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Background Serial screening is critical for restricting spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by facilitating timely identification of infected individuals to interrupt transmission. Variation in sensitivity of different diagnostic tests at different stages of infection has not been well documented. Methods In a longitudinal study of 43 adults newly infected with SARS-CoV-2, all provided daily saliva and nasal swabs for quantitative reverse transcription polymerase chain reaction (RT-qPCR), Quidel SARS Sofia antigen fluorescent immunoassay (FIA), and live virus culture. Results Both RT-qPCR and Quidel SARS Sofia antigen FIA peaked in sensitivity during the period in which live virus was detected in nasal swabs, but sensitivity of RT-qPCR tests rose more rapidly prior to this period. We also found that serial testing multiple times per week increases the sensitivity of antigen tests. Conclusions RT-qPCR tests are more effective than antigen tests at identifying infected individuals prior to or early during the infectious period and thus for minimizing forward transmission (given timely results reporting). All tests showed >98% sensitivity for identifying infected individuals if used at least every 3 days. Daily screening using antigen tests can achieve approximately 90% sensitivity for identifying infected individuals while they are viral culture positive.

Background Cancer cells are known to display varying degrees of metastatic propensity, but the molecular basis underlying such heterogeneity remains unclear. Our aims in this study were to (i) elucidate prognostic subtypes in primary tumors based on an epithelial-to-mesenchymal-to-amoeboid transition (EMAT) continuum that captures the heterogeneity of metastatic propensity and (ii) to more comprehensively define biologically informed subtypes predictive of breast cancer metastasis and survival in lymph node-negative (LNN) patients. Methods We constructed a novel metastasis biology-based gene signature (EMAT) derived exclusively from cancer cells induced to undergo either epithelial-to-mesenchymal transition (EMT) or mesenchymal-to-amoeboid transition (MAT) to gauge their metastatic potential. Genome-wide gene expression data obtained from 913 primary tumors of lymph node-negative breast cancer (LNNBC) patients were analyzed. EMAT gene signature-based prognostic stratification of patients was performed to identify biologically relevant subtypes associated with distinct metastatic propensity. Results Delineated EMAT subtypes display a biologic range from less stem-like to more stem-like cell states and from less invasive to more invasive modes of cancer progression. Consideration of EMAT subtypes in combination with standard clinical parameters significantly improved survival prediction. EMAT subtypes outperformed prognosis accuracy of receptor or PAM50-based BC intrinsic subtypes even after adjusting for treatment variables in 3 independent, LNNBC cohorts including a treatment-naïve patient cohort. Conclusions EMAT classification is a biologically informed method that provides prognostic information beyond that which can be provided by traditional cancer staging or PAM50 molecular subtype status and may improve metastasis risk assessment in early stage, LNNBC patients, who may otherwise be perceived to be at low metastasis risk.

Sepsis is a leading cause of death and is the most expensive condition to treat in U.S. hospitals. Despite targeted efforts to automate earlier detection of sepsis, current techniques rely exclusively on using either standard clinical data or novel biomarker measurements. In this study, we apply machine learning techniques to assess the predictive power of combining multiple biomarker measurements from a single blood sample with electronic medical record data (EMR) for the identification of patients in the early to peak phase of sepsis in a large community hospital setting. Combining biomarkers and EMR data achieved an area under the receiver operating characteristic (ROC) curve (AUC) of 0.81, while EMR data alone achieved an AUC of 0.75. Furthermore, a single measurement of six biomarkers (IL-6, nCD64, IL-1ra, PCT, MCP1, and G-CSF) yielded the same predictive power as collecting an additional 16 hours of EMR data(AUC of 0.80), suggesting that the biomarkers may be useful for identifying these patients earlier. Ultimately, supervised learning using a subset of biomarker and EMR data as features may be capable of identifying patients in the early to peak phase of sepsis in a diverse population and may provide a tool for more timely identification and intervention.

Sepsis is a leading cause of death and is the most expensive condition to treat in U.S. hospitals. Despite targeted efforts to automate earlier detection of sepsis, current techniques rely exclusively on using either standard clinical data or novel biomarker measurements. In this study, we apply machine learning techniques to assess the predictive power of combining multiple biomarker measurements from a single blood sample with electronic medical record data (EMR) for the identification of patients in the early to peak phase of sepsis in a large community hospital setting. Combining biomarkers and EMR data achieved an area under the receiver operating characteristic (ROC) curve (AUC) of 0.81, while EMR data alone achieved an AUC of 0.75. Furthermore, a single measurement of six biomarkers (IL-6, nCD64, IL-1ra, PCT, MCP1, and G-CSF) yielded the same predictive power as collecting an additional 16 hours of EMR data(AUC of 0.80), suggesting that the biomarkers may be useful for identifying these patients earlier. Ultimately, supervised learning using a subset of biomarker and EMR data as features may be capable of identifying patients in the early to peak phase of sepsis in a diverse population and may provide a tool for more timely identification and intervention.

