Mechanisms of skin vascular maturation and maintenance captured by longitudinal imaging of live mice.

A functional network of blood vessels is essential for organ growth and homeostasis, yet how the vasculature matures and maintains homeostasis remains elusive in live mice. By longitudinally tracking the same neonatal endothelial cells (ECs) over days to weeks, we found that capillary plexus expansion is driven by vessel regression to optimize network perfusion. Neonatal ECs rearrange positions to evenly distribute throughout the developing plexus and become positionally stable in adulthood. Upon local ablation, adult ECs survive through a plasmalemmal self-repair response, while neonatal ECs are predisposed to die. Furthermore, adult ECs reactivate migration to assist vessel repair. Global ablation reveals coordinated maintenance of the adult vascular architecture that allows for eventual network recovery. Lastly, neonatal remodeling and adult maintenance of the skin vascular plexus are orchestrated by temporally restricted, neonatal VEGFR2 signaling. Our work sheds light on fundamental mechanisms that underlie both vascular maturation and adult homeostasis in vivo.

Positional Stability and Membrane Occupancy Define Skin Fibroblast Homeostasis In Vivo.

Fibroblasts are an essential cellular and structural component of our organs. Despite several advances, the critical behaviors that fibroblasts utilize to maintain their homeostasis in vivo have remained unclear. Here, by tracking the same skin fibroblasts in live mice, we show that fibroblast position is stable over time and that this stability is maintained despite the loss of neighboring fibroblasts. In contrast, fibroblast membranes are dynamic during homeostasis and extend to fill the space of lost neighboring fibroblasts in a Rac1-dependent manner. Positional stability is sustained during aging despite a progressive accumulation of gaps in fibroblast nuclei organization, while membrane occupancy continues to be maintained. This work defines positional stability and cell occupancy as key principles of skin fibroblast homeostasis in vivo, throughout the lifespan of mice, and identifies membrane extension in the absence of migration as the core cellular mechanism to carry out these principles.

Hardwiring Stem Cell Communication through Tissue Structure.

Adult stem cells across diverse organs self-renew and differentiate to maintain tissue homeostasis. How stem cells receive input to preserve tissue structure and function largely relies on their communication with surrounding cellular and non-cellular elements. As such, how tissues are organized and patterned not only reflects organ function, but also inherently hardwires networks of communication between stem cells and their environment to direct tissue homeostasis and injury repair. This review highlights how different methods of stem cell communication reflect the unique organization and function of diverse tissues.

Stem Cells Show Parental Control.

Stem cells interact with their niche to maintain an undifferentiated state. The study by Pardo-Saganta et al. shows that airway basal stem cells maintain secretory daughter cells in airway epithelia through forward regulation, suggesting that stem cells may serve as a niche for their progeny.

Injury prevents Ras mutant cell expansion in mosaic skin.

Healthy skin is a mosaic of wild-type and mutant clones1,2. Although injury can cooperate with mutated Ras family proteins to promote tumorigenesis3-12, the consequences in genetically mosaic skin are unknown. Here we show that after injury, wild-type cells suppress aberrant growth induced by oncogenic Ras. HrasG12V/+ and KrasG12D/+ cells outcompete wild-type cells in uninjured, mosaic tissue but their expansion is prevented after injury owing to an increase in the fraction of proliferating wild-type cells. Mechanistically, we show that, unlike HrasG12V/+ cells, wild-type cells respond to autocrine and paracrine secretion of EGFR ligands, and this differential activation of the EGFR pathway explains the competitive switch during injury repair. Inhibition of EGFR signalling via drug or genetic approaches diminishes the proportion of dividing wild-type cells after injury, leading to the expansion of HrasG12V/+ cells. Increased proliferation of wild-type cells via constitutive loss of the cell cycle inhibitor p21 counteracts the expansion of HrasG12V/+ cells even in the absence of injury. Thus, injury has a role in switching the competitive balance between oncogenic and wild-type cells in genetically mosaic skin.

