Understanding the Interplay Between Skin, Fascia, and Muscles of the Midface in Facial Aging.

BACKGROUND: Surgical, minimally-invasive, and non-invasive aesthetic procedures try to ameliorate the signs of facial aging, but also focus on enhancing various individual features of beauty in each patient. Herein, the midface plays a central role due to its location but also its importance for the aesthetic perception and facial expression. OBJECTIVE: To date, no study has investigated the interplay between facial muscles and its connecting subdermal architecture during facial aging to provide a more comprehensive understanding of the middle face. MATERIALS AND METHODS: A total of 76 subjects, consisting of 30 males (39.5%) and 46 females (60.5%) with a mean age of 42.2 (18.7) years [range 19-80] and a mean BMI of 24.6 (3.7) kg/m2 [range 18-35], were enrolled in this investigation. Cutometry (skin aging), 3D skin displacement analyses (subdermal connective tissue aging), and sEMG (muscle aging) analyses were utilized. RESULTS: The results revealed that overall skin firmness increased, and skin elasticity decreased (p < 0.001), sEMG signal of the investigated muscles decreased (p < 0.001), whereas midfacial mobility remained unaltered (p = 0.722). CONCLUSION: The results of this study indicate that midfacial aging is a measurable effect when utilizing individual measurement modalities for assessing skin, subdermal fascia, and midfacial muscles. The function of midfacial muscles revealed a potential threshold effect, which is not reached during midfacial aging due to the unchanged soft tissue mobility at older age. However, to understand its clinical presentation all midfacial soft tissues need to be factored in and a holistic picture needs to be created. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes review articles, book reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to authors www.springer.com/00266 .

Synthesis of Chiral 1,2-Amino Alcohol-Containing Compounds Utilizing Ruthenium-Catalyzed Asymmetric Transfer Hydrogenation of Unprotected α-Ketoamines.

Herein, we disclose a facile synthetic strategy to access an important class of drug molecules that contain chiral 1,2-amino alcohol functionality utilizing highly effective ruthenium-catalyzed asymmetric transfer hydrogenation of unprotected α-ketoamines. Recently, the COVID-19 pandemic has caused a crisis of shortage of many important drugs, especially norepinephrine and epinephrine, for the treatment of anaphylaxis and hypotension because of the increased demand. Unfortunately, the existing technologies are not fulfilling the worldwide requirement due to the existing lengthy synthetic protocols that require additional protection and deprotection steps. We identified a facile synthetic protocol via a highly enantioselective one-step process for epinephrine and a two-step process for norepinephrine starting from unprotected α-ketoamines 1b and 1a, respectively. This newly developed enantioselective ruthenium-catalyzed asymmetric transfer hydrogenation was extended to the synthesis of many 1,2-amino alcohol-containing drug molecules such as phenylephrine, denopamine, norbudrine, and levisoprenaline, with enantioselectivities of >99% ee and high isolated yields.

Practical Synthesis of 6-Amino-1-hydroxy-2,1-benzoxaborolane: A Key Intermediate of DNDI-6148.

Visceral leishmaniasis (VL), a parasitic, poverty-linked, neglected disease, is endemic across multiple regions of the world and fatal if untreated. There is an urgent need for a better and more affordable treatment for VL. DNDI-6148 is a promising drug candidate being evaluated for the treatment of VL; however, the current process for producing the key intermediate of DNDI-6148, 6-amino-1-hydroxy-2,1-benzoxaborolane, is expensive and difficult to scale up. Herein, we describe two practical approaches to synthesizing 6-amino-1-hydroxy-2,1-benzoxaborolane from inexpensive and readily available raw materials. Starting with 4-tolunitrile, the first approach is a five-step sequence involving a Hofmann rearrangement, resulting in an overall yield of 40%. The second approach utilizes 2-methyl-5-nitroaniline as the starting material and features borylation of aniline and continuous flow hydrogenation as the key steps, with an overall yield of 46%. Both routes bypass the nitration of 1-hydroxy-2,1-benzoxaborolane, which is challenging and expensive to scale. In particular, the second approach is more practical and scalable because of the mild operating conditions and facile isolation process.

Molecular dissection of an immunodominant epitope in Kv1.2-exclusive autoimmunity.