β-Hydroxy β-methylbutyrate (HMB) is a metabolite of the essential amino acid leucine. Recent studies demonstrate a decline in plasma HMB concentrations in humans across the lifespan, and HMB supplementation may be able to preserve muscle mass and strength in older adults. However, the impact of HMB supplementation on hippocampal neurogenesis and cognition remains largely unexplored. The purpose of this study was to simultaneously evaluate the impact of HMB on muscle strength, neurogenesis and cognition in young and aged mice. In addition, we evaluated the influence of HMB on muscle-resident mesenchymal stem/stromal cell (Sca-1+CD45-; mMSC) function to address these cells potential to regulate physiological outcomes. Three month-old (n=20) and 24 month-old (n=18) female C57BL/6 mice were provided with either Ca-HMB or Ca-Lactate in a sucrose solution twice per day for 5.5weeks at a dose of 450mg/kg body weight. Significant decreases in relative peak and mean force, balance, and neurogenesis were observed in aged mice compared to young (age main effects, p≤0.05). Short-term HMB supplementation did not alter activity, balance, neurogenesis, or cognitive function in young or aged mice, yet HMB preserved relative peak force in aged mice. mMSC gene expression was significantly reduced with age, but HMB supplementation was able to recover expression of select growth factors known to stimulate muscle repair (HGF, LIF). Overall, our findings demonstrate that while short-term HMB supplementation does not appear to affect neurogenesis or cognitive function in young or aged mice, HMB may maintain muscle strength in aged mice in a manner dependent on mMSC function.

Over the past several years, biomaterials loaded with mesenchymal stem cells (MSCs) have increasingly been used to reduce the myocardial fate of postinfarction collagen deposition and scar tissue formation. Despite successful gains, therapeutic efficacy has remained limited because of restricted transport of cell-secreting factors at the site of implantation. We hypothesized that an MSC-laden hydrogel patch with multiple microchannels would retain transplanted cells on target tissue and support transport of cell-secreting factors into tissue. By doing so, the gel patch will improve the therapeutic potential of the cells and minimize the degradation of myocardial tissue postinfarction. To examine this hypothesis, a stereolithographic apparatus (SLA) was used to introduce microchannels of controlled diameters (e.g., 500 and 1000 μm) during in situ cross-linking reaction of poly(ethylene glycol)dimethacrylate solution suspended with cells. Placement of the MSC-laden, microchanneled gel patch on the occluded left coronary artery in a murine model showed significant improvement in the ejection fraction, fractional shortening, and stroke volume, compared with gel patches without MSCs and MSC-laden gel patches without microchannels. In particular, the microchannels significantly reduced the number of cells required to recover cardiac function, while minimizing cardiac remodeling. In sum, the microchanneled gel patch would provide a means to prevent abnormal fibrosis resulting from acute ischemic injury.

Adipose tissue expansion during periods of excess nutrient intake requires significant turnover of the extracellular matrix (ECM) to allow for maximal lipid filling. Recent data suggest that stromal cells may be a primary contributor to ECM modifications in visceral adipose. The purpose of this study was to investigate the capacity for high fat diet (HFD)-induced obesity to alter adipose-derived stromal cell (ADSC) relative quantity and ECM gene expression, and determine the extent to which exercise training can mitigate such changes. Male C57BL/6J mice were placed on control or HFD for 8weeks prior to and following initiation of a 16week treadmill exercise program. ADSCs (Sca-1+CD45−) were isolated from epididymal adipose tissue and mRNA was evaluated using high throughput qPCR. Stromal cells were also obtained from skeletal muscle (MDSC). HFD decreased the quantity of ADSCs and markedly altered gene expression related to ECM remodeling (Col1α1, MMP2, MMP9, Timp1). Exercise did not reverse these changes. MDSCs were minimally altered by HFD or exercise. Overall, the data from this study suggest that ADSCs decrease in quantity and contribute to adipose ECM remodeling in response to obesity, and exercise training does not significantly impact these outcomes.

Perivascular stromal cells, including mesenchymal stem/stromal cells (MSCs), secrete paracrine factor in response to exercise training that can facilitate improvements in muscle remodeling. This study was designed to test the capacity for muscle-resident MSCs (mMSCs) isolated from young mice to release regenerative proteins in response to mechanical strain in vitro, and subsequently determine the extent to which strain-stimulated mMSCs can enhance skeletal muscle and cognitive performance in a mouse model of uncomplicated aging. Protein arrays confirmed a robust increase in protein release at 24h following an acute bout of mechanical strain in vitro (10%, 1Hz, 5h) compared to non-strain controls. Aged (24month old), C57BL/6 mice were provided bilateral intramuscular injection of saline, non-strain control mMSCs, or mMSCs subjected to a single bout of mechanical strain in vitro (4×104). No significant changes were observed in muscle weight, myofiber size, maximal force, or satellite cell quantity at 1 or 4wks between groups. Peripheral perfusion was significantly increased in muscle at 4wks post-mMSC injection (p