The art of observation: bridging science and art to see the unexpected.

Observation is the heart of research, but it can be challenging to observe deeply and go beyond expected observations. Here, we describe activities designed for scientists to enhance their observational skills by engaging with art. In collaboration with an art gallery at our university, our lab practiced observing representational paintings in a systematic way, separating the act of observation from interpretation. Applying this skill to our microscopy images allowed us to access information in the data that may otherwise have been overlooked. In addition, these activities highlighted the power of collecting observations from multiple observers before generating interpretations, as well as the value of discussing the creative and emotional aspects of data collection and interpretation. We provide concrete examples of how we will incorporate these skills into our research processes, as well as details that other groups can use to engage in similar art-based training activities to enhance their own observational skills.

Subjective Shoulder Value for Sport Is a Simple, Reliable, and Valid Score to Assess Shoulder Function in Athletes.

PURPOSE: To validate the subjective shoulder value for sport (SSV-Sport) by measuring its correlation with existing patient-reported outcome measures (PROMs) and defining its psychometric properties. METHODS: Between May 2021 and May 2022, we established 2 patient groups. Group 1 included those (1) aged 18 years or older, who were (2) consulting for the first time for any shoulder condition, (3) regularly participated in sports, and were capable of accessing a questionnaire independently. There were asked to rate their SSV and SSV-Sport at admission and 2 weeks later; they also were asked to answer a questionnaire including other PROMS. Group 2 comprised patients who had (1) undergone shoulder stabilization surgery and had (2) a minimum follow-up period of 6 months. RESULTS: For the shoulder disability patients (group 1, n = 62), there was a strong and significant correlation between SSV-Sport and other PROMs: Quick Disabilities of the Arm, Shoulder and Hand Sport (r = 0.84), Walch-Duplay (r = 0.65), Rowe (r = 0.74), Western Ontario Shoulder Instability (r = 0.78), and SSV (r = 0.75) (P = .0001). The SSV-Sport was reliable at baseline and 2 weeks after (0.91, 95% confidence interval 0.85-0.94), and was responsive to change (P < .001). For the anterior instability patients (group 2, n = 83), SSV was on average 50 points greater than SSV-Sport (29.2 vs 79.4, P < .001) for preoperative values. In both groups, the values of SSV were constantly and significantly higher than the values of SSV-Sport (81.9 ± 21.3 vs 54.8 ± 30.9; P < .001). CONCLUSIONS: The SSV-Sport is an easily administered, reliable, responsive, and valid measure of shoulder function in athletes that is highly correlated with other PROMs. SSV-Sport is better adapted than SSV to quantify pre- and postoperative shoulder deficiency in athletes. LEVEL OF EVIDENCE: Level III, cohort study (diagnosis).

EGCG Disrupts the LIN28B/Let-7 Interaction and Reduces Neuroblastoma Aggressiveness.

Neuroblastoma (NB) is the most commonly diagnosed extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. Although the 5-year survival rate of patients with a high-risk disease has increased in recent decades, NB remains a challenge in pediatric oncology, and the identification of novel potential therapeutic targets and agents is an urgent clinical need. The RNA-binding protein LIN28B has been identified as an oncogene in NB and is associated with a poor prognosis. Given that LIN28B acts by negatively regulating the biogenesis of the tumor suppressor let-7 miRNAs, we reasoned that selective interference with the LIN28B/let-7 miRNA interaction would increase let-7 miRNA levels, ultimately leading to reduced NB aggressiveness. Here, we selected (-)-epigallocatechin 3-gallate (EGCG) out of 4959 molecules screened as the molecule with the best inhibitory activity on LIN28B/let-7 miRNA interaction and showed that treatment with PLC/PLGA-PEG nanoparticles containing EGCG (EGCG-NPs) led to an increase in mature let-7 miRNAs and a consequent inhibition of NB cell growth. In addition, EGCG-NP pretreatment reduced the tumorigenic potential of NB cells in vivo. These experiments suggest that the LIN28B/let-7 miRNA axis is a good therapeutic target in NB and that EGCG, which can interfere with this interaction, deserves further preclinical evaluation.

