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Objective: To evaluate whether micronutrients (vitamins/minerals) benefit attention-deficit/hyperactivity disorder (ADHD) and irritability in a North American pediatric sample., Method: A 3-site, 8-week, placebo-controlled, randomized clinical trial of micronutrients was conducted in nonmedicated children aged 6 to 12 years with ADHD and at least 1 impairing irritability symptom by parent report on the Child and Adolescent Symptom Inventory-5 (CASI-5). A priori-defined primary outcomes were Clinical Global Impression-Improvement (CGI-I) (CGI-I of 1 or 2 = treatment responder) and parent-rated CASI-5 composite score of ADHD, oppositional defiant, disruptive mood dysregulation, and peer conflict symptoms, including impairment scores., Results: Of 135 randomized (mean age 9.8 years), 126 youths (93%) comprised the modified intention-to-treat population. Blinding was maintained. For the CGI-I, 54% of the micronutrient and 18% of the placebo group were responders (risk ratio = 2.97, 97.5% CI = 1.50, 5.90, p < .001). CASI-5 composite scores improved significantly for both groups (p < .01), with a mean change of -0.31 (95% CI = -0.39, -0.23) in the micronutrient group and a mean change of -0.28 (95% CI = -0.38, -0.19) in the placebo group. However, the between-group difference was not significant (mean change = -0.02; 97.5% CI = -0.16, 0.12, effect size = 0.07, p = .70). The micronutrient group grew 6 mm more than the placebo group (p = .002). No serious adverse events or clinically significant changes from baseline in blood and urine tests occurred., Conclusion: Micronutrients showed global benefit over placebo by blinded clinician rating, but not by parent-report CASI-5 composite rating in a population with ADHD and irritability. Micronutrients showed greater height growth. Micronutrients were well tolerated, and the majority of participants adhered to the number of capsules prescribed. This randomized controlled trial replicates safety and efficacy reported for ADHD in 2 smaller trials of a similar formula containing all vitamins and known essential minerals in amounts between the Recommended Dietary Allowance and Upper Tolerable Intake Level., Clinical Trial Registration Information: Micronutrients for ADHD in Youth (MADDY) Study; https://clinicaltrials.gov; NCT03252522., (Copyright © 2021 American Academy of Child and Adolescent Psychiatry. Published by Elsevier Inc. All rights reserved.)

Connections between students and faculty on campus may influence students' sense of belonging, and a greater sense of belonging has a positive effect on student success. We developed a low-cost, faculty-led program of community-building events and implemented the program in the biology department at a small liberal-arts institution with the goal of improving students' sense of community. Student responses to surveys indicated that the majority of students felt connected to faculty and students in the department; however, Black or African American students initially felt a lower level of connection to faculty than did white students. After implementing our series of community-building events, students surveyed reported high levels of satisfaction with the events. Furthermore, there was a trend toward a higher percentage of Black or African American students than white students reporting that they were more likely to reach out to faculty after participating in the community-building events. Thus, our low-cost program improved connections between students and faculty in the biology department. Collectively, our results suggest that academic departments can implement community-building programs to improve students' sense of belonging., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Schafer et al.)

The aim of this paper is to present several popular methods for in vitro culture of osteocytes and osteocyte cell lines. Osteocytes are located extremely suitably within the calcified bone matrix to sense mechanical signals, and are equipped with a multitude of molecular features that allow mechanosensing. However, osteocytes are more than specialized mechanosensing cells. Several signaling molecules are preferentially produced by osteocytes, and osteocytes hold a tight reign over osteoblast and osteoclast formation and activity, but also have a role as endocrine cell, communicating with muscles or organs as remote as the kidneys. In order to facilitate further research into this fascinating cell type, three protocols will be provided in this paper. The first protocol will be on the culture of mouse (early) osteocyte cell lines, the second on the isolation and culture of primary mouse bone cells, and the third on the culture of fully embedded human osteocytes within their own three-dimensional bone matrix., Competing Interests: The authors declare no conflict of interest.

The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38. The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease.

