RANK Ligand Modulation of Autophagy in Oral Squamous Cell Carcinoma Tumor Cells.

Autophagy is a cellular process to recycle nutrients and has been implicated in cancer treatment. Oral squamous cell carcinoma (OSCC) is the most common oral cancer which ranks 3% of cancers in men and 2% in women. In this study, immunohistochemical staining of OSCC tumor specimens from human subjects and an athymic mouse model demonstrated high levels of autophagy markers LC3-II and ATG5 expression. Further, we identified high levels LC3-II expression in OSCC tumor cell lines (SCC-1, SCC-12, and SCC-14a) compared to normal human epithelial (RWPE-1) cells. OSCC cells express high levels of RANK ligand (RANKL); however, a functional role in autophagy is unknown. Interestingly, RANKL stimulation significantly increased autophagosome-related gene expressions such as LC3, ATG5, BECN1, and PI3KC3 mRNA expression in OSCC cells. Further, Western blot analysis of total cell lysates demonstrated a dose-dependent increase in LC3-II and ATG5 expression in RANKL-stimulated cells. In addition, RANKL increased expression of LC3-I and LC3-II, essential for autophagosome formation. Confocal microscopy analysis of LC3-II and localization with lysosome further confirms autophagosome formation in response to RANKL treatment in OSCC cells. Collectively, our results indicate a novel function of RANKL to induce autophagosome formation, and could be a potential therapeutic target to control OSCC tumor progression.

mTOR Complexes Repress Hypertrophic Agonist-Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells.

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 µM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium.

Yogic breathing when compared to attention control reduces the levels of pro-inflammatory biomarkers in saliva: a pilot randomized controlled trial.

BACKGROUND: Self-report measures indicate that Yoga practices are perceived to reduce stress; however, molecular mechanisms through which YB affects stress are just beginning to be understood. While invasive sampling such as blood has been widely used to measure biological indicators such as pro-inflammatory biomarkers, the use of saliva to measure changes in various biomolecules has been increasingly recognized. As Yoga practice stimulates salivary secretion, and saliva is considered a source of biomarkers, changes in salivary cytokines before and after Yogic breathing exercise as specified in an ancient Tamil script, Thirumanthiram, were examined using a Cytokine Multiplex to compare to Attention Control (AC) group. METHODS: Twenty healthy volunteers were randomized into two groups stratified by gender (N = 10 per YB and AC groups); The YB group performed two YB exercises, each for ten minutes, for a total of twenty minutes in a single session as directed by a trained Yoga instructor. The AC group read a text of their choice for 20 min. Saliva was collected immediately after YB training at 0, 5, 10, 15 and 20 min and analyzed by Multiplex enzyme linked immunosorbent assay (ELISA). RESULTS: The levels of interleukin (IL)-1ß, IL-8, and monocyte chemotactic protein -1 (MCP-1) were significantly reduced in YB group when compared to AC group. The level of reduction of IL-8 was significant at all time points tested, whereas IL-1ß showed reduction at 15 and 20 min time points (p < 0.05), and MCP-1 level was marginally different at 5-20 min. There were no significant differences between YB and AC groups in the salivary levels of IL-1RA, IL-6, IL-10, IL-17, IP-10, MIP-1b, and TNF-α. CONCLUSIONS: These data are the first to demonstrate the feasibility of detecting salivary cytokines using multiplex assay in response to a Yoga practice. This study was registered in Clinical Trials.gov # NCT02108769.

Inhibition of caspases protects mice from radiation-induced oral mucositis and abolishes the cleavage of RNA-binding protein HuR.