Introduction: Subgroups of autoantibodies directed against voltage-gated potassium channel (Kv) complex components have been associated with immunotherapy-responsive clinical syndromes. The high prevalence and the role of autoantibodies directly binding Kv remain, however, controversial. Our objective was to determine Kv autoantibody binding requirements and to clarify their contribution to the observed immune response. Methods: Binding epitopes were studied in sera (n = 36) and cerebrospinal fluid (CSF) (n = 12) from a patient cohort positive for Kv1.2 but negative for 32 common neurological autoantigens and controls (sera n = 18 and CSF n = 5) by phospho and deep mutational scans. Autoantibody specificity and contribution to the observed immune response were resolved on recombinant cells, cerebellum slices, and nerve fibers. Results: 83% of the patients (30/36) within the studied cohort shared one out of the two major binding epitopes with Kv1.2-3 reactivity. Eleven percent (4/36) of the serum samples showed no binding. Fingerprinting resolved close to identical sequence requirements for both shared epitopes. Kv autoantibody response is directed against juxtaparanodal regions in peripheral nerves and the axon initial segment in central nervous system neurons and exclusively mediated by the shared epitopes. Discussion: Systematic mapping revealed two shared autoimmune responses, with one dominant Kv1.2-3 autoantibody epitope being unexpectedly prevalent. The conservation of the molecular binding requirements among these patients indicates a uniform autoantibody repertoire with monospecific reactivity. The enhanced sensitivity of the epitope-based (10/12) compared with that of the cell-based detection (7/12) highlights its use for detection. The determined immunodominant epitope is also the primary immune response visible in tissue, suggesting a diagnostic significance and a specific value for routine screening.

Ostarine blunts the effect of endurance training on submaximal endurance in rats.

The purpose of this study is to study the effects of ostarine alone and in combination with endurance training in sexually mature, male Wistar rats. The rats were divided into a treadmill-trained group and a sedentary group. Half of each group received either ostarine or vehicle for 8 weeks (n = 10 each, in total n = 40). We examined some functional, hormonal, and anthropometric parameters and the myogenic gene expression of myostatin, insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor-A (VEGF-A) in m. gastrocnemius. Ostarine decreased submaximal endurance and increased myogenic gene expression of myostatin but had no effect on maximal time to exhaustion and grip strength. Training increased submaximal endurance, maximal time to exhaustion, and grip strength. Our results indicate that both exercise and ostarine treatment had no significant effects on serum levels of luteinizing hormone, follicle-stimulating hormone, and testosterone, or on the myogenic gene expression of IGF-1 and VEGF-A. Neither ostarine nor the training had a significant effect on the testis, liver, and heart weights. In conclusion, ostarine had no effect on anthropometric and hormonal parameters but increased the myostatin gene expression in muscle. The SARM treatment decreased submaximal endurance without affecting maximal time to exhaustion, and training increased both metrics.

Outbreak of severe community-acquired bacterial infections among children in North Rhine-Westphalia (Germany), October to December 2022.

PURPOSE: In late 2022, a surge of severe S. pyogenes infections was reported in several European countries. This study assessed hospitalizations and disease severity of community-acquired bacterial infections with S. pyogenes, S. pneumoniae, N. meningitidis, and H. influenzae among children in North Rhine-Westphalia (NRW), Germany, during the last quarter of 2022 compared to long-term incidences. METHODS: Hospital cases due to bacterial infections between October and December 2022 were collected in a multicenter study (MC) from 59/62 (95%) children's hospitals in NRW and combined with surveillance data (2016-2023) from the national reference laboratories for streptococci, N. meningitidis, and H. influenzae. Overall and pathogen-specific incidence rates (IR) from January 2016 to March 2023 were estimated via capture-recapture analyses. Expected annual deaths from the studied pathogens were calculated from national death cause statistics. RESULTS: In the MC study, 153 cases with high overall disease severity were reported with pneumonia being most common (59%, n = 91). IRs of bacterial infections declined at the beginning of the COVID-19 pandemic and massively surged to unprecedented levels in late 2022 and early 2023 (overall hospitalizations 3.5-fold), with S. pyogenes and S. pneumoniae as main drivers (18-fold and threefold). Observed deaths during the study period exceeded the expected number for the entire year in NRW by far (7 vs. 0.9). DISCUSSION: The unprecedented peak of bacterial infections and deaths in late 2022 and early 2023 was caused mainly by S. pyogenes and S. pneumoniae. Improved precautionary measures are needed to attenuate future outbreaks.