Background: Distant metastatic spread of cancer cells to other organs from the primary site of origin currently constitutes the most significant contributor to cancer-related morbidity and mortality. Epithelial-to-mesenchymal transition (EMT) is a biologic transformation of cancer cells from a non-migratory phenotype to a migratory one, and is thought to initiate the metastatic cascade in cancer. EMT has also been reported to trigger acquisition of stem cell traits in breast cancer. Transcription factor (TF) Forkhead box C1 (FOXC1), strongly associated with the basal-like and claudin-low breast cancer molecular subtypes, is a powerful EMT inducer and is also a marker of stem/progenitor cells. In contrast, TF forkhead box A1 (FOXA1), strongly associated with luminal subtypes, is an EMT repressor and a luminal differentiation marker, thus seemingly exerting reciprocally opposite transcriptional effects to that of FOXC1. We hypothesized that effective EMT program activation status in breast cancer might be better predicted by examining the expression ratio of an EMT inducer and EMT repressor, such as FOXC1/FOXA1, theoretically being more reflective of net transcriptional effect than either component alone. Methods: Herein we utilize RNA-Seq profiling of the HRAS-transformed MCF10A cell series, a well characterized and widely accepted in vitro model of breast cancer progression and metastasis, to correlate measured FOXC1/FOXA1 ratios to dynamic shifts in EMT marker expression in 3D matrigel cultures and to stem cell traits observed in primary and secondary mammosphere suspension cultures. We further test the ability of the FOXC1/FOXA1 expression ratio to predict lymph node independent breast cancer metastasis and death in independent human breast cancer gene expression datasets. Results: RNA-Seq and qRT-PCR profiling confirmed progressive increase in FOXC1/FOXA1 ratio to correlate with a progressive loss of E-cadherin expression and synchronous gain of EMT markers N-cadherin, Fibronectin, and Vimentin. FOXC1/FOXA1 ratio was found to be directly proportional to mammosphere formation efficiency, a surrogate indicator of stem cell enrichment. In patients without any evidence of nodal metastasis, elevated FOXC1/FOXA1 ratio was associated with significantly decreased 10 year Overall Survival (HR 2.58;95%CI 1.39 to 4.80, p = 0.003, 295 patient Van de Vijver dataset), 10 year Disease-specific Survival (HR 1.74;95%CI 1.16 to 2.61, p = 0.008,1992 patient Curtis dataset) and predicted the development of lung metastasis. Conclusion: Elevated FOXC1/FOXA1 expression ratio indicates EMT program activation in breast cancer, and predicts the associated occurrence of lymph-node-independent distant metastasis and death in human patients. These findings may allow for the early (pre-symptomatic) diagnosis of clinically occult (node negative) metastasis by using the FOXC1/FOXA1 ratio as a biomarker of metastasis and permit institution of appropriate therapy earlier than currently possible. The current study improves our understanding of EMT and highlights the importance of future studies geared towards unraveling mechanisms involved in regulating FOXC1 and FOXA1 expression in breast cancer. Citation Format: Partha S Ray, Tor W Jensen, Tania Ray, Omid Gholamalamdari, Connie Y Tsai, Bomy Kim, Rohit Bhargava, K V Prasanth. FOXC1/FOXA1 transcriptional balance in breast cancer: From acquisition of mesenchymal and stem cell traits to occult lymph node independent breast cancer metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-30.

Cancer stem cells (CSCs) are considered to be an important contributing factor towards treatment failure, cancer recurrence, and mortality. CSCs are known to be more enriched in the basal-like and claudin-low subtypes of breast cancer. The Wnt/β catenin signaling pathway is well known as a regulator of embryonic development and stem cell biology, and is prominently active in basal-like and claudin-low breast cancers. We have previously reported that transcription factor FOXC1 plays a critical role in mediating aggressive cell traits in basal-like/claudin-low breast cancer. In this study, we sought to investigate the link between Wnt signaling and FOXC1 and its potential in regulating CSC biology in basal-Like/claudin-low breast cancer. We observed that exposure of the MDA-MB-231 basal-like/claudin-low cell line (low constitutive FOXC1 expressor) to Wnt3a (a canonical Wnt signaling ligand), resulted in increased expression of FOXC1. Reciprocally, overexpression of FOXC1 in MCF10A human mammary epithelial cells led to a pronounced increase in Wnt signaling activity, strongly suggestive of a direct or indirect positive feedback loop between Wnt signaling and FOXC1. More importantly, BT549 and HS578t basal-like/claudin-low cells (high constitutive FOXC1 expressors) proved to be more sensitive to treatment with the Wnt inhibitor ICRT3 as evidenced by decreased cell viability when compared to MCF7 (luminal) or SKBR3 (HER2) breast cancer cell lines. Furthermore the decrease in cell viability appeared to be proportionate to the level of FOXC1 expression. Upon pharmacological inhibition with ICRT3 and biological inhibition with siRNA knockdown of LRP6, (a canonical Wnt signaling cell surface receptor) a decrease in FOXC1 expression level was observed in a dose and time dependent manner. This effect was particularly pronounced in mammosphere cultures enriched for BT549 cancer stem-like cells. Inhibition of Wnt signaling reduced mammosphere formation efficiency of BT549 cells, suggesting that Wnt inhibition targets cancer stem cells (CSCs) in the basal-like/claudin-low breast cancer subtype. More importantly, however, after an initial 4 day incubation period, some cells are observed to persist and later display renewed enhancement of mammosphere formation ability. Profiling of such cells interestingly revealed depletion of FOXC1+ve cells but persistence of cells displaying pronounced up regulation of stereotypical embryonic stem cell Transcription Factors OCT4, SOX2 and NANOG, strongly suggestive of a potential primitive stem cell/quiescent cell state escape mechanism. qRT-PCR based pathway activation analysis revealed marked activation of NFκB signaling in the residual cells that withstood Wnt inhibition. Simultaneous pharmacologic inhibition with Wnt inhibitor ICRT3 and the proteasome inhibitor Bortezomib (known to inhibit NFκB signaling) effectively targeted BT549 cancer stem cells in mammosphere culture and prevented the persistence/emergence of any residual cells. Taken together, our findings suggest that combination therapy approaches are likely required to effectively target breast cancer stem cells. Citation Format: Partha S Ray, Connie Y Tsai, Tania Ray, Bomy Kim, Tor W Jensen. Proteosome inhibitor bortezomib inhibits NFκB and effectively overcomes cancer stem cell escape triggered by Wnt inhibitor therapy in FOXC1+ basal-Like/claudin-low breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-04.