The oral mucosal epithelium is typically insulted during chemotherapy and ionizing radiation (IR) therapy and disposed to mucositis, which creates painful inflammation and ulceration in the oral cavity. Oral mucositis alters gene expression patterns, inhibits cellular growth, and initiates cell death in the oral epithelial compartments. Such alterations are governed by several different factors, including transcription factors, RNA-binding proteins, and microRNAs. IR-induced post-transcriptional regulation of RNA-binding proteins exists but is poorly studied in clinically relevant settings. We herein report that the RNA-binding protein human antigen R (HuR) undergoes cleavage modification by caspase-3 following IR-induced oral mucositis and subsequently promotes the expression of the pro-apoptotic factor BAX (Bcl-2-associated X protein), as well as cell death. Further analyses revealed that the HuR cleavage product-1 (HuR-CP1) directly associates and stabilizes the BAX mRNA and concurrently activates the apoptotic pathway. On the other hand, a noncleavable isoform of HuR promotes the clonogenic capacity of primary oral keratinocytes and decreases the effect of IR-induced cell death. Additionally, specific inhibition of caspase-3 by a compound, NSC321205, increases the clonogenic capacity of primary oral keratinocytes and causes increased basal layer cellularity, thickened mucosa, and elevated epithelial cell growth in the tongues of mice with oral mucositis. This protective effect of NSC321205 is mediated by a decrease in caspase-3 activity and the consequent inhibition of HuR cleavage, which reduces the expression of BAX in mice with IR-induced oral mucositis. Thus, we have identified a new molecular mechanism of HuR in the regulation of mRNA turnover and apoptosis in oral mucositis, and our data suggest that blocking the cleavage of HuR enhances cellular growth in the oral epithelial compartment.

Feasibility and Preliminary Impact of a Web-Based Mind Body Intervention for Older Dementia Caregivers.

BACKGROUND: Mind-body interventions focused on intentional breathing and movement have been found to mitigate the negative effects of caregiving such as such as stress, psychosocial distress, and emotional distress associated for persons living with Alzheimer's disease and related dementias. OBJECTIVE: The objective was to assess the feasibility and acceptability and preliminary impacts of our 12-week mind body intervention "Gentle Yoga and Yogic Breathing" for caregivers of persons living with dementia on health outcomes including mutuality, depression and anxiety, loneliness and social support, quality of life, and physical function. METHODS: We conducted a single-group cohort study in which 20 caregivers were enrolled. Data were collected at baseline and at the 12-week post-intervention endpoint. RESULTS: The intervention was acceptable; 75% (n = 15/20) completed the study; 16 completed post-study questionnaires. Very few experienced technical issues; 31% (n = 3) most commonly reported as poor internet connectivity, 75% (n = 12/16) perceived a health benefit, 88% (n = 14/16) perceived improved day-to-day mood, and 100% (n = 16/16) would recommend the intervention to other caregivers. Although there was minimal change from baseline to 12 weeks, for health outcomes, there were very small improvements noted in anxiety and overall health. There were no reported adverse events. CONCLUSION: The intervention was well received and is feasible and acceptable for future studies of stress and health management interventions for caregivers of persons living with dementia.Registered with https://www.ClinicalTrials.gov (NCT03853148).

Overexpression of RNA-binding protein CELF1 prevents apoptosis and destabilizes pro-apoptotic mRNAs in oral cancer cells.

CELF1 RNA-binding protein, otherwise called CUGBP1, associates and coordinates the degradation of GU-rich element (GRE) containing mRNA's encoding factors important for cell growth, migration and apoptosis. Although many substrates of CELF1 have been identified, the biological significance of CELF1-mediated mRNA decay remains unclear. As the processes modulated by CELF1 are frequently disrupted in cancer, we investigated the expression and role of CELF1 in oral squamous cancer cells (OSCCs). We determined that CELF1 is reproducibly overexpressed in OSCC tissues and cell lines. Moreover, depletion of CELF1 reduced proliferation and increased apoptosis in OSCCs, but had negligible effect in non-transformed cells. We found that CELF1 associates directly with the 3'UTR of mRNAs encoding the pro-apoptotic factors BAD, BAX and JunD and mediates their rapid decay. Specifically, 3'UTR fragment analysis of JunD revealed that the GRE region is critical for binding with CELF1 and expression of JunD in oral cancer cells. In addition, silencing of CELF1 rendered BAD, BAX and JunD mRNAs stable and increased their protein expression in oral cancer cells. Taken together, these results support a critical role for CELF1 in modulating apoptosis and implicate this RNA-binding protein as a cancer marker and potential therapeutic target.

Self-Administered Gentle Yoga and Yoga Breathing Intervention Improves Burden and Stress Biomarkers in Caregivers of Persons Living with Dementia.