MARTin-an open-source platform for microarray analysis.

Background: Microarray technology has brought significant advancements to high-throughput analysis, particularly in the comprehensive study of biomolecular interactions involving proteins, peptides, and antibodies, as well as in the fields of gene expression and genotyping. With the ever-increasing volume and intricacy of microarray data, an accurate, reliable and reproducible analysis is essential. Furthermore, there is a high level of variation in the format of microarrays. This not only holds true between different sample types but is also due to differences in the hardware used during the production of the arrays, as well as the personal preferences of the individual users. Therefore, there is a need for transparent, broadly applicable and user-friendly image quantification techniques to extract meaningful information from these complex datasets, while also addressing the challenges posed by specific microarray and imager formats, which can flaw analysis and interpretation. Results: Here we introduce MicroArray Rastering Tool (MARTin), as a versatile tool developed primarily for the analysis of protein and peptide microarrays. Our software provides state-of-the-art methodologies, offering researchers a comprehensive tool for microarray image quantification. MARTin is independent of the microarray platform used and supports various configurations including high-density formats and printed arrays with significant x and y offsets. This is made possible by granting the user the ability to freely customize parts of the application to their specific microarray format. Thanks to built-in features like adaptive filtering and autofit, measurements can be done very efficiently and are highly reproducible. Furthermore, our tool integrates metadata management and integrity check features, providing a straightforward quality control method, along with a ready-to-use interface for in-depth data analysis. This not only promotes good scientific practice in the field of microarray analysis but also enhances the ability to explore and examine the generated data. Conclusion: MARTin has been developed to empower its users with a reliable, efficient, and intuitive tool for peptidomic and proteomic array analysis, thereby facilitating data-driven discovery across disciplines. Our software is an open-source project freely available via the GNU Affero General Public License licence on GitHub.

Highly Regioselective Protecting-Group-Free Synthesis of the Antimalarial Drug MMV693183.

MMV693183 is a promising antimalarial drug candidate that works for uncomplicated malaria treatment and resistance management. Herein, we report an efficient and highly regioselective synthesis of MMV693183. This novel synthetic method highlights a three-step route with an overall yield of 46% from readily available starting materials. The key to the success lies in (1) utilizing the subtle difference of the two amino groups in the starting material (S)-propane-1,2-diamine dihydrochloride without amino protection and (2) identifying the L-(+)-tartaric acid as the counter acid for the organic salt formation, yielding the desired regioisomer up to 100:0. The efficient and scalable three-step protocol operates under mild conditions with a high chemo/regioselectivity, providing effective access to MMV693183.

Minimizing Interfacial Recombination in 1.8 eV Triple-Halide Perovskites for 27.5% Efficient All-Perovskite Tandems.

All-perovskite tandem solar cells show great potential to enable the highest performance at reasonable costs for a viable market entry in the near future. In particular, wide-bandgap (WBG) perovskites with higher open-circuit voltage (VOC ) are essential to further improve the tandem solar cells' performance. Here, a new 1.8 eV bandgap triple-halide perovskite composition in conjunction with a piperazinium iodide (PI) surface treatment is developed. With structural analysis, it is found that the PI modifies the surface through a reduction of excess lead iodide in the perovskite and additionally penetrates the bulk. Constant light-induced magneto-transport measurements are applied to separately resolve charge carrier properties of electrons and holes. These measurements reveal a reduced deep trap state density, and improved steady-state carrier lifetime (factor 2.6) and diffusion lengths (factor 1.6). As a result, WBG PSCs achieve 1.36 V VOC , reaching 90% of the radiative limit. Combined with a 1.26 eV narrow bandgap (NBG) perovskite with a rubidium iodide additive, this enables a tandem cell with a certified scan efficiency of 27.5%.

Therapeutic Consequences of Targeting the IGF-1/PI3K/AKT/FOXO3 Axis in Sarcopenia: A Narrative Review.

