Impairment of an Endothelial NAD+-H2S Signaling Network Is a Reversible Cause of Vascular Aging.

A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.

Effects of Cocooning on Coronavirus Disease Rates after Relaxing Social Distancing.

As coronavirus disease spreads throughout the United States, policymakers are contemplating reinstatement and relaxation of shelter-in-place orders. By using a model capturing high-risk populations and transmission rates estimated from hospitalization data, we found that postponing relaxation will only delay future disease waves. Cocooning vulnerable populations can prevent overwhelming medical surges.

Comparison of sodium hypochlorite extrusion by five irrigation systems using an artificial root socket model and a quantitative chemical method.

OBJECTIVES: This is to compare the volumes of irrigant apically extruded by five irrigation systems in an artificial socket model simulating clinical conditions. MATERIALS AND METHODS: Twenty extracted human single-rooted teeth were enlarged to size 40/04 and then embedded in silicone impression material. The root canal space was irrigated with nominal 3% sodium hypochlorite (NaOCl) using standard needle irrigation (SNI) with a 30-gauge notched needle, EndoActivator (EA), XP Endo Finisher (XP Endo), EndoVac (EV), and photon-induced photoacoustic streaming (PIPS). Extruded NaOCl was collected, reacted with taurine to form taurine-monochloramine, and absorbance of taurine-monochloramine was measured at 252 nm using a spectrophotometer. The five irrigation systems were compared with repeated measures ANOVA and pairwise comparisons. RESULTS: The EV group had very low extrusion (mean ± SD = 0.12 ± 0.2 µL) and differed significantly from the other four groups (P ≤ 0.001). Larger volumes of irrigant were extruded in the other irrigation groups. There were no significant differences in the extruded volumes among the SNI (7.4 ± 3.4 µL), EA (7.0 ± 6.1 µL), and XP Endo (7.8 ± 4.1 µL) groups (P = 1). The PIPS group had the highest mean extruded volume (12.9 ± 6.8 µL) and differed significantly from SNI (P = 0.030), EV (P < 0.0005), and EA (P = 0.02), but not XP Endo (P = 0.154). CONCLUSION: Under the in vitro conditions of this study, irrigant extrusion appears unavoidable unless negative pressure irrigation such as EV is used. PIPS extrudes more irrigant than other systems, while SNI, EA, and XP Endo extrude similar volumes of irrigant. CLINICAL RELEVANCE: The findings help clinicians select the optimal irrigation system to avoid irrigant extrusion.

Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment.

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1+/CD146+, STRO-1-/CD146+ and STRO-1-/CD146- subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were downregulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration.

Glaucarubulone glucoside from Castela macrophylla suppresses MCF-7 breast cancer cell growth and attenuates benzo[a]pyrene-mediated CYP1A gene induction.

Quassinoids often exhibit antioxidant and antiproliferative activity. Emerging evidence suggests that these natural metabolites also display chemopreventive actions. In this study, we investigated the potential for the quassinoid glaucarubulone glucoside (Gg), isolated from the endemic Jamaican plant Castela macrophylla (Simaroubaceae), to display potent cytotoxicity and inhibit human cytochrome P450s (CYPs), particularly CYP1A enzymes, known to convert polyaromatic hydrocarbons into carcinogenic metabolites. Gg reduced the viability of MCF-7 breast adenocarcinoma cells (IC50 = 121 nm) to a greater extent than standard of care anticancer agents 5-fluorouracil, tamoxifen (IC50 >10 µm) and the tamoxifen metabolite 4-hydroxytamoxifen (IC50 = 2.6 µm), yet was not cytotoxic to non-tumorigenic MCF-10A breast epithelial cells. Additionally, Gg induced MCF-7 breast cancer cell death. Gg blocked increases in reactive oxygen species in MCF-10A cells mediated by the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) metabolite B[a]P 1,6-quinone, yet downregulated the expression of genes that promote antioxidant activity in MCF-7 cells. This implies that Gg exhibits antioxidant and cytoprotective actions in non-tumorigenic breast epithelial cells and pro-oxidant, cytotoxic actions in breast cancer cells. Furthermore, Gg inhibited the activities of human CYP1A according to non-competitive kinetics and attenuated the ability of B[a]P to induce CYP1A gene expression in MCF-7 cells. These data indicate that Gg selectively suppresses MCF-7 breast cancer cell growth without impacting non-tumorigenic breast epithelial cells and blocks B[a]P-mediated CYP1A induction. Taken together, our data provide a rationale for further investigations of Gg and similar plant isolates as potential agents to treat and prevent breast cancer. Copyright © 2017 John Wiley & Sons, Ltd.