Family caregivers of adults with Alzheimer's disease and related dementias (ADRD) are the fastest growing group of informal healthcare providers in the United States. These individuals experience high levels of stress and physical and psychosocial symptoms related to their caregiving role. We provided 20 ADRD caregivers (≥ 55 years of age) with training and access to a mobile health app for at-home practice of gentle yoga and yoga breathing exercises. Here, we report secondary outcomes, including changes in caregiver physical and mental function, burden, fatigue, sleep, and biological stress markers (salivary cortisol and 1,5-anhydroglucitol [1,5-AG] assays) from Baseline (Week 1) to Study End (Week 12). Self-reported questionnaire data were collected online via the Research Electronic Data Capture system, and biomarker data were collected via saliva samples. Results supported indications for slight improvement in outcomes of caregiving burden among ADRD caregivers using the gentle yoga and yoga breathing app. Furthermore, slight improvements in mental health were observed; the greatest gains were made by those reporting higher burden and having family members with more severe dementias. Findings indicate that those experiencing the greatest improvement in caregiver burden scores spent the most time doing gentle yoga and yoga breathing exercises. Salivary stress biomarkers moved in a favorable direction (decreased cortisol; increased 1,5-AG) among caregivers practicing the exercises most often. This is among the first studies of gentle yoga and yoga breathing exercises for relieving ADRD caregiver burden and the first we are aware of to incorporate stress biomarker measurement.

A quality improvement study on the feasibility and potential benefits of a yogic breathing program for cancer survivors and caregivers during treatment in a lodging facility.

Background: Complementary and integrative health approaches with a focus on relieving side effects of cancer treatment are popular among cancer patients. Previous studies have investigated the combined effects of yoga postures, breathing, and meditation, but the specific effects of the breathing component are under-reported. Our previous studies indicate that yogic breathing can improve salivary biomarker expression related to stress, immune response, and tumor suppression. We aim to assess the acceptability and feasibility of a yogic breathing program in cancer patients and caregivers during the treatment period. Methods: In this quality improvement study, we designed a 20-minute yogic breathing regimen and introduced them to all-site cancer patients and their caregivers during the cancer treatment period at a lodging facility, Hope Lodge in Charleston, SC. All interested participants were included as there were no eligibility criteria set for the study. The availability of the class was advertised via intercom, displays, and word of mouth. Participants were taught five different breathing exercises, and after completion of the exercises in a single session, a self-reported quality improvement questionnaire was administered assessing sociodemographic/clinical factors, expectations about the session, and ratings of satisfaction with the session. Results: During the nine months of the data collection period, 52 participants provided feedback of which patients and caregivers were almost equal numbers. Participants' perception of intervention acceptance, symptom management, satisfaction with the sessions, and future needs for practice indicate that the yogic breathing sessions help improve some of the key symptoms of cancer experience such as stress. Conclusion: Findings indicate that yogic breathing is acceptable to patients and caregivers and may help alleviate some of the side effects resulting from cancer treatment, and the intervention is feasible at lodging facilities during treatment. Currently, the yogic breathing sessions are conducted on a weekly basis by Hope Lodge volunteers trained by the study team.

Protocol for the Pilot Study of Group Video Yogic Breathing App in Breast Cancer Survivors.

INTRODUCTION: Breast cancer remains a leading cause of cancer deaths; however, recent improvements in treatment have improved survivorship. As a result of this improvement, more individuals are living with the long-term side effects of cancer treatment. Therefore, methods that incorporate lifestyle and mind-body approaches are becoming increasingly used in the patient treatment pathway. METHODS: In this study, PranaScience Institute will develop and test a group video mobile application for Yogic Breathing (YB). YB is shown to reduce symptomatic conditions associated with several conditions including breast cancer. For this initial feasibility study, PranaScience will collaborate with the Medical University of South Carolina to implement the study app-based program in breast cancer survivors. This research is aimed to understand if the YB could be delivered via an app, if participants are able to practice it satisfactorily, and if there is any symptom relief by the YB practice. In the control group, participants will be directed to the Attention Control (AC) feature of the app, which guides users to focus on a mindfulness activity not involving YB. Participants will be randomly assigned to the YB or AC study plan (N = 20 per group). Breast cancer survivors who have completed radiation therapy within last 2 months will be recruited for this study and provided access to the app for a 12-weeks program. The study app will record total practice times. Virtual visits by a study yoga instructor during group video sessions will measure participant compliance with proper technique. Feasibility will be examined by evaluating intervention delivery factors and resource needs. Acceptability of using the mobile study app to support symptom management will be evaluated using a satisfaction and system usability scale. Behavioral survey measures will help guide effect sizes and power calculations for the next larger-scale study. Biomarkers in the saliva (tumor suppressors, cytokines), and fingernails (cortisol, differential proteomics) will be measured at baseline and end of study at 12 weeks. DISCUSSION: All findings from this pilot study will be synthesized to refine the mobile study app in preparation for large-scale evaluation in Phase II involving all-study site participants with cancer. ClinicalTrials.gov Identifier NCT05161260.

