Basement membrane dynamics in living animals: Insights and pitfalls.

Recent studies with fluorophore-tagged basement membrane (BM) components have led to remarkable discoveries about BMs but also inconsistent interpretations. Here, we review types of BM dynamics, discuss how we conduct and interpret fluorophore-tagged BM studies, and highlight experimental conditions that are important to consider.

Deposition of collagen III and alterations in basement membrane integrity as candidate prognostic markers in prostate cancer.

The extracellular matrix surrounding the tumor undergoes changes in its organization during the metastasis process. The present study aims to quantify total collagen, collagen I (Col I) and collagen III (Col III), analyze the alignment of collagen fibers and assess the basement membrane integrity in samples from patients with metastatic and non-metastatic prostate cancer. Tissue samples from 60 patients were classified into groups based on prognostic parameters: better prognosis (n = 20), worse prognosis without metastasis (n = 23) and metastatic (n = 17). Picrosirius red with further analysis under polarizing microscope was used to quantify (with validation using immunohistochemistry) and analyze collagen alignment, and Periodic Acid Schiff staining was used to analyze the basement membrane integrity. The Col I/Col III ratio was found to be higher in the metastatic group than in the groups with better prognosis (p = 0.012) and worse prognosis without metastasis (p = 0.018). Basement membrane integrity constitution in malignant tumor tissue differed from that of adjacent non-tumor tissue (p < 0.001). Moreover, the worsening in the tumor tissue integrity was positively correlated with worse prognostic parameters. All in all, absence of Col III and basement membrane integrity might be indicators of poor prognosis in prostate cancer.

Comparative Study of Basement-membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

Outcomes of epiretinal proliferation embedding technique in the surgery for full-thickness macular hole.

To compare visual and anatomical outcomes between peeling and embedding of epiretinal proliferation in patients with full-thickness macular holes (FTMH) with epiretinal proliferation (EP), this retrospective cohort study classified patients into two groups based on whether EP was completely peeled (peeling group, n = 25 eyes), or embedded into the hole (embedding group, n = 31 eyes) during surgery. Preoperative characteristics and postoperative outcomes, including best-corrected visual acuity and the length of the disrupted external limiting membrane and ellipsoid zone, were compared. Preoperative features including visual acuity and hole size did not differ between the two groups. All studied eyes achieved closure of the macular hole postoperatively. Visual acuity significantly improved at 3, 6, and 12 months postoperatively in both groups. The visual acuity 1-month after surgery was better in the embedding group than that in the peeling group (0.28 ± 0.29 vs. 0.50 ± 0.42 logarithm of the minimum angle of resolution, P = 0.016), although the difference was not noted after 3 months postoperatively. The embedding group showed shorter disruption of the external limiting membrane than the peeling group postoperatively (62.6 ± 40.2 µm vs. 326.2 ± 463.9 µm at postoperative 12 months, P = 0.045). In conclusion, the embedding technique during surgical repair of a FTMH with EP facilitates recovery of the outer foveal layers and promotes earlier restoration of visual function.

Flow in temporally and spatially varying porous media: a model for transport of interstitial fluid in the brain.

Flow in a porous medium can be driven by the deformations of the boundaries of the porous domain. Such boundary deformations locally change the volume fraction accessible by the fluid, creating non-uniform porosity and permeability throughout the medium. In this work, we construct a deformation-driven porous medium transport model with spatially and temporally varying porosity and permeability that are dependent on the boundary deformations imposed on the medium. We use this model to study the transport of interstitial fluid along the basement membranes in the arterial walls of the brain. The basement membrane is modeled as a deforming annular porous channel with the compressible pore space filled with an incompressible, Newtonian fluid. The role of a forward propagating peristaltic heart pulse wave and a reverse smooth muscle contraction wave on the flow within the basement membranes is investigated. Our results identify combinations of wave amplitudes that can induce either forward or reverse transport along these transport pathways in the brain. The magnitude and direction of fluid transport predicted by our model can help in understanding the clearance of fluids and solutes along the Intramural Periarterial Drainage route and the pathology of cerebral amyloid angiopathy.