Divergence Compensatory Optical Flow Method for Blood Velocimetry.

Detailed blood velocity map in the vascular system can be obtained by applying the optical flow method (OFM) in processing fluoroscopic digital subtracted catheter angiographic images; however, there are still challenges with the accuracy of this method. In the present study, a divergence compensatory optical flow method (DC-OFM), in which a nonzero divergence of velocity is assumed due to the finite resolution of the image, was explored and applied to the digital subtraction angiography (DSA) images of blood flow. The objective of this study is to examine the applicability and evaluate the accuracy of DC-OFM in assessing the blood flow velocity in vessels. First, an Oseen vortex flow was simulated on the standard particle image to generate an image pair. Then, the DC-OFM was applied on the particle image pair to recover the velocity field for validation. Second, DSA images of intracranial arteries were used to examine the accuracy of the current method. For each set of images, the first image is the in vivo DSA image, and the second image is generated by superimposing a given flow field. The recovered velocity map by DC-OFM agrees well with the exact velocity for both the particle images and the angiographic images. In comparison with the traditional OFM, the present method can provide more accurate velocity estimation. The accuracy of the velocity estimation can also be improved by implementing preprocess techniques including image intensification, Gaussian filtering, and "image-shift."

The Influence of Normal and Early Vascular Aging on Hemodynamic Characteristics in Cardio- and Cerebrovascular Systems.

Age-associated alterations in cardiovascular structure and function induce cardiovascular disease in elderly subjects. To investigate the effects of normal vascular aging (NVA) and early vascular aging (EVA) on hemodynamic characteristics in the circle of Willis (CoW), a closed-loop one-dimensional computational model was developed based on fluid mechanics in the vascular system. The numerical simulations revealed that higher central pulse pressure and augmentation index (AIx) appear in the EVA subjects due to early arrival of reflected waves, resulted in the increase of cardiac afterload compared with the NVA subjects. Moreover, the hemodynamic characteristics in the CoW show that the EVA subjects in an older age display a higher blood pressure than that of the NVA with a complete CoW. Herein, the increased blood pressure and flow rate coexist in the subjects with an incomplete CoW. In conclusion, the hemodynamic characteristics in the aortic tree and CoW related to aging appear to play an important role in causing cardiovascular and intravascular disease.

What is Missing for Stem Cell-Based Therapies in Dentistry.

There have been significant advancements in regenerative dentistry demonstrated in large animal studies, particularly in the areas of endodontics and periodontics. By using dental stem cells, pulp tissues can be regenerated in empty root canal space, and periodontal pockets can be regenerated to its normal state. To further test its clinical applications and perhaps to provide such patient care service if proven safe and successful, a facility called good manufacturing practice where stem cells are processed is needed. However, such facilities are rare.

Increased glycolysis and cellular crosstalk in eosinophilic chronic rhinosinusitis with nasal polyps.

Introduction: Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the sinonasal mucosa with distinct endotypes including type 2 (T2) high eosinophilic CRS with nasal polyps (eCRSwNP), T2 low non-eosinophilic CRS with nasal polyps (neCRSwNP), and CRS without nasal polyps (CRSsNP). Methods: Given the heterogeneity of disease, we hypothesized that assessment of single cell RNA sequencing (scRNA-seq) across this spectrum of disease would reveal connections between infiltrating and activated immune cells and the epithelial and stromal populations that reside in sinonasal tissue. Results: Here we find increased expression of genes encoding glycolytic enzymes in epithelial cells (EpCs), stromal cells, and memory T-cell subsets from patients with eCRSwNP, as compared to healthy controls. In basal EpCs, this is associated with a program of cell motility and Rho GTPase effector expression. Across both stromal and immune subsets, glycolytic programming was associated with extracellular matrix interactions, proteoglycan generation, and collagen formation. Furthermore, we report increased cell-cell interactions between EpCs and stromal/immune cells in eCRSwNP compared to healthy control tissue, and we nominate candidate receptor-ligand pairs that may drive tissue remodeling. Discussion: These findings support a role for glycolytic reprograming in T2-elicited tissue remodeling and implicate increased cellular crosstalk in eCRSwNP.