There is currently no optimal system to expand and maintain the function of human adult hepatocytes in culture. Recent studies have demonstrated that specific tissue-derived extracellular matrix (ECM) can serve as a culture substrate and that cells tend to proliferate and differentiate best on ECM derived from their tissue of origin. The goal of this study was to investigate whether three-dimensional (3D) ECM derived from porcine liver can facilitate the growth and maintenance of physiological functions of liver cells. Optimized decellularization/oxidation procedures removed up to 93% of the cellular components from porcine liver tissue and preserved key molecular components in the ECM, including collagen-I, -III, and -IV, proteoglycans, glycosaminoglycans, fibronectin, elastin, and laminin. When HepG2 cells or human hepatocytes were seeded onto ECM discs, uniform multi-layer constructs of both cell types were formed. Dynamic culture conditions yielded better cellular infiltration into the ECM discs. Human hepatocytes cultured on ECM discs expressed significantly higher levels of albumin over a 21-day culture period compared to cells cultured in traditional polystyrene cultureware or in a collagen gel "sandwich". The culture of hepatocytes on 3D liver-specific ECM resulted in considerably improved cell growth and maintained cell function; therefore, this system could potentially be used in liver tissue regeneration, drug discovery or toxicology studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Skeletal muscle relies upon regeneration to maintain homeostasis and repair injury. This process involves the recruitment of the tissue's resident stem cell, the muscle progenitor cell, and a subsequent proliferative response by newly generated myoblasts, which must then align and fuse to generate new muscle fibers. During regeneration, cells rely on environmental input for direction. Extracellular matrix (ECM) represents a crucial component of a cell's microenvironment that aids in guiding muscle regeneration. We hypothesized that ECM extracted from skeletal muscle would provide muscle progenitor cells and myoblasts with an ideal substrate for growth and differentiation ex vivo. To test this hypothesis, we developed a method to extract ECM from the large thigh muscles of adult rats and present it to cells as a surface coating. Myogenic cells cultured on ECM extract experienced enhanced proliferation and differentiation relative to standard growth surfaces. As the methodology can be applied to any size muscle, these results demonstrate that bioactive ECM can be readily obtained from skeletal muscle and used to develop biomaterials that enhance muscle regeneration. Furthermore, the model system demonstrated here can be applied to the study of interactions between the ECM of a particular tissue and a cell population of interest.

Transdifferentiation has become a common claim for somatic stem cells, yet how such cells can be directed toward a specified cell lineage has not been well investigated. We previously demonstrated that when isolated epidermal stem cells were placed into an embryonic environment, their potential was extended beyond the keratinocyte lineage. Here, we present evidence that cells isolated using a modification of our published method for epidermal stem cells can be specifically directed to differentiate into B lymphocyte precursors. We found that these isolated cells co-cultured with S17 bone marrow stromal cells in cytokine-supplemented medium changed their cell surface marker profile and gene expression pattern to one characteristic of B lymphocyte precursors. Such cells also underwent variable, diversity, joining rearrangement at the immunoglobulin heavy-chain locus, a permanent genetic change unique to lymphocytes. This feature is limited to the cells isolated using the modified epidermal stem cell method, as cells isolated using the modified transit amplifying cell method could not be re-directed or reprogrammed. Such results demonstrate that cells from the epidermis can be directed to cross lineage boundaries to become mesodermally derived lymphocytes.

The epidermis of the skin, acting as the primary physical barrier between self and environment, is a dynamic tissue whose maintenance is critical to the survival of an organism. Like most other tissues and organs, the epidermis is maintained and repaired by a population of resident somatic stem cells. The epidermal stem cells reside in the proliferative basal cell layer and are believed to persist for the lifetime of an individual. Acting through intermediaries known as transit amplifying cells, epidermal stem cells ensure that the enormous numbers of keratinocytes required for epidermal homeostasis to be maintained are generated. This continual demand for new cell production must be met over the entire lifetime of an individual. Breakdown of the epidermal barrier would have catastrophic consequences. This leads us to question whether or not epidermal stem cells represent a unique population of cells which, by necessity, might be resistant to cellular aging. We hypothesized that the full physiologic functional capacity of epidermal stem cells is maintained over an entire lifetime. Using murine skin epidermis as our model system, we compared several properties of young and old adult epidermal stem cells. We found that, over an average mouse's lifetime, there was no measurable loss in the physiologic functional capacity of epidermal stem cells, leading us to conclude that murine epidermal stem cells resist cellular aging.

Homeostasis of continuously renewing tissues, such as the epidermis, is maintained by somatic undifferentiated, self-renewing stem cells, which are thought to persist throughout life. Through a series of labeling experiments, we previously showed that stem cells from mouse skin did not divide often, but they did divide at a steady rate in vivo. Using our recently redefined sorting method, we isolated epidermal stem and transit amplifying (TA) cells from mouse skin. When injected into a developing blastocyst or into damaged tissues, the stem cells, but not the TA cells, could participate in the formation of new tissues. We hypothesize that all tissues contain reserved undifferentiated stem cells that are primed to react if needed. These reserve stem cells could restore the tissue in which they reside or they could be called upon to help restore another tissue that was severely damage.

The keratinocyte cell compartment in the continuously renewing epidermis of the skin is maintained by undifferentiated, self-renewing stem cells. We show that a small subpopulation of epidermal stem cells (EpiSCs) have the capacity to integrate into multiple tissues. These EpiSCs can change their phenotype in direct response of changes in cytokines in vitro, changes in cocultured cells, after injection into damaged environments in vivo. These changes appear to be unrelated to the age of the EpiSC. Even though we can isolate these cells and show that the age of thses cells appears to be irrelevant to this multipotent function, we still do not know how such cells are defined within a tissue or what the life span of a multipotent stem cell is.