The high prevalence of sarcopenia in an aging population has an underestimated impact on quality of life by increasing the risk of falls and subsequent hospitalization. Unfortunately, the application of the major established key therapeutic-physical activity-is challenging in the immobile and injured sarcopenic patient. Consequently, novel therapeutic directions are needed. The transcription factor Forkhead-Box-Protein O3 (FOXO3) may be an option, as it and its targets have been observed to be more highly expressed in sarcopenic muscle. In such catabolic situations, Foxo3 induces the expression of two muscle specific ubiquitin ligases (Atrogin-1 and Murf-1) via the PI3K/AKT pathway. In this review, we particularly evaluate the potential of Foxo3-targeted gene therapy. Foxo3 knockdown has been shown to lead to increased muscle cross sectional area, through both the AKT-dependent and -independent pathways and the reduced impact on the two major downstream targets Atrogin-1 and Murf-1. Moreover, a Foxo3 reduction suppresses apoptosis, activates satellite cells, and initiates their differentiation into muscle cells. While this indicates a critical role in muscle regeneration, this mechanism might exhaust the stem cell pool, limiting its clinical applicability. As systemic Foxo3 knockdown has also been associated with risks of inflammation and cancer progression, a muscle-specific approach would be necessary. In this review, we summarize the current knowledge on Foxo3 and conceptualize a specific and targeted therapy that may circumvent the drawbacks of systemic Foxo3 knockdown. This approach presumably would limit the side effects and enable an activity-independent positive impact on skeletal muscle.

Radiofrequency and pulsed electromagnetic field application following liposuction-Clinical evaluation of performance and safety.

OBJECTIVE: With more than 1.5 million performed procedures, liposuction was the second most performed esthetic surgical procedure all over the world in males and in females in the year 2020. The objective of this open-label, evaluator-blinded study was to assess the efficacy and safety of an energy-based device that combines multipolar radiofrequency with pulsed electromagnetic field (PEMF) in patients that underwent liposuction. MATERIALS AND METHODS: A total of 30 subjects, of whom 24 were females (80%), with a mean age of 48.4 ± 11.0 years (range: 27-69 years) and a mean weight of 69.5 ± 11.7 kg underwent unilateral treatment with radiofrequency and application of PEMF after bilateral liposuction. One month after the last of a total of six weekly treatments, skin elasticity, visco-elasticity and net elasticity, as well as subject reported satisfaction on a 5-point scale ranging from -1 (worse) to 3 (very much improved) and esthetic improvement ranging from 0 (very unsatisfied) to 4 (very satisfied) rated by three independent reviewers were assessed. RESULTS: Three independent and blinded reviewers rated the improvement of the treated side on average as 1.17 + 0.07 compared with baseline. Subjects were very satisfied with their treatment, with a mean score of 3.24 (0.03) out of 4 at the 1-month follow-up visit. Cutometer measurements showed no significant changes. CONCLUSION: This study revealed that the subjective satisfaction with the results of the application of RF and PEMF after liposuction is high, while at the same time the esthetic appearance as rated by independent raters improved on the treated side.

Application of Chiral Transfer Reagents to Improve Stereoselectivity and Yields in the Synthesis of the Antituberculosis Drug Bedaquiline.

Bedaquiline (BDQ) is an important drug for treating multidrug-resistant tuberculosis (MDR-TB), a worldwide disease that causes more than 1.6 million deaths yearly. The current synthetic strategy adopted by the manufacturers to assemble this molecule relies on a nucleophilic addition reaction of a quinoline fragment to a ketone, but it suffers from low conversion and no stereoselectivity, which subsequently increases the cost of manufacturing BDQ. The Medicines for All Institute (M4ALL) has developed a new reaction methodology to this process that not only allows high conversion of starting materials but also results in good diastereo- and enantioselectivity toward the desired BDQ stereoisomer. A variety of chiral lithium amides derived from amino acids were studied, and it was found that lithium (R)-2-(methoxymethyl)pyrrolidide, obtained from d-proline, results in high assay yield of the desired syn-diastereomer pair (82%) and with considerable stereocontrol (d.r. = 13.6:1, e.r. = 3.6:1, 56% ee), providing BDQ in up to a 64% assay yield before purification steps toward the final API. This represents a considerable improvement in the BDQ yield compared to previously reported conditions and could be critical to further lowering the cost of this life-saving drug.