Development of a Pipeline for Removing Allergy Labels in Patients Undergoing Hematopoietic Stem Cell Transplantation.

Penicillin allergy is reported by 10% to 20 % of patients, but when evaluated only 1% to 2% may have a true allergy. Patients undergoing hematopoietic stem cell transplantation (HSCT) have a high likelihood of requiring beta-lactam antibiotics due to increased infection risk, which can be limited by a penicillin allergy label. When a penicillin allergy is recorded, alternatives are needed, including more expensive broader-spectrum antibiotics, with increases in drug-resistant bacteria, longer hospital stays, higher expenditures, and increases in nosocomial infections, such as Clostridium difficile colitis. This group of patients already undergoes extensive pretreatment testing and would especially benefit from allergy delabeling. This study aimed to develop a self-sustaining, low-cost pipeline between an HSCT clinic and an allergy clinic to identify and successfully delabel low-risk patients who endorse an allergy to penicillin, amoxicillin, amoxicillin-clavulanate, piperacillin-tazobactam, or ampicillin before admission to the hospital. We developed a survey to triage allergy risk, identified key stakeholders in building the pipeline, and underwent 4 plan, do, study, act (PDSA) cycles. Changes were made in each of the PDSA cycles to minimize cost and uncompensated provider time, as well as to increase patient retention throughout the pipeline by increasing appointment availability and decreasing reliance on patients to independently progress through the pathway. Of the 410 patients with planned HSCT who were screened over 11 months, 89 (21.7%) were listed as having a penicillin and/or beta lactam allergy. All but 1 (66 of 67; 98.5%) of the participants completed the survey accurately when confirmed by an allergist, and the survey was 100% accurate in predicting delabeling success in low-risk patients. Of eligible patients, 43.8% (n = 39) were successfully delabeled before their transplant date, and 97.4% of these (n = 38) have undergone HSCT to date. This pipeline is maintained by approximately 5 hours of work per week (1 hour of allergy physician time, 4 hours of nurse and/or clinical coordinator time), with no other direct costs. There is an estimated direct savings of at least $1914.93 per patient delabeled. We successfully designed and implemented a pipeline between the HSCT clinic and the allergy clinic as a quality improvement initiative to identify and address high rates of reported beta-lactam allergies. We identified and addressed patient-based factors, logistical, temporal, and financial barriers that impacted patient retention and sustainability. This model is expected to yield significant and sustained cost savings for the healthcare system as well as to improve patient outcomes, and this hypothesis is currently undergoing formal analysis. We anticipate that this model can be used to create a similar pipeline in other healthcare systems for HSCT recipients, as well as patients in other clinical settings, such as oncology and chimeric antigen receptor T cell therapy.

Rhinovirus infection of airway epithelial cells uncovers the non-ciliated subset as a likely driver of genetic susceptibility to childhood-onset asthma.