AB Purpose: The [alpha]7[beta]1 integrin is a transmembrane protein expressed in the skeletal muscle that can link the actin cytoskeleton to the surrounding basal lamina. We have previously demonstrated that transgenic mice overexpressing the [alpha]7B integrin in the skeletal muscle (MCK:[alpha]7B; [alpha]7Tg) mount an enhanced satellite cell and growth response to single or multiple bouts of eccentric exercise. In addition, interstitial stem cells characterized as mesenchymal stem cells (MSCs) accumulate in [alpha]7Tg muscle (mMSCs) in the sedentary state and after exercise. The results from these studies prompted us to determine the extent to which mMSC underlie the beneficial adaptive responses observed in [alpha]7Tg skeletal muscle after exercise. Methods: mMSCs (Sca-1+CD45-) were isolated from [alpha]7Tg mice, dye-labeled, and intramuscularly injected into adult wild type recipient mice. After injection of mMSCs or saline, mice remained sedentary (SED) or were subjected to eccentric exercise training (TR) (downhill running) on a treadmill (three times per week) for 2 or 4 wk. Gastrocnemius-soleus complexes were collected 24 h after the last bout of exercise. Results: mMSCs did not directly fuse with existing fibers; however, mMSCs injection enhanced Pax7+ satellite cell number and myonuclear content compared with all other groups at 2 wk after exercise. Mean CSA, percentage of larger caliber fibers (>3000 [mu]m2), and grip strength were increased in mMSCs/TR compared with saline/SED and mMSCs/SED at 4 wk. mMSC transplantation did not enhance repair or growth in the absence of exercise. Conclusions: The results from this study demonstrate that mMSCs contribute to beneficial changes in satellite cell expansion and growth in [alpha]7Tg muscle after eccentric exercise. Thus, MSCs that naturally accumulate in the muscle after eccentric contractions may enhance the adaptive response to exercise.

We describe for the first time a therapeutic strategy to target stem-like cancer cells via STAT-3 modulation using a nanomedicine approach. Niclocelle, a niclosamide loaded rigid core mixed micelle, was synthesized from a self-assembled well-defined amphiphilic diblock copolymer and an FDA-approved signal transducer and activator of transcription factor 3. Followed by a rigorous physico-chemical characterization, niclocelles were evaluated biologically for cytotoxicity and apoptosis in human melanoma (C32) and breast cancer (MDA-MB231 and MCF-7) cells. Niclocelles were found to selectively reduce the CD44+ stem cell population in C32 cells via STAT-3 modulation.

Herein we report a bi-modal cancer treatment technique using therapeutic ultrasound in the presence of a novel sorafenib loaded nanobubble for hepatocellular carcinoma. Therapeutic ultrasound was used for improving fusion-triggered drug release from nanobubbles leading to enhanced cytotoxicity in HepG2 cells by greater than two folds improvement in IC50 values. This strategy could also be made compatible with catheter-based devices to derive a highly localized treatment approach, a concept that might be extendable to metastatic cancers as well.

Cancer cells within a tumor are known to display varying degrees of metastatic propensity but the molecular basis underlying such heterogeneity remains unclear. We analyzed genome-wide gene expression data obtained from primary tumors of lymph node-negative breast cancer patients using a novel metastasis biology-based Epithelial-Mesenchymal-Amoeboid Transition (EMAT) gene signature, and identified molecularly distinct subtypes associated with distinct prognostic profiles. EMAT subtype status improved prognosis accuracy of clinical parameters and statistically outperformed traditional breast cancer intrinsic subtypes. Additionally, analysis of 3D spheroids from an in vitro isogenic model of breast cancer progression reveals that EMAT subtypes display progression from premalignant to malignant and pre-invasive to invasive cancer. EMAT classification is a biologically informed method to assess metastasis risk in early stage, lymph node-negative breast cancer patients and appears to molecularly reconcile conflicting paradigms of breast cancer metastasis.