Foxo3 Knockdown Mediates Decline of Myod1 and Myog Reducing Myoblast Conversion to Myotubes.

Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization, subsequently increasing healthcare costs while limiting quality of life. In sarcopenic muscle fibers, the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32) is expressed at substantially higher levels, driving ubiquitin-proteasomal muscle protein degradation. As one of the key regulators of muscular equilibrium, the transcription factor Forkhead Box O3 (FOXO3) can increase the expression of Fbxo32, making it a possible target for the regulation of this detrimental pathway. To test this hypothesis, murine C2C12 myoblasts were transduced with AAVs carrying a plasmid for four specific siRNAs against Foxo3. Successfully transduced myoblasts were selected via FACS cell sorting to establish single clone cell lines. Sorted myoblasts were further differentiated into myotubes and stained for myosin heavy chain (MHC) by immunofluorescence. The resulting area was calculated. Myotube contractions were induced by electrical stimulation and quantified. We found an increased Foxo3 expression in satellite cells in human skeletal muscle and an age-related increase in Foxo3 expression in older mice in silico. We established an in vitro AAV-mediated FOXO3 knockdown on protein level. Surprisingly, the myotubes with FOXO3 knockdown displayed a smaller myotube size and a lower number of nuclei per myotube compared to the control myotubes (AAV-transduced with a functionless control plasmid). During differentiation, a lower level of FOXO3 reduced the expression Fbxo32 within the first three days. Moreover, the expression of Myod1 and Myog via ATM and Tp53 was reduced. Functionally, the Foxo3 knockdown myotubes showed a higher contraction duration and time to peak. Early Foxo3 knockdown seems to terminate the initiation of differentiation due to lack of Myod1 expression, and mediates the inhibition of Myog. Subsequently, the myotube size is reduced and the excitability to electrical stimulation is altered.

Short and Efficient Synthesis of the Antituberculosis Agent Pretomanid from (R)-Glycidol.

An efficient gram-scale synthesis of the antituberculosis agent pretomanid using straightforward chemistry, mild reaction conditions, and readily available starting materials is reported. Four different protecting groups on the glycidol moiety were investigated for their technical feasibility and ability to suppress side reactions. Starting from readily available protected (R)-glycidols and 2-bromo-4-nitro-1H-imidazole, pretomanid could be prepared in a linear three-step synthesis in up to 40% isolated yield. In contrast to most syntheses reported so far, deprotection and cyclization were performed in a one-pot fashion without any hazardous steps or starting materials.

Safety considerations for treating the parotid and submandibular glands with neuromodulators for facial slimming.

BACKGROUND: Neuromodulators have predominantly been used for the treatment of upper facial lines, but their use has expanded to include lower face and neck treatments. However, the injection sites for these treatments are based on skin surface landmarks, which may pose risks to nearby structures and result in undesired outcomes. OBJECTIVE: To investigate the spatial relationship between the FDA-approved skin surface landmarks for neuromodulator injections in the parotid and submandibular glands and the topographical anatomy of critical facial structures such as the facial artery, facial vein, external carotid artery, and retromandibular vein. MATERIALS AND METHODS: A cross-sectional retrospective analysis was conducted on contrast-enhanced cranial CT scans. The scans were analyzed for the morphology and location of the parotid and submandibular glands. Measurements were taken for gland volume, craniocaudal extent, anterior-posterior extent, and distances between the skin surface and gland capsule or nearby structures such as arteries. RESULTS: The study sample consisted of 53 subjects, including 7 males and 46 females, with a mean age of 36.91 years and a mean BMI of 23.28 kg/m2 . The mean volume of the parotid gland was 31.9 ± 3.0 cc in males and 28.5 ± 3.6 cc in females with p < 0.001, while the mean volume of the submandibular gland was 18.2 ± 2.0 cc in males and 14.5 ± 3.4 cc in females with p < 0.001. The mean distances between skin surface and the gland capsule were 5.98 ± 2.2 and 8.84 ± 4.0 mm for the parotid and submandibular gland, respectively. This distance increased with higher age and higher BMI values in a statistically significant manner with p < 0.001. CONCLUSION: The distances between FDA-approved skin surface landmarks and the parotid and submandibular glands varied significantly depending on gender, age, and BMI. Optimal injection depth and location for neuromodulator treatments cannot be generalized based on these landmarks alone, emphasizing the need for real-time ultrasound imaging guidance.