Asthma is a complex disease caused by genetic and environmental factors. Epidemiological studies have shown that in children, wheezing during rhinovirus infection (a cause of the common cold) is associated with asthma development during childhood. This has led scientists to hypothesize there could be a causal relationship between rhinovirus infection and asthma or that RV-induced wheezing identifies individuals at increased risk for asthma development. However, not all children who wheeze when they have a cold develop asthma. Genome-wide association studies (GWAS) have identified hundreds of genetic variants contributing to asthma susceptibility, with the vast majority of likely causal variants being non-coding. Integrative analyses with transcriptomic and epigenomic datasets have indicated that T cells drive asthma risk, which has been supported by mouse studies. However, the datasets ascertained in these integrative analyses lack airway epithelial cells. Furthermore, large-scale transcriptomic T cell studies have not identified the regulatory effects of most non-coding risk variants in asthma GWAS, indicating there could be additional cell types harboring these "missing regulatory effects". Given that airway epithelial cells are the first line of defense against rhinovirus, we hypothesized they could be mediators of genetic susceptibility to asthma. Here we integrate GWAS data with transcriptomic datasets of airway epithelial cells subject to stimuli that could induce activation states relevant to asthma. We demonstrate that epithelial cultures infected with rhinovirus significantly upregulate childhood-onset asthma-associated genes. We show that this upregulation occurs specifically in non-ciliated epithelial cells. This enrichment for genes in asthma risk loci, or 'asthma heritability enrichment' is also significant for epithelial genes upregulated with influenza infection, but not with SARS-CoV-2 infection or cytokine activation. Additionally, cells from patients with asthma showed a stronger heritability enrichment compared to cells from healthy individuals. Overall, our results suggest that rhinovirus infection is an environmental factor that interacts with genetic risk factors through non-ciliated airway epithelial cells to drive childhood-onset asthma.

Synchronization of Shop-Floor Logistics and Manufacturing Under IIoT and Digital Twin-Enabled Graduation Intelligent Manufacturing System.

Logistics interfaces with manufacturing throughout the entire production process need synchronous operations. For achieving integrated organization and operations between manufacturing and logistics, this article introduces the concept of shop-floor logistics and manufacturing synchronization with four principles, including: 1) synchronization-oriented manufacturing system; 2) synchronized information sharing; 3) synchronized decision making; and 4) synchronized operations. The marriage of the principles rendered the development of an overall framework of the Industrial Internet of Things (IIoT) and digital twin-enabled graduation intelligent manufacturing system (GiMS). A mixed-integer programming-based synchronization mechanism is proposed under GiMS. To meet the requirement of fast decision making in real-life shop-floor logistics and manufacturing synchronization problems, an equivalent constraint programming model is developed and tested. The observation and analysis of the case company show the advantage of the proposed concept and approach with the best performance regarding key performance indicators. The concept of synchronization provides an insight for understanding the interaction of logistics and manufacturing at the operational level. This article potentially enables manufacturers to reevaluate and develop their manufacturing planning and control strategies in the IIoT and digital twin-based manufacturing environment.

Internet of Everything and Digital Twin enabled Service Platform for Cold Chain Logistics.

The proliferation of the e-commerce market has posed challenges to staff safety, product quality, and operational efficiency, especially for cold chain logistics (CCL). Recently, the logistics of vaccine supply under the worldwide COVID-19 pandemic rearouses public attention and calls for innovative solutions to tackle the challenges remaining in CCL. Accordingly, this study proposes a cyber-physical platform framework applying the Internet of Everything (IoE) and Digital Twin (DT) technologies to promote information integration and provide smart services for different stakeholders in the CCL. In the platform, reams of data are generated, gathered, and leveraged to interconnect and digitalize physical things, people, and processes in cyberspace, paving the way for digital servitization. Deep learning techniques are used for accident identification and indoor localization based on Bluetooth Low Energy (BLE) to actualize real-time staff safety supervision in the cold warehouse. Both algorithms are designed to take advantage of the IoE infrastructure to achieve online self-adapting in response to surrounding evolutions. Besides, with the help of mobile and desktop applications, paperless operation for shipment, remote temperature and humidity (T&H) monitoring, anomaly detection and warning, and customer interaction are enabled. Thus, information traceability and visibility are highly fortified in this way. Finally, a real-life case study is conducted in a pharmaceutical distribution center to demonstrate the feasibility and practicality of the proposed platform and methods. The dedicated hardware and software are developed and deployed on site. As a result, the effectiveness of staff safety management, operational informatization, product quality assurance, and stakeholder loyalty maintenance shows a noticeable improvement. The insights and lessons harvested in this study may spark new ideas for researchers and inspire practitioners to meet similar needs in the industry.