Laminin-111 (α1, β1, γ1; LM-111) is an important component of the extracellular matrix that is required for formation of skeletal muscle during embryonic development. Recent studies suggest that LM-111 supplementation can enhance satellite cell proliferation and muscle function in mouse models of muscular dystrophy. The purpose of this study was to determine the extent to which LM-111 can alter satellite and nonsatellite stem cell quantity following eccentric exercise-induced damage in young adult, healthy mice. One week following injection of LM-111 or saline, mice either remained sedentary or were subjected to a single bout of downhill running (EX). While one muscle was preserved for evaluation of satellite cell number, the other muscle was processed for isolation of mesenchymal stem cells (MSCs; Sca-1+CD45−) via FACS at 24 hours postexercise. Satellite cell number was approximately twofold higher in LM-111/EX compared with all other groups (p < .05), and the number of satellite cells expressing the proliferation marker Ki67 was 50% to threefold higher in LM-111/EX compared with all other groups (p < .05). LM-111 also increased the quantity of embryonic myosin heavy chain-positive (eMHC+) fibers in young mice after eccentric exercise (p < .05). Although MSC percentage and number were not altered, MSC proinflammatory gene expression was decreased, and hepatocyte growth factor gene expression was increased in the presence of LM-111 (p < .05). Together, these data suggest that LM-111 supplementation provides a viable solution for increasing skeletal muscle stem cell number and/or function, ultimately allowing for improvements in the regenerative response to eccentric exercise.

Since stem cells emerged as a new generation of medicine, there are increasing efforts to deliver the stem cells to a target tissue via intravascular injection. However, the therapeutic stem cells lack a capacity to detect and adhere to the target tissue. Therefore, this study presents synthesis of a bioactive hyper-branched polyglycerol (HPG) which can non-invasively associate with stem cells and further guide them to target sites, such as inflamed endothelium. The overall process is analogous to the way in which leukocytes are mobilized to the injured endothelium.

Impaired skin wound healing is a significant comorbid condition of diabetes, which often results in nonhealing diabetic ulcers due to poor peripheral microcirculation, among other factors. The effectiveness of the regeneration of adipose-derived stem cells (ADSCs) and muscle-derived stem cells (MDSCs) was assessed using an integrated multimodal microscopy system equipped with two-photon fluorescence and second-harmonic generation imaging. These imaging modalities, integrated in a single platform for spatial and temporal coregistration, allowed us to monitor in vivo changes in the collagen network and cell dynamics in a skin wound. Fluorescently labeled ADSCs and MDSCs were applied topically to the wound bed of wild-type and diabetic (db/db) mice following punch biopsy. Longitudinal imaging demonstrated that ADSCs and MDSCs provided remarkable capacity for improved diabetic wound healing, and integrated microscopy revealed a more organized collagen remodeling in the wound bed of treated mice. The results from this study verify the regenerative capacity of stem cells toward healing and, with multimodal microscopy, provide insight regarding their impact on the skin microenvironment. The optical method outlined in this study, which has the potential for in vivo human use, may optimize the care and treatment of diabetic nonhealing wounds.

// Mahdieh Jadaliha 1 , Xinying Zong 1 , Pushkar Malakar 2 , Tania Ray 3 , Deepak K. Singh 1 , Susan M. Freier 4 , Tor Jensen 5 , Supriya G. Prasanth 1 , Rotem Karni 2 , Partha S. Ray 6, 7 , Kannanganattu V. Prasanth 1 1 Department of Cell and Developmental Biology, University of Illinois, Urbana, IL, USA 2 Department of Biochemistry and Molecular Biology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel 3 Onconostic Technologies, Champaign, IL, USA 4 Ionis Pharmaceuticals Inc., Carlsbad, CA, USA 5 Interdisciplinary Health Sciences Initiative, University of Illinois, Urbana, IL, USA 6 Department of Surgery, University of Illinois College of Medicine, Champaign, IL, USA 7 Carle Cancer Center, Urbana, IL, USA Correspondence to: Kannanganattu V. Prasanth, email: kumarp@illinois.edu Partha S. Ray, email: psray@illinois.edu Rotem Karni, email: rotemka@ekmd.huji.ac.il Keywords: breast cancer, nuclear speckle, triple negative breast cancer, lncRNA, basal-like breast cancer cells Received: February 05, 2016 Accepted: May 09, 2016 Published: May 26, 2016 ABSTRACT MALAT1 (metastasis associated lung adenocarcinoma transcript1) is a conserved long non-coding RNA, known to regulate gene expression by modulating transcription and post-transcriptional pre-mRNA processing of a large number of genes. MALAT1 expression is deregulated in various tumors, including breast cancer. However, the significance of such abnormal expression is yet to be fully understood. In this study, we demonstrate that regulation of aggressive breast cancer cell traits by MALAT1 is not predicted solely based on an elevated expression level but is context specific. By performing loss- and gain-of-function studies, both under in vitro and in vivo conditions, we demonstrate that MALAT1 facilitates cell proliferation, tumor progression and metastasis of triple-negative breast cancer (TNBC) cells despite having a comparatively lower expression level than ER or HER2-positive breast cancer cells. Furthermore, MALAT1 regulates the expression of several cancer metastasis-related genes, but displays molecular subtype specific correlations with such genes. Assessment of the prognostic significance of MALAT1 in human breast cancer (n=1992) revealed elevated MALAT1 expression was associated with decreased disease-specific survival in ER negative, lymph node negative patients of the HER2 and TNBC molecular subtypes. Multivariable analysis confirmed MALAT1 to have independent prognostic significance in the TNBC lymph node negative patient subset (HR=2.64, 95%CI 1.35- 5.16, p=0.005). We propose that the functional significance of MALAT1 as a metastasis driver and its potential use as a prognostic marker is most promising for those patients diagnosed with ER negative, lymph node negative breast cancer who might otherwise mistakenly be stratified to have low recurrence risk.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

