therapy in Incontinent Rat Injured Urethra.

Periurethral human mesenchymal stem cell (hMSC) injections are associated with functional improvement in animal models of postpartum stress urinary incontinence (SUI). However, limited data exist on the role of hMSCs in modulating gene expression in tissue repair after urethral injury. To this end, we quantified temporal gene expression modulation in hMSCs, and in injured rat urethral tissue, using RNA-seq in an animal model of SUI, over a 3-day period following urethral injury, and local hMSC injection. We injected PKH fluorescent-labeled hMSC into the periurethral space of rats following a 4 h vaginal distention (VD) (three rats per time point). Control rats underwent VD injury only, and all animals were euthanized at 12, 24, 36, 72 h postinjury. Rat urethral and vaginal tissues were frozen and sectioned. Fluorescent labeled hMSCs were distinguished from adjacent, unlabeled rat urethral tissue. RNA was prepared from hMSCs and urethral tissue obtained by laser dissection of frozen tissue sections and sequenced on an Illumina HiSeq 2500. Differentially expressed genes (DEGs) over 72 h were evaluated using a two-group t-test (p < 0.05). Our transcriptional analyses identified candidate genes involved in tissue injury that were broadly sorted by injury and exposure to hMSC throughout the first 72 h of acute phase of injury. DEGs in treated urethra, compared with untreated urethra, were functionally associated with tissue repair, angiogenesis, neurogenesis, and oxidative stress suppression. DEGs included a variety of cytokines, extracellular matrix stabilization and regeneration genes, cytokine signaling modification, cell cycle regulation, muscle differentiation, and stabilization. Moreover, our results revealed DEG changes in hMSCs (PKH-labeled) harvested from injured urethra. The expressions are related to DNA damage repair, transcription activation, stem cell regulation, cell survival, apoptosis, self-renewal, cell proliferation, migration, and injury response. Impact statement Stress urinary incontinence (SUI) affects nearly half of women over 40, resulting in reduced quality of life and increased health care cost. Development of SUI is multifactorial and strongly associated with vaginal delivery. While stem cell therapy in animal models of SUI and limited preliminary clinical trials demonstrate functional improvement of SUI, the role of stem cell therapy in modulating tissue repair is unclear impeding advanced clinical trials. Our work provides a new understanding of the transcriptional mechanisms with which human mesenchymal stem cells improve acute injury repair thus guiding the development of cell-based therapies for women with nonacute established SUI.

PET imaging of hepatocellular carcinoma with anti-1-amino-3-[18F]fluorocyclobutanecarboxylic acid in comparison with L-[S-methyl-11C]methionine.

PURPOSE: [11C]methionine ([11C]Met) was used for cancer imaging based on upregulated amino acid transport and protein synthesis in different tumor types. However, the short half-life of 11C decay limited further clinical development of [11C]Met. Synthetic amino acid analog anti-1-amino-3-[18F]fluoro-cyclobutyl-1-carboxylic acid ([18F]FCABC) was developed and FDA-approved for PET imaging of recurrent prostate cancer. This study investigated "repurposed" [18F]FACBC for PET imaging of primary liver cancer such as hepatocellular carcinoma (HCC) in comparison with [11C]Met. METHODS: [11C]Met was synthesized in the lab, and [18F]FACBC was purchased from a commercial outlet. A clinically relevant animal model of spontaneously developed HCC in the woodchucks was used for PET imaging. Bioinformatics analysis was performed for the expression of amino acid transporters responsible for radiotracer uptake and validated by PCR. Dynamic PET scans of [11C]Met and [18F]FACBC were acquired within 1 week. Standardized uptake value (SUV) was calculated for regions of interest (ROIs) defined over HCC and a liver background region. H&E staining and immunohistochemical (IHC) staining were performed with harvested tissues post-imaging. RESULTS: Higher expression of ACST2 and LAT1 was found in HCC than in the surrounding liver tissues. PCR validated this differential expression. [11C]Met and [18F]FACBC displayed some differences in their uptake and retention in HCC. Both peaked in HCC with an SUV of 3.5 after 10 min post-injection. Met maintained a plateaued contrast uptake in HCC to that in the liver while [18F]FCABC declined in HCC and liver after peak uptake. The pathological assessment revealed the liver tumor as moderately differentiated similar to the human HCC and proliferative. CONCLUSION: Both [18F]FACBC and [11C]Met showed uptake in HCC through the use of a clinically relevant animal model of woodchuck HCC. The uptake and retention of [18F]FACBC and [11C]Met depend on their metabolism and also rely on the distribution of their principal amino acid transporters.

