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.

Oncogenic Kras induces spatiotemporally specific tissue deformation through converting pulsatile into sustained ERK activation.

Tissue regeneration and maintenance rely on coordinated stem cell behaviours. This orchestration can be impaired by oncogenic mutations leading to cancer. However, it is largely unclear how oncogenes perturb stem cells' orchestration to disrupt tissue. Here we used intravital imaging to investigate the mechanisms by which oncogenic Kras mutation causes tissue disruption in the hair follicle. Through longitudinally tracking hair follicles in live mice, we found that KrasG12D, a mutation that can lead to squamous cell carcinoma, induces epithelial tissue deformation in a spatiotemporally specific manner, linked with abnormal cell division and migration. Using a reporter mouse capture real-time ERK signal dynamics at the single-cell level, we discovered that KrasG12D, but not a closely related mutation HrasG12V, converts ERK signal in stem cells from pulsatile to sustained. Finally, we demonstrated that interrupting sustained ERK signal reverts KrasG12D-induced tissue deformation through modulating specific features of cell migration and division.

BDNF- and VEGF-Responsive Stimulus to an NGF Mimic Cyclic Peptide with Copper Ionophore Capability and Ctr1/CCS-Driven Signaling.

Neurotrophins are a family of growth factors that play a key role in the development and regulation of the functioning of the central nervous system. Their use as drugs is made difficult by their poor stability, cellular permeability, and side effects. Continuing our effort to use peptides that mimic the neurotrophic growth factor (NGF), the family model protein, and specifically the N-terminus of the protein, here we report on the spectroscopic characterization and resistance to hydrolysis of the 14-membered cyclic peptide reproducing the N-terminus sequence (SSSHPIFHRGEFSV (c-NGF(1-14)). Far-UV CD spectra and a computational study show that this peptide has a rigid conformation and left-handed chirality typical of polyproline II that favors its interaction with the D5 domain of the NGF receptor TrkA. c-NGF(1-14) is able to bind Cu2+ with good affinity; the resulting complexes have been characterized by potentiometric and spectroscopic measurements. Experiments on PC12 cells show that c-NGF(1-14) acts as an ionophore, influencing the degree and the localization of both the membrane transporter (Ctr1) and the copper intracellular transporter (CCS). c-NGF(1-14) induces PC12 differentiation, mimics the protein in TrkA phosphorylation, and activates the kinase cascade, inducing Erk1/2 phosphorylation. c-NGF(1-14) biological activities are enhanced when the peptide interacts with Cu2+ even with the submicromolar quantities present in the culture media as demonstrated by ICP-OES measurements. Finally, c-NGF(1-14) and Cu2+ concur to activate the cAMP response element-binding protein CREB that, in turn, induces the brain-derived neurotrophic factor (BDNF) and the vascular endothelial growth factor (VEGF) release.

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).

Copper(II) Complexes with Carnosine Conjugates of Hyaluronic Acids at Different Dipeptide Loading Percentages Behave as Multiple SOD Mimics and Stimulate Nrf2 Translocation and Antioxidant Response in In Vitro Inflammatory Model.

A series of copper(II) complexes with the formula [Cu2+Hy(x)Car%] varying the molecular weight (MW) of Hyaluronic acid (Hy, x = 200 or 700 kDa) conjugated with carnosine (Car) present at different loading were synthesized and characterized via different spectroscopic techniques. The metal complexes behaved as Cu, Zn-superoxide dismutase (SOD1) mimics and showed some of the most efficient reaction rate values produced using a synthetic and water-soluble copper(II)-based SOD mimic reported to date. The increase in the percentage of Car moieties parallels the enhancement of the I50 value determined via the indirect method of Fridovich. The presence of the non-functionalized Hy OH groups favors the scavenger activity of the copper(II) complexes with HyCar, recalling similar behavior previously found for the copper(II) complexes with Car conjugated using ß-cyclodextrin or trehalose. In keeping with the new abilities of SOD1 to activate protective agents against oxidative stress in rheumatoid arthritis and osteoarthritis diseases, Cu2+ interaction with HyCar promotes the nuclear translocation of erythroid 2-related factor that regulates the expressions of target genes, including Heme-Oxigenase-1, thus stimulating an antioxidant response in osteoblasts subjected to an inflammatory/oxidative insult.

Organ function is preserved despite reorganization of niche architecture in the hair follicle.