BACKGROUND Diagnosis of basal-like breast cancer (BLBC) remains a bottleneck to conducting effective clinical trials for this aggressive subtype. We postulated that elevated expression of Forkhead Box transcription factor C1 (FOXC1) is a simple and accurate diagnostic biomarker for BLBC. METHODS Accuracy of FOXC1 expression in identifying BLBC was compared with the PAM50 gene expression panel in gene expression microarray (GEM) (n = 1992) and quantitative real-time polymerase chain reaction (qRT-PCR) (n = 349) datasets. A FOXC1-based immunohistochemical (IHC) assay was developed and assessed in 96 archival formalin-fixed, paraffin-embedded (FFPE) breast cancer samples that also underwent PAM50 profiling. All statistical tests were two-sided. RESULTS A FOXC1-based two-tier assay (IHC +/- qRT-PCR) accurately identified BLBC (AUC = 0.88) in an independent cohort of FFPE samples, validating the accuracy of FOXC1-defined BLBC in GEM (AUC = 0.90) and qRT-PCR (AUC = 0.88) studies, when compared with platform-specific PAM50-defined BLBC. The hazard ratio (HR) for disease-specific survival in patients having FOXC1-defined BLBC was 1.71 (95% CI = 1.31 to 2.23, P < .001), comparable to PAM50 assay-defined BLBC (HR = 1.74, 95% CI = 1.40 to 2.17, P < .001). FOXC1 expression also predicted the development of brain metastasis. Importantly, unlike triple-negative or Core Basal IHC definitions, a FOXC1-based definition is able to identify BLBC in both ER+ and HER2+ patients. CONCLUSION A FOXC1-based two-tier assay, by virtue of being rapid, simple, accurate, and cost-effective may emerge as the diagnostic assay of choice for BLBC. Such a test could substantially improve clinical trial enrichment of BLBC patients and accelerate the identification of effective chemotherapeutic options for this aggressive disease.

Agarose hydrogels of varied elastic modulus can be patterned into 100-microm-wide channels with wall heights of 60 microm. After modifying the hydrogels with chloroacetic acid (acid gels), they are amenable to modification with amine-containing ligands using EDC-NHS chemistry. Using both rheometry and atomic force microscopy (AFM) nanoindentation measurements, the elastic modulus of unmodified hydrogels increases linearly from 3.6 +/- 0.5 kPa to 45.2 +/- 5.5 kPa for 0.5 to 2.0 wt/vol % hydrogel, respectively. The elastic modulus of acid gels is 2.2 +/- 0.3 kPa to 16.2 +/- 1.6 kPa for 0.5 to 2.0 wt/vol %, respectively. No further changes were measured after further modifying the acid gels with fibronectin. Confocal images of rhodamine-modified acid gels show that the optimal filling viscosity of the agarose solutions is between 1 and 4 cP. This new method of patterning allows for the creation of substrates that take advantage of both micron-scale patterns and variably elastic hydrogels.

Increases in pH between 7.1 and 7.7 increase the efficiency of polybrene (Pb)- and protamine sulfate (PS)-aided retroviral transduction of NIH-3T3 cells in a serum-lot-dependent manner. The increase in Pb-aided transduction efficiency at pH 7.7, relative to the value at pH 7.33, ranged from 13% to 49% for three serum lots. For a constant Moloney murine leukemia virus (MMLV) vector dilution at pH 7.33, three different serum lots resulted in absolute transduction efficiencies ranging from 29% to 53% using Pb. At the same vector dilution, PS-aided transduction was less effective on an absolute basis than Pb-aided transduction, but the benefit of elevated pH was more pronounced with PS. There was a similar enhancement with PS at elevated pH for a murine stem cell virus (MSCV) vector as for the MMLV vector. The benefit at pH 7.7 for PS-aided transduction was partially due to greater PS stability at elevated pH. Heat inactivating the serum supplement or adding protease inhibitors helped to stabilize PS. This increased the absolute transduction efficiency but decreased the relative benefit of elevated pH to a level similar to that for Pb-aided transduction. Incubating Pb with the vector at pH 7.1 for 10 min, prior to readjusting to pH 7.7 and transducing the cells, was sufficient to abrogate the beneficial effects of transduction at pH 7.7. In contrast, prior exposure of PS with vector at pH 7.1 did not affect subsequent transduction at pH 7.7. These results indicate that pH is an important variable in retroviral transduction and that the relative benefits of Pb or PS on retroviral vector transduction will vary with the pH, polymer addition method, and serum lot.

