There is no evidence for bone remodeling caused by transdermal calcium loss in sweat during Bikram Hot Yoga in premenopausal women

It has been hypothesized that sweat loss during exercise causes a disruption in calcium homeostasis leading to bone resorption and low bone mineral density. There is substantial water and electrolyte loss in sweat during hot yoga, an exercise that is becoming increasingly popular with premenopausal women. A Bikram Hot Yoga session typically consists of 26 Hatha Yoga postures that are performed in 90 minutes inside of a room that is set to 105 °F and 40 % humidity. We measured sweat and plasma, electrolyte and water balance before and after a hot yoga session in a population of premenopausal women. Sweat was collected during the final yoga asana by saturating filter paper with sweat from the participants’ thighs to estimate the total amount of electrolyte loss. There was no change in serum sodium or serum osmolarity before or after Bikram Hot Yoga. Mean calcium concentration in serum increased after the hot yoga session. The concentration of parathyroid hormone (PTH) did not change from before to after the hot yoga session; however, a substantial amount of water was lost. This implies that the amount of parathyroid hormone in circulation decreased from before to after. Calcium loss in sweat loss did not trigger an increase in PTH secretion and did not initiate bone resorption. A disruption in calcium homeostasis was not observed in a bout of excessive sweating during a 90-minute Bikram Hot Yoga session. Bikram yoga appears to be a safe practice for premenopausal women as long as fluid and electrolytes are appropriately replenished after the Bikram Hot Yoga Session

TMOD-21. A NOVEL IN-VITRO METHOD TO MODEL MACROPHAGES IN GLIOBLASTOMA

Abstract BACKGROUND The GBM tumor microenvironment (TME) is comprised of a plethora of cancerous and non-cancerous cells that contribute to GBM growth, invasion, and chemoresistance. In-vitro models of GBM typically fail to incorporate multiple cell types. Others have addressed this problem by employing 3D bioprinting to incorporate astrocytes and macrophages in an extracellular matrix; however, they used serum-containing media and classically polarized anti-inflammatory macrophages. Serum has been shown to cause GBM brain-tumor initiating cells to lose their stem-like properties, highlighting the importance of excluding it from these models. Additionally, tumor-associated macrophages (TAMs) do not adhere to the traditional M2 phenotype. METHODS THP-1 monocytes and normal human astrocytes (NHAs) were transitioned into serum-free HL-1 and neurobasal-based media, respectively. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing them with GBM patient-derived xenograft(PDX) lines, using a transwell insert. CD206 expression was used to validate polarization and a cytokine array was used to characterize the cells. RESULTS There was no difference in proliferation rates at 72 hours for THP-1 monocytes grown in serum-free HL-1 media compared to serum-containing RPMI 1640 (p &amp;gt; 0.95). Macrophages polarized via transwell inserts expressed the lymphocyte chemoattractant protein, CCL2, whereas resting(M0), pro-inflammatory(M1), and anti-inflammatory(M2) macrophages did not. Additionally, these macrophages expressed more CXCL1 and IL-1ß relative to M1 macrophages. We have also demonstrated a method to maintain a tri-culture model of GBM PDX cells, NHAs, and TAMs in a serum-free media that supports the growth/maintenance of all cell types. CONCLUSIONS We have demonstrated a novel method by which we can polarize macrophages towards a tumor-supportive phenotype that differs in cytokine expression from traditionally polarized macrophages. This higher-fidelity method of modeling TAMs in GBM can aid in the development of targeted therapeutics that may one day enter the clinic in hopes of improving outcomes in GBM. </jats:sec

Developing and testing a smoking cessation app in Arabic language: A pilot randomized trial study protocol