Addressing Physical, Functional, and Physiological Outcomes in Older Adults using an Integrated mHealth Intervention "Active for Life": A Pilot Randomized Controlled Trial.

Objective: We evaluated components of an integrated, mobile health-based intervention "Activate for Life" (AFL) on health outcomes in lower-income older adults (≥ 60 years). Methods: AFL incorporates balance (Otago; OG), physical strength (Gentle Yoga and yogic Breathing; GYYB), and mental engagement (Behavioral Activation; BA) components. Thirty participants were randomly allocated to one of three study arms (n=10): OG (Arm 1), OG+GYYB (Arm 2), or OG+GYYB+BA (Arm 3; a.k.a. "full AFL"). Participants were evaluated for physical, functional, and physiological endpoints at baseline and post-intervention (12-weeks and/or 3-month follow up). Results: Improvements in pain interference and 1,5- anhydroglucitol biomarker levels over time were noted for all arms. No significant changes were observed for other physical, functional, or physiological measures. Discussion: This study illustrates potential benefits of the AFL intervention on the health of lower-income older adults. Lessons learned from this pilot trial will inform design improvements for a large-scale randomized controlled trial.

Rapamycin treatment augments both protein ubiquitination and Akt activation in pressure-overloaded rat myocardium.

Ubiquitin-mediated protein degradation is necessary for both increased ventricular mass and survival signaling for compensated hypertrophy in pressure-overloaded (PO) myocardium. Another molecular keystone involved in the hypertrophic growth process is the mammalian target of rapamycin (mTOR), which forms two distinct functional complexes: mTORC1 that activates p70S6 kinase-1 to enhance protein synthesis and mTORC2 that activates Akt to promote cell survival. Independent studies in animal models show that rapamycin treatment that alters mTOR complexes also reduces hypertrophic growth and increases lifespan by an unknown mechanism. We tested whether the ubiquitin-mediated regulation of growth and survival in hypertrophic myocardium is linked to the mTOR pathway. For in vivo studies, right ventricle PO in rats was conducted by pulmonary artery banding; the normally loaded left ventricle served as an internal control. Rapamycin (0.75 mg/kg per day) or vehicle alone was administered intraperitoneally for 3 days or 2 wk. Immunoblot and immunofluorescence imaging showed that the level of ubiquitylated proteins in cardiomyocytes that increased following 48 h of PO was enhanced by rapamycin. Rapamycin pretreatment also significantly increased PO-induced Akt phosphorylation at S473, a finding confirmed in cardiomyocytes in vitro to be downstream of mTORC2. Analysis of prosurvival signaling in vivo showed that rapamycin increased PO-induced degradation of phosphorylated inhibitor of κB, enhanced expression of cellular inhibitor of apoptosis protein 1, and decreased active caspase-3. Long-term rapamycin treatment in 2-wk PO myocardium blunted hypertrophy, improved contractile function, and reduced caspase-3 and calpain activation. These data indicate potential cardioprotective benefits of rapamycin in PO hypertrophy.

System transformation in palliative and end of life care: developing a model for excellence.

PURPOSE: The purpose of this study is to report how the palliative and end of life care community in one region of England worked together to create a new model for integrated palliative and end of life care to respond to the challenges of changing demography, the need to reduce unnecessary hospital admissions of people nearing the end of life and to improve the quality of provision in line with current policy. DESIGN/METHODOLOGY/APPROACH: A co-production approach to system transformation was adopted involving 73 members of the palliative and end of life care community in one region of England. FINDINGS: A new model for the delivery of integrated palliative and end of life care services was produced. The breadth of membership of the co-production working party and constructive/collaborative working helped ensure a viable model was produced. PRACTICAL IMPLICATIONS: Although systems' thinking perspectives can help address the challenges of large-scale transformation because they focus on promoting the value of relationships, recognise the nuances of context and the need to understand system behaviour over time, the potential for systems to benefit from this approach is limited by the complexity of the processes involved and the sheer number of issues to be addressed in practical terms by policy makers and change leaders. ORIGINALITY/VALUE: The paper explores the contribution that theories of large-scale transformation can make to the design of palliative and end of life care services in health and social care.