Background: The mesenchymal transcription factor Forkhead Box C1 (FOXC1), derepressed in hematopoietic stem progenitor cells (HSPCs) in the setting of acute myeloid leukemia (AML), but not in normal HSPCs, was recently demonstrated to play an important role in AML disease onset and progression by blocking myeloid lineage differentiation and enhancing clonogenic potential. Herein we sought to further examine the prognostic value of FOXC1 in AML and whether FOXC1 expression is a potential predictor of disease relapse and/or lack of response to induction chemotherapy in AML. Methods: Expression of FOXC1 mRNA in mixed karyotype AML bone marrow samples was examined using publicly available microarray datasets (n=521, n=244). The clinical significance of FOXC1 gene expression as a prognostic biomarker was evaluated using censored overall survival (OS) and event-free survival (EFS) data. Degree of statistical significance in the univariate and multivariate analyses performed was assessed using log-rank test and Cox regression model, respectively. Results: FOXC1 mRNA expression was a significant predictor of OS on univariate (hazard ratio [HR] 1.592 95% confidence interval [CI] 1.263-2.007, P = 0.0001) and multivariate (HR 1.755 95% CI 1.355-2.273, P < 0.0001) analyses. This effect on OS could be attributed to disease relapse and/or lack of response to induction chemotherapy as FOXC1 mRNA expression also proved to be a significant predictor of EFS on univariate analysis (hazard ratio [HR] 1.539 95% confidence interval [CI] 1.208-1.961, P = 0.0002) and multivariate analysis (HR 1.678 95% CI 1.280-2.201, P = 0.0001), independent of age, FLT3 ITD status, NPM1 status or cytogenetic risk status. Compared to patients who experienced disease remission, FOXC1 expression was significantly elevated in patients experiencing disease relapse following induction chemotherapy (P Conclusions: FOXC1 expression in AML is an independent prognostic predictor of decreased OS and EFS and is significantly associated with disease relapse and/or refractoriness to induction chemotherapy. A risk of relapse/non-response score that incorporates FOXC1 expression status may prove to be of value in identifying patients at the time of initial diagnosis who are likely to fail induction chemotherapy. Further studies are warranted to explore the prognostic and predictive utility of FOXC1 expression in the clinical management of AML. Figure Figure. Disclosures Ray: Onconostic Technologies, Inc.: Employment. Shah:Onconostic Technologies, Inc.: Employment. Ray:Onconostic Technologies, Inc.: Consultancy, Equity Ownership; 3N Diagnostics Ltd.: Consultancy, Equity Ownership.

Heat therapy has long been used for treatments in dermatology and sports medicine. The use of laser, RF, microwave, and more recently, ultrasound treatment, for psoriasis, collagen reformation, and skin tightening has gained considerable interest over the past several years. Numerous studies and commercial devices have demonstrated the efficacy of these methods for treatment of skin disorders. Despite these promising results, current systems remain highly dependent on operator skill, and cannot effectively treat effectively because there is little or no control of the size, shape, and depth of the target zone. These limitations make it extremely difficult to obtain consistent treatment results. The purpose of this study was to determine the feasibility for using acoustic energy for controlled dose delivery sufficient to produce collagen modification for the treatment of skin tissue in the dermal and sub-dermal layers. We designed and evaluated a curvilinear focused ultrasound device for treating skin disorders such as psoriasis, stimulation of wound healing, tightening of skin through shrinkage of existing collagen and stimulation of new collagen formation, and skin cancer. Design parameters were examined using acoustic pattern simulations and thermal modeling. Acute studies were performed in 201 freshly-excised samples of young porcine underbelly skin tissue and 56 in-vivo treatment areas in 60- 80 kg pigs. These were treated with ultrasound (9-11MHz) focused in the deep dermis. Dose distribution was analyzed and gross pathology assessed. Tissue shrinkage was measured based on fiducial markers and video image registration and analyzed using NIH Image-J software. Comparisons were made between RF and focused ultrasound for five energy ranges. In each experimental series, therapeutic dose levels (60degC) were attained at 2-5mm depth. Localized collagen changes ranged from 1-3% for RF versus 8-15% for focused ultrasound. Therapeutic ultrasound applied at high frequencies can achieve temperatures and dose distributions which concentrate in a depth profile that coincides with the location of maximum structural collagen content in skin tissues. Using an appropriate transducer configuration produces coverage of significant lateral area, thus making this a practical approach for treatment of skin disorders.

Injectable and biodegradable hydrogels have been increasingly studied for sustained drug delivery in various molecular therapies. However, it remains a challenge to attain desired delivery rate at injection sites due to local tissue pressures exerted on the soft hydrogels. Furthermore, there is often limited controllability of stiffness and degradation rates, which are key factors required for achieving desired drug release rate and therapeutic efficacy. This study presents a stiff and metastable poly(ethylene glycol) diacrylate (PEGDA)-poly(ethylene imine) (PEI) hydrogel which exhibits an elastic modulus equivalent to bulk plastic materials, and controllable degradation rate independent of its initial elastic modulus. Such unique stiffness was attained from the highly branched architecture of PEI, and the decoupled controllability of degradation rate was achieved by tuning the non-equilibrium swelling of the hydrogel. Furthermore, a single intramuscular administration of granulocyte colony stimulating factor (GCSF)-encapsulated PEGDA-PEI hydrogel extended the mobilization of mononuclear cells to four days. A larger yield of expanded CD34+ and CD31+ endothelial progenitor cells (EPCs) was also obtained as compared to the daily bolus administration. Overall, the hydrogel created in this study will be useful for the controlled and sustained delivery of a wide array of drug molecules.