Analysis of -5p and -3p Strands of miR-145 and miR-140 During Mesenchymal Stem Cell Chondrogenic Differentiation.

The chondrogenic differentiation of mesenchymal stem cells (MSCs) is mediated by transcription factors and small noncoding RNAs such as microRNAs (miRNAs). Each miRNA is initially transcribed as a long transcript, which matures to produce -5p and -3p strands. It is widely believed that the mature and functional miRNA from any given pre-miRNA, usually the -5p strand, is functional, while the opposing -3p strand is degraded. However, recent cartilage literature started to show functional -3p strands for a few miRNAs. This study aimed at examining both -5p and -3p strands of two key miRNAs miR-140 and miR-145, known to be involved in the chondrogenic differentiation of MSCs. The level (copy number) of both -5p and -3p strands of miR-145 and miR-140 along the time line of MSC chondrogenic differentiation was determined by polymerase chain reaction. The gene expression profiles of several genes related to MSC chondrogenesis were compared with these miRNA profiles along the same timeline. While miR-145-3p is declining in step with miR-145-5p in pellet cultures during the process, the -3p strand is only 1-2% of the total miR-145 products. In contrast, the mature -3p and -5p products of miR-140 are found to increase with near-equal molar expression throughout chondrogenic differentiation. Numerous genes are expressed by cartilage progenitor cells during development. One such target gene, Sox9, is a regulatory target of the dominant miR-145-5p, consistent with the data. Further experimental validations are warranted to confirm that ACAN, FOXO1, and RUNX3 as direct targets of miR-145-5p in the context of MSC chondrogenesis. Similarly, TRSP1 and ACAN are worth further validation as direct targets of miR-145-3p. For miR-140, SOX4 shall be further validated as a direct target of miR-140-5p, while KLF4, PTHLH, and WNT5A can be validated as direct targets of miR-140-3p.

Injectable liquid polymers extend the delivery of corticosteroids for the treatment of osteoarthritis.

Drug delivery strategies generally use inert materials, such as high molecular weight polymers, to encapsulate and control the release rate of therapeutic drugs. Diffusion governs release and depends on the ease of permeation of the polymer alongside the device thickness. Yet in applications such as osteoarthritis, the physiological constraints and limited intra-articular joint space prevent the use of large, solid drug delivery implants. Other investigators have explored the use of micro- and nanoparticle drug delivery systems. However, the small size of the systems limits the total drug that may be encapsulated and its short diffusion distance causes rapid release. Ordinarily, the extremely low diffusivity of a polymer fluid would make this an unsuitable delivery system. Our technology takes advantage of specific molecular interactions between drug and polymer, which can control the rate of release beyond diffusion. With this "affinity-based drug delivery", we have shown that delivery rates from solid polymer can be prolonged from hours and days, to weeks and months. In this paper, we demonstrate that this affinity-based mechanism also applies to low diffusivity fluid-phase polymers. They show release rates that are substantially slower than chemically similar polymers incapable of forming those inclusion complexes. The similarity of this study's liquid polymers to the viscoelastic fluids used in current clinical practice makes it an ample delivery system for osteoarthritic application. We confirmed the capacity of anti-inflammatory delivery of corticosteroids: hydrocortisone, triamcinolone, and dexamethasone; from both solid implants and polymer fluids. Further, we demonstrated that viscoelastic properties are widely tunable, and within the range of native synovial fluid. Lastly, we determined these polymer fluids have no impact on the differentiation of mesenchymal stem cells to cartilage and are not cytotoxic to a common cell line.