The ability of stem cells to build and replenish tissues depends on support from their niche. Although niche architecture varies across organs, its functional importance is unclear. During hair follicle growth, multipotent epithelial progenitors build hair via crosstalk with their remodeling fibroblast niche, the dermal papilla, providing a powerful model to functionally interrogate niche architecture. Through mouse intravital imaging, we show that dermal papilla fibroblasts remodel individually and collectively to form a morphologically polarized, structurally robust niche. Asymmetric TGF-ß signaling precedes morphological niche polarity, and loss of TGF-ß signaling in dermal papilla fibroblasts leads them to progressively lose their stereotypic architecture, instead surrounding the epithelium. The reorganized niche induces the redistribution of multipotent progenitors but nevertheless supports their proliferation and differentiation. However, the differentiated lineages and hairs produced by progenitors are shorter. Overall, our results reveal that niche architecture optimizes organ efficiency but is not absolutely essential for organ function.

A Spatiotemporal Compartmentalization of Glucose Metabolism Guides Mammalian Gastrulation Progression.

Gastrulation is considered the sine qua non of embryogenesis, establishing a multidimensional structure and the spatial coordinates upon which all later developmental events transpire. At this time, the embryo adopts a heavy reliance on glucose metabolism to support rapidly accelerating changes in morphology, proliferation, and differentiation. However, it is currently unknown how this conserved metabolic shift maps onto the three-dimensional landscape of the growing embryo and whether it is spatially linked to the orchestrated cellular and molecular processes necessary for gastrulation. Here we identify that glucose is utilised during mouse gastrulation via distinct metabolic pathways to instruct local and global embryonic morphogenesis, in a cell type and stage-specific manner. Through detailed mechanistic studies and quantitative live imaging of mouse embryos, in parallel with tractable in vitro stem cell differentiation models and embryo-derived tissue explants, we discover that cell fate acquisition and the epithelial-to-mesenchymal transition (EMT) relies on the Hexosamine Biosynthetic Pathway (HBP) branch of glucose metabolism, while newly-formed mesoderm requires glycolysis for correct migration and lateral expansion. This regional and tissue-specific difference in glucose metabolism is coordinated with Fibroblast Growth Factor (FGF) activity, demonstrating that reciprocal crosstalk between metabolism and growth factor signalling is a prerequisite for gastrulation progression. We expect these studies to provide important insights into the function of metabolism in other developmental contexts and may help uncover mechanisms that underpin embryonic lethality, cancer, and congenital disease.

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.

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.

Cell cycle controls long-range calcium signaling in the regenerating epidermis.

Skin homeostasis is maintained by stem cells, which must communicate to balance their regenerative behaviors. Yet, how adult stem cells signal across regenerative tissue remains unknown due to challenges in studying signaling dynamics in live mice. We combined live imaging in the mouse basal stem cell layer with machine learning tools to analyze patterns of Ca2+ signaling. We show that basal cells display dynamic intercellular Ca2+ signaling among local neighborhoods. We find that these Ca2+ signals are coordinated across thousands of cells and that this coordination is an emergent property of the stem cell layer. We demonstrate that G2 cells are required to initiate normal levels of Ca2+ signaling, while connexin43 connects basal cells to orchestrate tissue-wide coordination of Ca2+ signaling. Lastly, we find that Ca2+ signaling drives cell cycle progression, revealing a communication feedback loop. This work provides resolution into how stem cells at different cell cycle stages coordinate tissue-wide signaling during epidermal regeneration.

Live imaging reveals chromatin compaction transitions and dynamic transcriptional bursting during stem cell differentiation in vivo.

Stem cell differentiation requires dramatic changes in gene expression and global remodeling of chromatin architecture. How and when chromatin remodels relative to the transcriptional, behavioral, and morphological changes during differentiation remain unclear, particularly in an intact tissue context. Here, we develop a quantitative pipeline which leverages fluorescently-tagged histones and longitudinal imaging to track large-scale chromatin compaction changes within individual cells in a live mouse. Applying this pipeline to epidermal stem cells, we reveal that cell-to-cell chromatin compaction heterogeneity within the stem cell compartment emerges independent of cell cycle status, and instead is reflective of differentiation status. Chromatin compaction state gradually transitions over days as differentiating cells exit the stem cell compartment. Moreover, establishing live imaging of Keratin-10 (K10) nascent RNA, which marks the onset of stem cell differentiation, we find that Keratin-10 transcription is highly dynamic and largely precedes the global chromatin compaction changes associated with differentiation. Together, these analyses reveal that stem cell differentiation involves dynamic transcriptional states and gradual chromatin rearrangement.