Effects of ligandrol as a selective androgen receptor modulator in a rat model for osteoporosis.

INTRODUCTION: The selective androgen receptor modulator ligandrol (LGD-4033 or VK5211) has been shown to improve muscle tissue. In the present study, the effect of ligandrol on bone tissue was investigated in ovariectomized rat model. MATERIALS AND METHODS: Three-month-old Sprague Dawley rats were either ovariectomized (OVX, n = 60) or left intact (NON-OVX, n = 15). After 9 weeks, OVX rats were divided into four groups: untreated OVX (n = 15) group and three OVX groups (each of 15 rats) treated with ligandrol orally at doses of 0.03, 0.3, or 3 mg/kg body weight. After five weeks, lumbar vertebral bodies (L), tibiae, and femora were examined using micro-computed tomographical, biomechanical, ashing, and gene expression analyses. RESULTS: In the 3-mg ligandrol group, bone structural properties were improved (trabecular number: 38 ± 8 vs. 35 ± 7 (femur), 26 ± 7 vs. 22 ± 6 (L), 12 ± 5 vs. 6 ± 3 (tibia) and serum phosphorus levels (1.81 ± 0.17 vs.1.41 ± 0.17 mmol/l), uterus (0.43 ± 0.04 vs. 0.11 ± 0.02 g), and heart (1.13 ± 0.11 vs. 1.01 ± 0.08 g) weights were increased compared to the OVX group. Biomechanical parameters were not changed. Low and medium doses did not affect bone tissue and had fewer side effects. Body weight and food intake were not affected by ligandrol; OVX led to an increase in these parameters and worsened all bone parameters. CONCLUSION: Ligandrol at high dose showed a subtle anabolic effect on structural properties without any improvement in biomechanical properties of osteoporotic bones. Considering side effects of ligandrol at this dose, its further investigation for the therapy of postmenopausal osteoporosis should be reevaluated.

Chondral/Desmal Osteogenesis in 3D Spheroids Sensitized by Psychostimulants.

Attention deficit hyperactivity disorder (ADHD) affects 6.4 million children in the United States of America. Children and adolescents, the main consumers of ADHD medication, are in the bone growth phase, which extends over a period of up to two decades. Thus, impaired proliferation and maturation of chondrocytes and osteoblasts can result in impaired bone formation. The aim of this study is to investigate, for the first time, the effects of the ADHD-medication modafinil, atomoxetine and guanfacine on bone growth and repair in vitro. Using two-dimensional and three-dimensional cell models, we investigated the chondrogenic/osteogenic differentiation, proliferation and viability of human mesenchymal progenitor cells. Real-time cell proliferation was measured by xCELLigence. Live/dead staining and size measurement of hMSC- and MG63 monolayer and spheroids were performed after administration of therapeutic plasma concentrations of modafinil, atomoxetine and guanfacine. Chondrogenic differentiation was quantified by RTqPCR. The chondrogenic and osteogenic differentiation was evaluated by histological cryo-sections. Modafinil, atomoxetine and guanfacine reduced chondrogenic and osteogenic differentiation terms of transcript expression and at the histological level. Cell viability of the MG63- and hMSC monolayer was not impeded by ADHD-medication. Our in vitro results indicate that modafinil, atomoxetine and guanfacine may impair chondrogenic and osteogenic differentiation in a 3D model reflecting the in vivo physiologic condition.

Psychostimulants Modafinil, Atomoxetine and Guanfacine Impair Bone Cell Differentiation and MSC Migration.