Lymph vessels find a hairy niche.

Lymphatic vessels play important roles in coordinating fluid homeostasis and local immune responses of the tissues they reside in. A new study shows that in addition, lymphatic vessels of the skin are intimately associated with hair follicles and control HF development, cycling, and organization.

Trehalose-Carnosine Prevents the Effects of Spinal Cord Injury Through Regulating Acute Inflammation and Zinc(II) Ion Homeostasis.

Spinal cord injury (SCI) leads to long-term and permanent motor dysfunctions, and nervous system abnormalities. Injury to the spinal cord triggers a signaling cascade that results in activation of the inflammatory cascade, apoptosis, and Zn(II) ion homeostasis. Trehalose (Tre), a nonreducing disaccharide, and L-carnosine (Car), (ß-alanyl-L-histidine), one of the endogenous histidine dipeptides have been recognized to suppress early inflammatory effects, oxidative stress and to possess neuroprotective effects. We report on the effects of the conjugation of Tre with Car (Tre-car) in reducing inflammation in in vitro and in vivo models. The in vitro study was performed using rat pheochromocytoma cells (PC12 cell line). After 24 h, Tre-car, Car, Tre, and Tre + Car mixture treatments, cells were collected and used to investigate Zn2+ homeostasis. The in vivo model of SCI was induced by extradural compression of the spinal cord at the T6-T8 levels. After treatments with Tre, Car and Tre-Car conjugate 1 and 6 h after SCI, spinal cord tissue was collected for analysis. In vitro results demonstrated the ionophore effect and chelating features of L-carnosine and its conjugate. In vivo, the Tre-car conjugate treatment counteracted the activation of the early inflammatory cascade, oxidative stress and apoptosis after SCI. The Tre-car conjugate stimulated neurotrophic factors release, and influenced Zn2+ homeostasis. We demonstrated that Tre-car, Tre and Car treatments improved tissue recovery after SCI. Tre-car decreased proinflammatory, oxidative stress mediators release, upregulated neurotrophic factors and restored Zn2+ homeostasis, suggesting that Tre-car may represent a promising therapeutic agent for counteracting the consequences of SCI.

Probing the rules of cell coordination in live tissues by interpretable machine learning based on graph neural networks.

Robustness in developing and homeostatic tissues is supported by various types of spatiotemporal cell-to-cell interactions. Although live imaging and cell tracking are powerful in providing direct evidence of cell coordination rules, extracting and comparing these rules across many tissues with potentially different length and timescales of coordination requires a versatile framework of analysis. Here we demonstrate that graph neural network (GNN) models are suited for this purpose, by showing how they can be applied to predict cell fate in tissues and utilized to infer the cell interactions governing the multicellular dynamics. Analyzing the live mammalian epidermis data, where spatiotemporal graphs constructed from cell tracks and cell contacts are given as inputs, GNN discovers distinct neighbor cell fate coordination rules that depend on the region of the body. This approach demonstrates how the GNN framework is powerful in inferring general cell interaction rules from live data without prior knowledge of the signaling involved.

Correction of aberrant growth preserves tissue homeostasis.

Cells in healthy tissues acquire mutations with surprising frequency. Many of these mutations are associated with abnormal cellular behaviours such as differentiation defects and hyperproliferation, yet fail to produce macroscopically detectable phenotypes. It is currently unclear how the tissue remains phenotypically normal, despite the presence of these mutant cells. Here we use intravital imaging to track the fate of mouse skin epithelium burdened with varying numbers of activated Wnt/ß-catenin stem cells. We show that all resulting growths that deform the skin tissue architecture regress, irrespective of their size. Wild-type cells are required for the active elimination of mutant cells from the tissue, while utilizing both endogenous and ectopic cellular behaviours to dismantle the aberrant structures. After regression, the remaining structures are either completely eliminated or converted into functional skin appendages in a niche-dependent manner. Furthermore, tissue aberrancies generated from oncogenic Hras, and even mutation-independent deformations to the tissue, can also be corrected, indicating that this tolerance phenomenon reflects a conserved principle in the skin. This study reveals an unanticipated plasticity of the adult skin epithelium when faced with mutational and non-mutational insult, and elucidates the dynamic cellular behaviours used for its return to a homeostatic state.