Beta3 integrin-mediated ubiquitination activates survival signaling during myocardial hypertrophy.

Identifying the molecular mechanisms activated in compensatory hypertrophy and absent during decompensation will provide molecular targets for prevention of heart failure. We have previously shown enhanced ubiquitination (Ub) during the early growth period of pressure overload (PO) hypertrophy near intercalated discs of cardiomyocytes, where integrins are important for mechanotransduction. In this study, we tested the role of integrins upstream of Ub, whether enhanced Ub contributes to survival signaling in early PO, and if loss of this mechanism could lead to decreased ventricular function. The study used a beta(3) integrin (-/-) mouse and a wild-type mouse as a control for in vivo PO by transverse aortic constriction (TAC) and for cultured cardiomyocytes in vitro, stimulated with the integrin-activating peptide RGD. We demonstrate beta(3) integrin mediates transient Ub of targeted proteins during PO hypertrophy, which is necessary for cardiomyocyte survival and to maintain ventricular function. Prosurvival signaling proceeds by initiation of NF-kappaB transcription of the E3 ligase, cIAP1. In PO beta(3)(-/-) mice, absence of this mechanism correlates with increased TUNEL staining and decreased ventricular mass and function by 4 wk. This is the first study to show that a beta(3) integrin/Ub/NF-kappaB pathway contributes to compensatory hypertrophic growth.

Lack of beta3 integrin signaling contributes to calpain-mediated myocardial cell loss in pressure-overloaded myocardium.

Although cardiac hypertrophy initially ensues as a compensatory mechanism, it often culminates in congestive heart failure. Based on our earlier studies that calpain and beta3 integrin play cell death and survival roles, respectively, during pressure-overload (PO) hypertrophy, we investigated if the loss of beta3 integrin signaling is a potential mechanism for calpain-mediated cardiomyocyte death during PO. beta3 Integrin knockout (beta3) and wild-type mice were used to induce either moderate or severe PO in vivo for short-term (72-hour) and long-term (4-week) transverse aortic constriction. Whereas wild-type mice showed no changes during moderate PO at both time points, beta3 mice exhibited both enrichment of the mu-calpain isoform and programmed cell death of cardiomyocytes after 4-week PO. However, with severe PO that caused increased mortality in both mice groups, cell death was observed in wild-type mice also. To study calpain's role, calpeptin, a specific inhibitor of calpain, was administered through an osmotic mini-pump at 2.5 mg/kg per day beginning 3 days before moderate transverse aortic constriction or sham surgery. Calpeptin administration blocked both calpain enrichment and myocardial cell death in the 4-week PO beta3 mice. Because beta3 integrin contributes to cardioprotective signaling, these studies indicate that the loss of specific integrin function could be a key mechanism for calpain-mediated programmed cell death of cardiomyocytes in PO myocardium.

Calpain inhibition preserves myocardial structure and function following myocardial infarction.

Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that often trigger programmed cell death and contribute to maladaptive changes in myocardial structure and function. To test whether inhibition of calpain, a Ca(2+)-dependent cysteine protease, would prevent these changes, we used a mouse MI model. Calpeptin, an aldehydic inhibitor of calpain, was intravenously administered at 0.5 mg/kg body wt before MI induction and then at the same dose subcutaneously once per day. Both calpeptin-treated (n = 6) and untreated (n = 6) MI mice were used to study changes in myocardial structure and function after 4 days of MI, where end-diastolic volume (EDV) and left ventricular ejection fraction (EF) were measured by echocardiography. Calpain activation and programmed cell death were measured by immunohistochemistry, Western blotting, and TdT-mediated dUTP nick-end labeling (TUNEL). In MI mice, calpeptin treatment resulted in a significant improvement in EF [EF decreased from 67 + or - 2% pre-MI to 30 + or - 4% with MI only vs. 41 + or - 2% with MI + calpeptin] and attenuated the increase in EDV [EDV increased from 42 + or - 2 microl pre-MI to 73 + or - 4 microl with MI only vs. 55 + or - 4 microl with MI + calpeptin]. Furthermore, calpeptin treatment resulted in marked reduction in calpain- and caspase-3-associated changes and TUNEL staining. These studies indicate that calpain contributes to MI-induced alterations in myocardial structure and function and that it could be a potential therapeutic target in treating MI patients.