An IL-12-Based Nanocytokine Safely Potentiates Anticancer Immunity through Spatiotemporal Control of Inflammation to Eradicate Advanced Cold Tumors.

Treatment of immunologically cold tumors is a major challenge for immune checkpoint inhibitors (ICIs). Interleukin 12 (IL-12) can invigorate ICIs against cold tumors by establishing a robust antitumor immunity. However, its toxicity and systemic induction of counteracting immunosuppressive signals have hindered translation. Here, IL-12 activity is spatiotemporally controlled for safely boosting efficacy without the stimulation of interfering immune responses by generating a nanocytokine that remains inactive at physiological pH, but unleashes its full activity at acidic tumor pH. The IL-12-based nanocytokine (Nano-IL-12) accumulate and release IL-12 in tumor tissues, eliciting localized antitumoral inflammation, while preventing systemic immune response, counteractive immune reactions, and adverse toxicities even after repeated intravenous administration. The Nano-IL-12-mediated spatiotemporal control of inflammation prompt superior anticancer efficacy, and synergize with ICIs to profoundly inflame the tumor microenvironment and completely eradicate ICI-resistant primary and metastatic tumors. The strategy could be a promising approach toward safer and more effective immunotherapies.

mScarlet and split fluorophore mScarlet resources for plasmid-based CRISPR/Cas9 knock-in in C. elegans.

Fluorescent proteins allow the expression of a gene and the behavior of its protein product to be observed in living animals. The ability to create endogenous fluorescent protein tags via CRISPR genome engineering has revolutionized the authenticity of this expression, and mScarlet is currently our first-choice red fluorescent protein (RFP) for visualizing gene expression in vivo . Here, we have cloned versions of mScarlet and split fluorophore mScarlet previously optimized for C. elegans into the SEC-based system of plasmids for CRISPR/Cas9 knock-in. Ideally, an endogenous tag will be easily visible while not interfering with the normal expression and function of the targeted protein. For low molecular weight proteins that are a fraction of the size of a fluorescent protein tag (e.g. GFP or mCherry) and/or proteins known to be non-functional when tagged in this way, split fluorophore tagging could be an alternative. Here, we used CRISPR/Cas9 knock-in to tag three such proteins with split-fluorophore wrmScarlet: HIS-72, EGL-1, and PTL-1. Although we find that split fluorophore tagging does not disrupt the function of any of these proteins, we were unfortunately unable to observe the expression of most of these tags with epifluorescence, suggesting that split fluorophore tags are often very limited as endogenous reporters. Nevertheless, our plasmid toolkit provides a new resource that enables straightforward knock-in of either mScarlet or split mScarlet in C. elegans.

Increased epithelial mTORC1 activity in chronic rhinosinusitis with nasal polyps.

Background: The airway epithelium plays a central role in the pathogenesis of chronic respiratory diseases such as asthma and chronic rhinosinusitis with nasal polyps (CRSwNP), but the mechanisms by which airway epithelial cells (EpCs) maintain inflammation are poorly understood. Objective: We hypothesized that transcriptomic assessment of sorted airway EpCs across the spectrum of differentiation would allow us to define mechanisms by which EpCs perpetuate airway inflammation. Methods: Ethmoid sinus EpCs from adult patients with CRS were sorted into 3 subsets, bulk RNA sequenced, and analyzed for differentially expressed genes and pathways. Single cell RNA-seq (scRNA-seq) datasets from eosinophilic and non-eosinophilic CRSwNP and bulk RNA-seq of EpCs from mild/moderate and severe asthma were assessed. Immunofluorescent staining and ex vivo functional analysis of sinus EpCs were used to validate our findings. Results: Analysis within and across purified EpC subsets revealed an enrichment in glycolytic programming in CRSwNP vs CRSsNP. Correlation analysis identified mammalian target of rapamycin complex 1 (mTORC1) as a potential regulator of the glycolytic program and identified EpC expression of cytokines and wound healing genes as potential sequelae. mTORC1 activity was upregulated in CRSwNP, and ex vivo inhibition demonstrated that mTOR is critical for EpC generation of CXCL8, IL-33, and CXCL2. Across patient samples, the degree of glycolytic activity was associated with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in severe asthma. Conclusions: Together, these findings highlight a metabolic axis required to support epithelial generation of cytokines critical to both chronic T2 and non-T2 inflammation in CRSwNP and asthma.