Expression of miR-145-5p During Chondrogenesis of Mesenchymal Stem Cells.

Assessing the quality of tissue engineered (TE) cartilage has historically been performed by endpoint measurements including marker gene expression. Until the adoption of promoter-driven reporter constructs capable of quantitative and real time non-destructive expression analysis, temporal gene expression assessments along a timeline could not be performed on TE constructs. We further exploit this technique to utilize microRNA (miRNA or miR) through the use of firefly luciferase reporter (Luc) containing a 3' UTR perfect complementary target sequence to the mature miR-145-5p. We report the development and testing of a firefly luciferase (Luc) reporter responsive to miR-145-5p for longitudinal tracking of miR-145-5p expression throughout MSC chondrogenic differentiation. Plasmid reporter vectors containing a miR-145-5p responsive reporter (Luc reporter with a perfect complementary target sequence to the mature miR-145-5p sequence in the 3'UTR), a Luc reporter driven by a truncated Sox9 (one of the targets of miR-145-5p) promoter, or the Luc backbone (control) vector without a specific miRNA target were transfected into MSCs by electroporation. Transfected MSCs were mixed with untransfected MSC to generate chondrogenic pellets. Pellets were imaged by bioluminescent imaging (BLI) and harvested along a preset time line. The imaging signals from miR-145-5p responsive reporter and Sox9 promoter-driven reporter showed correlated time-courses (measured by BLI and normalized to Luc-control reporter; Spearman r=0.93, p=0.0002) during MSC chondrogenic differentiation. Expression analysis by qRT-PCR suggests an inverse relationship between miR-145-5p and Sox9 gene expression during MSC chondrogenic differentiation. Non-destructive cell-pellet imaging is capable of supplementing histological analyses to characterize TE cartilage. The miR-145-5p responsive reporter is relatively simple to construct and generates a consistent imaging signal responsive to miR-145-5p during MSC chondrogenesis in parallel to certain molecular and cellular events.

Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing.

Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1, an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from -938 to -337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1. We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34+ selected hematopoietic stem and progenitor cells.

Local release from affinity-based polymers increases urethral concentration of the stem cell chemokine CCL7 in rats.

The protein chemokine (C-C motif) ligand 7 (CCL7) is significantly over-expressed in urethral and vaginal tissues immediately following vaginal distention in a rat model of stress urinary incontinence. Further evidence, in this scenario and other clinical scenarios, indicates CCL7 stimulates stem cell homing for regenerative repair. This CCL7 gradient is likely absent or compromised in the natural repair process of women who continue to suffer from SUI into advanced age. We evaluated the feasibility of locally providing this missing CCL7 gradient by means of an affinity-based implantable polymer. To engineer these polymers we screened the affinity of different proteoglycans, to use them as CCL7-binding hosts. We found heparin to be the strongest binding host for CCL7 with a 0.323 nM dissociation constant. Our experimental approach indicates conjugation of heparin to a polymer backbone (using either bovine serum albumin or poly (ethylene glycol) as the base polymer) can be used as a delivery system capable of providing sustained concentrations of CCL7 in a therapeutically useful range up to a month in vitro. With this approach we are able to detect, after polymer implantation, significant increase in CCL7 in the urethral tissue directly surrounding the polymer implants with only trace amounts of human CCL7 present in the blood of the animals. Whole animal serial sectioning shows evidence of retention of locally injected human mesenchymal stem cells (hMSCs) only in animals with sustained CCL7 delivery, 2 weeks after affinity-polymers were implanted.