NFAM1 signaling enhances osteoclast formation and bone resorption activity in Paget's disease of bone.

Paget's disease of bone (PDB) is marked by the focal activity of abnormal osteoclasts (OCLs) with excess bone resorption. We previously detected measles virus nucleocapsid protein (MVNP) transcripts in OCLs from patients with PDB. Also, MVNP stimulates pagetic OCL formation in vitro and in vivo. However, the mechanism by which MVNP induces excess OCLs/bone resorption activity in PDB is unclear. Microarray analysis identified MVNP induction of NFAM1 (NFAT activating protein with ITAM motif 1) expression. Therefore, we hypothesize that MVNP induction of NFAM1 enhances OCL differentiation and bone resorption in PDB. MVNP transduced normal human PBMC showed an increased NFAM1 mRNA expression without RANKL treatment. Further, bone marrow cells from patients with PDB demonstrated elevated levels of NFAM1 mRNA expression. Interestingly, shRNA suppression of NFAM1 inhibits MVNP induced OCL differentiation and bone resorption activity in mouse bone marrow cultures. Live cell widefield fluorescence microscopy analysis revealed that MVNP induced intracellular Ca2+ oscillations and levels were significantly reduced in NFAM1 suppressed preosteoclasts. Further, western blot analysis demonstrates that shRNA against NFAM1 inhibits MVNP stimulated PLCγ, calcineurin, and Syk activation in preosteoclast cells. Furthermore, NFAM1 expression controls NFATc1, a critical transcription factor expression and nuclear translocation in MVNP transuded preosteoclast cells. Thus, our results suggest that MVNP modulation of the NFAM1 signaling axis plays an essential role in pagetic OCL formation and bone resorption activity.

Microgravity Induction of TRAIL Expression in Preosteoclast Cells Enhances Osteoclast Differentiation.

Evidence indicates that astronauts experience significant bone loss in space. We previously showed that simulated microgravity (µXg) using the NASA developed rotary cell culture system (RCCS) enhanced bone resorbing osteoclast (OCL) differentiation. However, the mechanism by which µXg increases OCL formation is unclear. RANK/RANKL signaling pathway is critical for OCL differentiation. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been shown to increase osteoclastogenesis. We hypothesize that TRAIL may play an important role in µXg enhanced OCL differentiation. In this study, we identified by RT profiler PCR array screening that µXg induces high levels of TRAIL expression in murine preosteoclast cells in the absence of RANKL stimulation compared to ground based (Xg) cultures. We further identified that µXg elevated the adaptor protein TRAF-6 and fusion genes OC-STAMP and DC-STAMP expression in preosteoclast cells. Interestingly, neutralizing antibody against TRAIL significantly reduced µXg induced OCL formation. We further identified that over-expression of pTRAIL in RAW 264.7 cells enhanced OCL differentiation. These results indicate that TRAIL signaling plays an important role in the µXg increased OCL differentiation. Therefore, inhibition of TRAIL expression could be an effective countermeasure for µXg induced bone loss.

Alterations in Salivary Proteome following Single Twenty-Minute Session of Yogic Breathing.

Yogic breathing (YB) has been suggested to reduce stress and blood pressure and increase cognitive processes. However, alterations after YB at the molecular level are not well established. Twenty healthy volunteers were randomized into two groups (N = 10 per group): YB or attention controls (AC). The YB group performed two YB exercises, each for ten minutes, for a total of twenty minutes in a single session. AC group read a text of their choice for 20 minutes. Saliva was collected at baseline and at 5, 10, 15, and 20 minutes. Using Mass Spectrometry (MS), we initially found that 22 proteins were differentially expressed and then validated deleted in malignant brain tumor-1 (DMBT1) and Ig lambda-2 chain C region (IGLC2) using Western Blotting. DMBT1 was elevated in 7 of YB group by 10-fold and 11-fold at 10 and 15 minutes, respectively, whereas it was undetectable in the time-matched AC group (P < 0.05). There was a significant interaction between groups and time assessed by two-way ANOVA (P < 0.001). IGLC2 also showed a significant increase in YB group as measured by Western Blotting. These data are the first to demonstrate the feasibility of stimulating and detecting salivary protein biomarkers in response to an acute Yoga exercise. This trial is registered with ClincalTrials.gov NCT02108769.