Bone marrow stromal cells produce long-term pain relief in rat models of persistent pain.

Chronic pain conditions are difficult to treat and are major health problems. Bone marrow stromal cells (BMSCs) have generated considerable interest as a candidate for cell-based therapy. BMSCs are readily accessible and are easy to isolate and expand ex vivo. Clinical studies show that direct injection of BMSCs does not produce unwanted side effects and is well tolerated and safe. Here, we show that a single systemic (intravenous) or local injection (into the lesion site) of rat primary BMSCs reversed pain hypersensitivity in rats after injury and that the effect lasted until the conclusion of the study at 22 weeks. The pain hypersensitivity was rekindled by naloxone hydrochloride, an opioid receptor antagonist that acts peripherally and centrally, when tested at 1-5 weeks after BMSC infusion. In contrast, naloxone methiodide, a peripherally acting opioid receptor antagonist, only rekindled hyperalgesia in the first 3 weeks of BMSC treatment. Focal downregulation of brainstem mu opioid receptors by RNA interference (RNAi) reversed the effect of BMSCs, when RNAi was introduced at 5- but not 1-week after BMSC transplantation. Thus, BMSCs produced long-term relief of pain and this effect involved activation of peripheral and central opioid receptors in distinct time domains. The findings prompt studies to elucidate the cellular mechanisms of the BMSC-induced pain relieving effect and translate these observations into clinical settings.

Effects of intentionally treated water on the growth of mesenchymal stem cells: An exploratory study.

OBJECTIVE: This study explored if human primary mesenchymal stem cells (MSCs), derived from two donors and cultivated in a medium made with intentionally treated water, would exhibit more growth and pluripotency than MSCs from the same source but grown in untreated (control) water. DESIGN: To create the treated water, three Buddhist monks directed their attention toward commercially bottled water while holding the intention that the water would enhance the growth of MSCs. Under double-blind conditions, cell culture growth mediums were prepared with the treated and untreated water, which was in turn used to grow the primary MSCs. Primary cells obtained from two donors were designated as Cells #1 and Cells #2. The prediction was that treated water would result in increased cell proliferation, that more cells would enter the cell cycle growth phase, and that there would be increased expression of genes (NANOG, OCT4 and SOX2) associated with improved cell growth and decreased expression of genes (p16, p21, and p53) associated with a decline in cell growth. The improved growth hypothesis was directional, thus one-tailed p-values were used to evaluate the results. RESULTS: Proliferation averaged across Cells #1 and #2 showed overall increased growth in treated as compared to control water (p = 0.0008). Cells #1 and #2 considered separately had differences in the same direction but only Cells #2 showed a significant difference on day 6 (p = 0.01). For cell cycle, there was a significantly greater percentage of Cells #2 in the S interphase with treated vs. control water (p = 0.04). For the gene expression analysis, when considering the average across the two donor cells, only the NANOG gene expression was in the predicted direction (p = 0.01); by contrast, the p16 gene expression was significantly opposite to the predicted direction (p = 0.005, one-tailed, post-hoc). For Cells #1 considered separately, no differences were significant except for p16, which resulted in an effect opposite to the predicted outcome (p = 0.05). For Cells #2, three genes were significantly in the predicted directions: NANOG (p = 0.0008), OCT4 (p = 0.005), and P53 (p = 0.05); p16 was significantly opposite to the prediction (p = 0.001). CONCLUSION: Intentionally treated water appeared to have some biological effects on the growth, pluripotency and senescence of human MSCs. This was especially the case in one of the two donor cells tested, but the effects were not consistently in the predicted direction. As an exploratory study, caution is warranted in interpreting these outcomes, and adjustment for multiple testing would likely reduce some of the weaker effects to nonsignificant. But given the double-blind protocol, as well as several more significant outcomes in the predicted directions, further research is warranted.