Background Tobacco consumption in the Middle East is very high. Many smoking cessation apps effectively reduce smoking rates, yet few smoking cessation apps are tailored to the region. To date, smoking cessation apps in Arabic are mostly translated from English, with no studies assessing their efficacy. Additionally, these apps often fail to address cultural differences. The purpose of this study is to develop a smoking cessation app in Arabic (iEndSmokin, أنهيت التدخين ) and to examine its feasibility and efficacy. The app will incorporate evidence-based 5As, 5Rs, and cognitive behavioral therapy. Method In this pilot randomized trial, adults who smoke will be randomized and allocated in a 1:1 ratio to either the app intervention group (using the app) or the control group. Participants in the control arm will attend educational sessions. The intervention will last for 3 months, and participants will be followed for 6 months. A total of 200 adults who smoke willing to quit will be recruited between October and November 2025 (or until recruitment is complete). The study will run from December 2025 to May 2026, with follow-ups at baseline, 1 month, 3 months, and 6 months. Data will be analyzed using an intention-to-treat approach. Results The primary outcome will be 30-day point prevalence of smoking abstinence with biochemically confirmed cessation via normal saliva cotinine levels at 3 months. Secondary outcomes include either a 7-day point prevalence abstinence verified by saliva cotinine levels, and a ≥ 20% reduction in smoking rate. Long-term abstinence will be assessed at 6 months’ post-intervention. Conclusion Developing a culturally sensitive smoking cessation app to be available for the majority of adults who smoke for free that adhere to clinical guideline is a promising low-cost solution to promote behavioral change. We hypothesize that the iEndSmokin App will be an effective, accessible, and feasible smoking cessation tool for Arabic-speaking populations. Clinical Trial Registration: ClinicalTrials.gov Identifier: NCT0676622

13109 Phosphorylation state of Myristoylated Alanine-Rich C-Kinase Substrate Effector Domain mimetics determines its cytotoxicity in glioblastoma and macrophage model

ABSTRACT IMPACT: This study provides insight into how MED2 impacts the immune cells surrounding glioblastoma that help it to grow and spread; having a more complete understanding of how MED2 works will help us better develop therapies that may one day enter the clinic to improve patient outcomes in glioblastoma. OBJECTIVES/GOALS: The purpose of this study was to determine whether the phosphorylation state of the MED2 peptide impacts its biological activity in GBM and macrophages. MED2 variants include the phosphorylatable wild-type (MED2), pseudo-phosphorylated (MED2-PP), non-phosphorylatable (MED2-NP) and control length (CTL2) peptides. METHODS/STUDY POPULATION: MED2, MED2-NP, MED2-PP, and CTL2 were screened against a panel of molecularly characterized glioblastoma patient derived xenografts and IL4/13 stimulated M2-like THP-1 macrophages. The luminescent cell viability assay, CellTiter-Glo, was used to determine viability. RESULTS/ANTICIPATED RESULTS: The proneural lines XD456 and X1441 were highly sensitive to 5 µM MED2 and 5 µM MED2NP compared to 5 µM MED2PP (p&lt;0.001). There was no statistically significant difference between untreated, 5 µM CTL2, and 5 µM MED2PP groups or between the MED2NP and MED2 treated groups. M2-like THP-1 macrophages were highly sensitive to 10 µM MED2NP compared to 10 µM CTL2 (p&lt;0.01) and 10 µM MED2PP (p&lt;0.01) No statistically significant difference was observed between untreated, 10 µM MED2, 10 µM MED2PP, and 10 µM CTL2 groups. DISCUSSION/SIGNIFICANCE OF FINDINGS: The phosphorylation state of MED2 determines its toxicity. When MED2 is phosphorylated, it is nontoxic to GBM or M2-like macrophages. The non-phosphorylatable version is toxic to both GBM and M2-like macrophages. The wild-type peptide is toxic to GBM but not M2-like macrophages, suggesting that MED2 may be phosphorylated in M2-like macrophages.</jats:p

A High-Throughput Neurosphere-Based Colony Formation Assay to Test Drug and Radiation Sensitivity of Different Patient-Derived Glioblastoma Lines

The gold standard assay for radiation response is the clonogenic assay, a normalized colony formation assay (CFA) that can capture a broad range of radiation-induced cell death mechanisms. Traditionally, this assay relies on two-dimensional (2D) cell culture conditions with colonies counted by fixing and staining protocols. While some groups have converted these to three-dimensional (3D) conditions, these models still utilize 2D-like media compositions containing serum that are incompatible with stem-like cell models such as brain tumor initiating cells (BTICs) that form self-aggregating spheroids in neural stem cell media. BTICs are the preferred patient-derived model system for studying glioblastoma (GBM) as they tend to better retain molecular and phenotypic characteristics of the original tumor tissue. As such, it is important that preclinical radiation studies should be adapted to BTIC conditions. In this study, we describe a series of experimental approaches for performing CFA experiments with BTIC cultures. Our results indicate that serum-free clonogenic assays are feasible for combination drug and radiation testing and may better facilitate translatability of preclinical findings