A two-site reactive platform in the synthesis of amino-functionalized amphiphilic molecules via sulfenic acids.

The synthesis of some bolaamphiphiles is described. It is a convergent approach that allows the linkage of a glucosyl derivative to a bis-functionalized platform, via a copper-free Sonogashira cross-coupling. The central core was obtained from the reaction of a suitably substituted bis-sulfoxide with diethynyl benzenes. The intermediates of such reaction are sulfenyl functions that are easily added to one triple bond of the unsaturated molecules. The functionalization at the central core, through the nucleophilic addition of ammonia or piperidine onto the two vinyl sulfonyl groups already present in the backbones of the molecules, opened the way to the preparation of more complex derivatives. The observation of the formation of new stereogenic carbons with an unexpected significantly high diastereoselectivity was justified and supported by preliminary theoretical calculations. The two ending glucosyl moieties were favourably deprotected to afford the amino-functionalized bolaamphiphilic molecules.

Mono- and dialdehyde of trehalose: new synthons to prepare trehalose bio-conjugates.

Trehalose, a non-reducing disaccharide of glucose, is a natural bioactive and non-toxic sugar. It is found in many organisms that synthesise it when their cells are exposed to stress conditions. While not produced by mammalian cells, this disaccharide and also some of its derivatives have been shown to have a number of interesting properties that indicate their importance in the treatment of certain human diseases. Differentiating the two glucosyl moieties in the trehalose molecule has often been a synthetic challenge. We report here an easy way to obtain the monoaldehyde of trehalose, as well as the relevant symmetrical dialdehyde. The reactivity of the aldehyde functionalities involved in the molecular structure of these synthons allows the easy preparation of the corresponding amino or carboxy derivatives of trehalose, as well the synthesis of some new trehalose conjugates useful for diagnostic or therapeutic purposes.

Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool.

Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression). In contrast to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration. Here we show by intravital microscopy in live mice that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbours. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through transforming growth factor (TGF)-ß activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool, as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviours and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis.

Relationship Between the Lateral Collateral Ligament of the Elbow and the Kocher Approach: A Cadaver Study.

PURPOSE: The lateral ulnar collateral ligament (LUCL) is considered to be the portion of the lateral collateral ligament playing the most important stabilizing role. Iatrogenic forms of posterolateral rotatory instability have been described. The Kocher approach is a popular approach to the lateral side of the elbow. The aim of this study was to describe the relationship between the LUCL and the Kocher interval. METHODS: The Kocher interval was identified and marked in 20 cadavers. The LUCL was identified and the distance between the LUCL insertion on the tubercle of the cresta supinatoris and the Kocher interval was calculated (TK distance). This distance was considered 0 if the Kocher interval was directly above the tubercle, as a positive value if it was anterior to the tubercle, and as a negative value if it was posterior. Finally, the Kocher interval was sharply opened, and elbow stability was tested using the posterolateral rotatory drawer test. RESULTS: A discrete LUCL was identified in 16 specimens. The mean TK distance was -2.3 ± 4.4 mm (range, -11 to +10). The median TK distance was -3 mm. The posterolateral rotatory drawer test was positive for subluxation after the sharp incision of the Kocher interval in 15 specimens. The median TK distance was significantly higher in the stable group (+2 mm) than in the unstable group (-3 mm). CONCLUSIONS: The LUCL often lies beneath the Kocher interval and is at risk during the Kocher approach. CLINICAL RELEVANCE: Iatrogenic forms of posterolateral rotatory instability could result from this approach.