Attention deficit hyperactivity disorder (ADHD) is one of the most common worldwide mental disorders in children, young and adults. If left untreated, the disorder can continue into adulthood. The abuse of ADHD-related drugs to improve mental performance for studying, working and everyday life is also rising. The potentially high number of subjects with controlled or uncontrolled use of such substances increases the impact of possible side effects. It has been shown before that the early ADHD drug methylphenidate influences bone metabolism negatively. This study focused on the influence of three more recent cognitive enhancers, modafinil, atomoxetine and guanfacine, on the differentiation of mesenchymal stem cells to osteoblasts and on their cell functions, including migration. Human mesenchymal stem cells (hMSCs) were incubated with a therapeutic plasma dosage of modafinil, atomoxetine and guanfacine. Gene expression analyses revealed a high beta-2 adrenoreceptor expression in hMSC, suggesting it as a possible pathway to stimulate action. In bone formation assays, all three cognitive enhancers caused a significant decrease in the mineralized matrix and an early slight reduction of cell viability without triggering apoptosis or necrosis. While there was no effect of the three substances on early differentiation, they showed differing effects on the expression of osterix (OSX), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in the later stages of osteoblast development, suggesting alternative modes of action. All three substances significantly inhibited hMSC migration. This effect could be rescued by a selective beta-blocker (Imperial Chemical Industries ICI-118,551) in modafinil and atomoxetine, suggesting mediation via beta-2 receptor stimulation. In conclusion, modafinil, atomoxetine and guanfacine negatively influence hMSC differentiation to bone-forming osteoblasts and cell migration through different intracellular pathways.

Culture-Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids.

BACKGROUND & AIMS: Human intestinal epithelial organoids (IEOs) are a powerful tool to model major aspects of intestinal development, health, and diseases because patient-derived cultures retain many features found in vivo. A necessary aspect of the organoid model is the requirement to expand cultures in vitro through several rounds of passaging. This is of concern because the passaging of cells has been shown to affect cell morphology, ploidy, and function. METHODS: Here, we analyzed 173 human IEO lines derived from the small and large bowel and examined the effect of culture duration on DNA methylation (DNAm). Furthermore, we tested the potential impact of DNAm changes on gene expression and cellular function. RESULTS: Our analyses show a reproducible effect of culture duration on DNAm in a large discovery cohort as well as 2 publicly available validation cohorts generated in different laboratories. Although methylation changes were seen in only approximately 8% of tested cytosine-phosphate-guanine dinucleotides (CpGs) and global cellular function remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokine exposure and withdrawal of Wnt agonists. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication, indicating similarities to malignant transformation. CONCLUSIONS: Our study shows distinct culture-associated epigenetic changes in mucosa-derived human IEOs, some of which appear to impact gene transcriptomic and cellular function. These findings highlight the need for future studies in this area and the importance of considering passage number as a potentially confounding factor.

Stereophotogrammetry can feasibly assess 'physiological' longitudinal three-dimensional head development of very preterm infants from birth to term.

Head development is a surrogate for brain development in infants and is related to neurocognitive outcome. There is only limited knowledge on early extra-uterine head shape and size assessment in very preterm infants. Here, 26 very preterm infants with a mean gestational age of 29.1 ± 2.2 weeks and a mean birth weight of 1273.8 ± 427.7 g underwent serial stereophotogrammetric 3D head imaging in weekly intervals from birth to term-equivalent age. The main outcome was the longitudinal assessment of the 'physiological' preterm head development with cephalometric size (head circumference, cranial volume) and shape parameters (cranial index, cranial vault asymmetry index) according to chronological and postmenstrual age (PMA). Potential clinical risk factors for the development of an abnormal low cranial index (dolichocephaly) were analysed. In serial measurements of 26 infants, the estimated head volume (95% confidence interval) increased from 244 (226-263) cm3 at 28 weeks PMA to 705 (688-721) cm3 at 40 weeks PMA. Moderate or severe dolichocephaly occurred in 21/26 infants (80.8%). Cranial index decreased over time (72.4%; 70.7-74 95% confidence interval). Brachycephaly and plagiocephaly were uncommon. No risk factors for severe dolichocephaly were identified. Our study shows that early detection of head shape and size anomalies utilizing 3D stereophotogrammetry is feasible and safe even in very preterm infants < 1500 g and/or < 32 weeks. 3D stereophotogrammetry could be used for timely identification of infants at risk for head shape anomalies. No specific risk factors for head shape anomalies were identified, especially not mode and duration of respiratory support.