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.

Double knockin mice show NF-κB trajectories in immune signaling and aging.

In vitro studies suggest that mapping the spatiotemporal complexity of nuclear factor κB (NF-κB) signaling is essential to understanding its function. The lack of tools to directly monitor NF-κB proteins in vivo has hindered such efforts. Here, we introduce reporter mice with the endogenous RelA (p65) or c-Rel labeled with distinct fluorescent proteins and a double knockin with both subunits labeled. Overcoming hurdles in simultaneous live-cell imaging of RelA and c-Rel, we show that quantitative features of signaling reflect the identity of activating ligands, differ between primary and immortalized cells, and shift toward c-Rel in microglia from aged brains. RelA:c-Rel heterodimer is unexpectedly depleted in the nuclei of stimulated cells. Trajectories of subunit co-expression in immune lineages reveal a reduction at key cell maturation stages. These results demonstrate the power of these reporters in gaining deeper insights into NF-κB biology, with the spectral complementarity of the labeled NF-κB proteins enabling diverse applications.

Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer.

Highly regenerative tissues continuously produce terminally differentiated cells to replace those that are lost. How they orchestrate the complex transition from undifferentiated stem cells towards post-mitotic, molecularly distinct and often spatially segregated differentiated populations is not well understood. In the adult skin epidermis, the stem cell compartment contains molecularly heterogeneous subpopulations1-4 whose relationship to the complete trajectory of differentiation remains unknown. Here we show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes with upregulation of differentiation genes preceding downregulation of typical stemness genes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit-amplifying programme but help to buffer the differentiating cell pool during heightened demand. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover.

Next Generation Sequencing Analysis of MODY-X Patients: A Case Report Series.

BACKGROUND: Classic criteria for a maturity-onset diabetes of the young (MODY) diagnosis are often unable to identify all subjects, and traditional Sanger sequencing, using a candidate gene approach, leads to a high prevalence of missed genetic diagnosis, classified as MODY-X. Next generation sequencing (NGS) panels provide a highly sensitive method even for rare forms. METHODS: We investigated 28 pediatric subjects suspected for MODY-X, utilizing a 15-gene NGS panel for monogenic diabetes (MD). RESULTS: NGS detected variants of uncertain significance (VUS), likely pathogenic or pathogenic for rarer subtypes of MODY, in six patients. We found variants in the wolframin gene (WFS1), traditionally not considered in MD genetic screening panels, in three patients; KCNJ11 gene mutation, typically responsible for neonatal diabetes and rarely causing isolated diabetes in adolescents; INS gene mutation; a variant in the HNF1B gene in a young male with diabetes on sulfonylurea treatment. CONCLUSION: In our cohort, the availability of an NGS panel for MD was determined for the correct identification of MD subtypes in six patients with MODY-X. Our study underlines how a precise diagnosis utilizing NGS may have an impact on the management of different forms of MODY and, thus, lead to a tailored treatment and enable genetic counselling of other family members.

Smartphone-Assisted Sensing of Trinitrotoluene by Optical Array.

Here we report the design and fabrication of an array-based sensor, containing functionalized Carbon Dots, Bodipy's and Naphthalimide probes, that shows high fluorescence emissions and sensitivity in the presence of low amounts of TNT explosive. In particular, we have fabricated the first sensor device based on an optical array for the detection of TNT in real samples by using a smartphone as detector. The possibility to use a common smartphone as detector leads to a prototype that can be also used in a real-life field application. The key benefit lies in the possibility of even a nonspecialist operator in the field to simply collect and send data (photos) to the trained artificial intelligence server for rapid diagnosis but also directly to the bomb disposal unit for expert evaluation. This new array sensor contains seven different fluorescent probes that are able to interact via noncovalent interactions with TNT. The interaction of each probe with TNT has been tested in solution by fluorescence titrations. The solid device has been tested in terms of selectivity and linearity toward TNT concentration. Tests performed with other explosives and other nitrogen-based analytes demonstrate the high selectivity for TNT molecules, thus supporting the reliability of this sensor. In addition, TNT can be detected in the range of 98 ng∼985 µg, with a clear different response of each probe to the different amounts of TNT.

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.