The pig is a recognized model for the onset of coronary heart disease and heart attacks. Previous studies have shown that serum cholesterol levels in the pig can be elevated using a high fat, high cholesterol (HFHC) diet. What has been lacking is a genetically defined model corresponding to human ApoE4 susceptibility that can be linked to diets capable of inducing atherosclerosis. This study used a cloned pig model to examine the impact of cholesterol levels with the development of aorta fatty deposits leading to atherosclerosis. Diets were formulated using vegetable sources of protein to provide similar intakes of metabolizable energy, calcium, phosphorous and principal amino acids in both control and HFHC groups. After 60 days, the HFHC group demonstrated a 40-fold increase in aortic fatty streak lesion area combined with 6- and 11-fold increases in total and LDL cholesterol, respectively, over control diet fed cloned pigs. Previous studies have suffered from either imbalanced total caloric intake, an overall imbalance in the nutrition of the control versus HFHC groups or genetic heterogeneity when evaluating dietary constraints related to atherosclerosis. This study demonstrated that cloned, genetically-defined ApoE4 pigs provided balanced nutrition diets provide an experimental system ideally suited to examining atherosclerosis and the onset of coronary heart disease.

The ability to present cell adhesion molecule (CAM) ligands in controlled amounts on a culture surface would greatly facilitate the control of cell growth and differentiation. Supported lipid monolayer/bilayer systems have previously been developed that allow for presentation of CAM ligands for cell interaction; however, these systems have employed peptide loadings much higher than those used in poly(ethylene glycol) (PEG)-based immobilization systems. We report the development of synthetic methods that can be used for the efficient and versatile creation of many linear and cyclic lipid-linked peptide moieties. Using RGD-based peptides for the alpha5beta1 integrin as a model system, we have demonstrated that these lipopeptides support efficient cell binding and spreading at CAM ligand loadings as low as 0.1 mol %, which is well below that previously reported for supported lipid systems. Engineered lipopeptide-based surfaces offer unique presentation options not possible with other immobilization systems, and the high activity at low loadings we have shown here may be extremely useful in presenting multiple CAM ligands for studying cell growth, differentiation, and signaling.

Ex vivo expansion of hematopoietic stem cells (HSCs) would greatly facilitate cell and gene therapies. However, HSC division in culture is associated with differentiation. This contrasts with sustained HSC expansion in vivo, and has led to the hypothesis that a stem cell niche supports self-renewal. It is likely that multiple aspects of the niche will have to be mimicked to substantially enhance HSC self-renewal. We are developing a defined culture surface for the presentation of cytokines and cell adhesion molecule (CAM) ligands that are thought to be in the HSC niche. Peptide mimics of CAM ligands and cytokines conjugated to dipalmitoyl glycerol via a polyethylene glycol tether are incorporated into dipalmitoylphosphatidylcholine (DPPC) vesicles and deposited onto a hydrophobic surface to create a lipid monolayer. We have previously shown that this system effectively presents adhesive peptide ligands (Jensen et al., JACS 126:15223, 2004). The strategy for immobilizing lipopeptides has been extended to the presentation of a peptide mimetic for the hematopoietic growth factor thrombopoietin (TPO). The lipopeptide mimetic of TPO is based on the branched dimer mimic (TPOm) developed by Cwirla et al. (Science 276:1696, 1997). We have synthesized two versions of TPOm lipopeptide, the first linked to a lipid at both of the amine termini (TPOm-2L) and the second is linked by a single lipid at the carboxy terminus (TPOm-1L). This immobilization strategy does not interfere with the bioactivity of the TPOm as evidenced by cell adhesion and signaling assays. Adhesion was measured with a normal force assay at 30g using the TPO-responsive M07e cell line. We observed a dose-dependent increase in adhesion, with

Introduction: Mesenchymal stem cells (MSCs) reside in a variety of tissues and provide a stromal role in regulating progenitor cell function. Current studies focus on identifying the specific factors in the niche that can alter the MSC secretome, ultimately determining the effectiveness and timing of tissue repair. The purpose of the present study was to evaluate the extent to which substrate and mechanical strain simultaneously regulate MSC quantity, gene expression, and secretome. Methods: MSCs (Sca-1 + CD45 � ) isolated from murine skeletal muscle (muscle-derived MSCs, or mMSCs) via fluorescence-activated cell sorting were seeded onto laminin (LAM)- or collagen type 1 (COL)-coated membranes and exposed to a single bout of mechanical strain (10%, 1 Hz, 5 hours). Results: mMSC proliferation was not directly affected by substrate or strain; however, gene expression of growth and inflammatory factors and extracellular matrix (ECM) proteins was downregulated in mMSCs grown on COL in a manner independent of strain. Focal adhesion kinase (FAK) may be involved in substrate regulation of mMSC secretome as FAK phosphorylation was significantly elevated 24 hours post-strain in mMSCs plated on LAM but not COL (P