Ionophore Ability of Carnosine and Its Trehalose Conjugate Assists Copper Signal in Triggering Brain-Derived Neurotrophic Factor and Vascular Endothelial Growth Factor Activation In Vitro.

l-carnosine (ß-alanyl-l-histidine) (Car hereafter) is a natural dipeptide widely distributed in mammalian tissues and reaching high concentrations (0.7-2.0 mM) in the brain. The molecular features of the dipeptide underlie the antioxidant, anti-aggregating and metal chelating ability showed in a large number of physiological effects, while the biological mechanisms involved in the protective role found against several diseases cannot be explained on the basis of the above-mentioned properties alone, requiring further research efforts. It has been reported that l-carnosine increases the secretion and expression of various neurotrophic factors and affects copper homeostasis in nervous cells inducing Cu cellular uptake in keeping with the key metal-sensing system. Having in mind this l-carnosine ability, here we report the copper-binding and ionophore ability of l-carnosine to activate tyrosine kinase cascade pathways in PC12 cells and stimulate the expression of BDNF. Furthermore, the study was extended to verify the ability of the dipeptide to favor copper signaling inducing the expression of VEGF. Being aware that the potential protective action of l-carnosine is drastically hampered by its hydrolysis, we also report on the behavior of a conjugate of l-carnosine with trehalose that blocks the carnosinase degradative activity. Overall, our findings describe a copper tuning effect on the ability of l-carnosine and, particularly its conjugate, to activate tyrosine kinase cascade pathways.

Calorimetric Evaluation of Glycyrrhetic Acid (GA)- and Stearyl Glycyrrhetinate (SG)-Loaded Solid Lipid Nanoparticle Interactions with a Model Biomembrane.

Glycyrrhetic acid (GA) and stearyl glycyrrhetinate (SG) are two interesting compounds from Glycyrrhiza glabra, showing numerous biological properties widely applied in the pharmaceutical and cosmetic fields. Despite these appreciable benefits, their potential therapeutic properties are strongly compromised due to unfavourable physical-chemical features. The strategy exploited in the present work was to develop solid lipid nanoparticles (SLNs) as carrier systems for GA and SG delivery. Both formulations loaded with GA and SG (GA-SLNs and SG-SLNs, respectively) were prepared by the high shear homogenization coupled to ultrasound (HSH-US) method, and we obtained good technological parameters. DSC was used to evaluate their thermotropic behaviour and ability to act as carriers for GA and SG. The study was conducted by means of a biomembrane model (multilamellar vesicles; MLVs) that simulated the interaction of the carriers with the cellular membrane. Unloaded and loaded SLNs were incubated with the biomembranes, and their interactions were evaluated over time through variations in their calorimetric curves. The results of these studies indicated that GA and SG interact differently with MLVs and SLNs; the interactions of SG-SLNs and GA-SLNs with the biomembrane model showed different variations of the MLVs calorimetric curve and suggest the potential use of SLNs as delivery systems for GA.

Spatial organization within a niche as a determinant of stem-cell fate.

Stem-cell niches in mammalian tissues are often heterogeneous and compartmentalized; however, whether distinct niche locations determine different stem-cell fates remains unclear. To test this hypothesis, here we use the mouse hair follicle niche and combine intravital microscopy with genetic lineage tracing to re-visit the same stem-cell lineages, from their exact place of origin, throughout regeneration in live mice. Using this method, we show directly that the position of a stem cell within the hair follicle niche can predict whether it is likely to remain uncommitted, generate precursors or commit to a differentiated fate. Furthermore, using laser ablation we demonstrate that hair follicle stem cells are dispensable for regeneration, and that epithelial cells, which do not normally participate in hair growth, re-populate the lost stem-cell compartment and sustain hair regeneration. This study provides a general model for niche-induced fate determination in adult tissues.