Selective internal limiting membrane peeling for prevention of secondary epiretinal membrane after vitrectomy for rhegmatogenous retinal detachment.

PURPOSE: We introduce selective internal limiting membrane (ILM) peeling, a guideline procedure to determine whether to remove the ILM during vitrectomy for rhegmatogenous retinal detachment (RRD). STUDY DESIGN: Retrospective case series METHODS: Patients who underwent pars plana vitrectomy for RRD and were followed up for 12 months or longer were included. When vitreous cortex remnants (VCRs) were detected with triamcinolone acetonide, the ILM was removed; otherwise, the ILM was preserved ("selective ILM peeling"). The factors associated with the presence of VCRs and incidence of secondary epiretinal membrane (ERM) were analyzed. RESULTS: VCRs were detected in 87 of 133 eyes (65.4%) in which the ILM was removed. Younger age, better preoperative visual acuity, and vitreous hemorrhage were negatively correlated with the presence of VCRs. No ERM occurred in the eyes after ILM peeling. Among the eyes with ILM preservation, subclinical ERM was noticed in 4 eyes (8.7%), and 1 eye (2.1%) required additional surgery owing to ERM. ERM occurred more commonly in eyes with the ILM preserved (P = .004). However, no differences in the rate of additional surgeries were found between the 2 groups. CONCLUSION: Selective ILM peeling offers an alternative option to reduce the burden of ILM peeling or additional surgery.

A brain-specific angiogenic mechanism enabled by tip cell specialization.

Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals3-5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-ß-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.

Integrating integrins with the hallmarks of cancer.

Epithelial cells adhere to a specialized extracellular matrix called the basement membrane which allows them to polarize and form epithelial tissues. The extracellular matrix provides essential physical scaffolding and biochemical and biophysical cues required for tissue morphogenesis, differentiation, function, and homeostasis. Epithelial cell adhesion to the extracellular matrix (i.e., basement membrane) plays a critical role in organizing epithelial tissues, separating the epithelial cells from the stroma. Epithelial cell detachment from the basement membrane classically results in death, though detachment or invasion through the basement membrane represents a critical step in carcinogenesis. Epithelial cells bind to the extracellular matrix via specialized matrix receptors, including integrins. Integrins are transmembrane receptors that form a mechanical linkage between the extracellular matrix and the intracellular cytoskeleton and are required for anchorage-dependent cellular functions such as proliferation, migration, and invasion. The role of integrins in the development, growth, and dissemination of multiple types of carcinomas has been investigated by numerous methodologies, which has led to great complexity. To organize this vast array of information, we have utilized the "Hallmarks of Cancer" from Hanahan and Weinberg as a convenient framework to discuss the role of integrins in the pathogenesis of cancers. This review explores this biology and how its complexity has impacted the development of integrin-targeted anti-cancer therapeutics.

Oct4 controls basement membrane development during human embryogenesis.

Basement membranes (BMs) are sheet-like structures of extracellular matrix (ECM) that provide structural support for many tissues and play a central role in signaling. They are key regulators of cell behavior and tissue functions, and defects in their assembly or composition are involved in numerous human diseases. Due to the differences between human and animal embryogenesis, ethical concerns, legal constraints, the scarcity of human tissue material, and the inaccessibility of the in vivo condition, BM regulation during human embryo development has remained elusive. Using the post-implantation amniotic sac embryoid (PASE), we delineate BM assembly upon post-implantation development and BM disassembly during primitive streak (PS) cell dissemination. Further, we show that the transcription factor Oct4 regulates the expression of BM structural components and receptors and controls BM development by regulating Akt signaling and the small GTPase Rac1. These results represent a relevant step toward a more comprehensive understanding of early human development.

Brain endothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

Three-dimensional imaging studies in mice identify cellular dynamics of skeletal muscle regeneration.

The function of many organs, including skeletal muscle, depends on their three-dimensional structure. Muscle regeneration therefore requires not only reestablishment of myofibers but also restoration of tissue architecture. Resident muscle stem cells (SCs) are essential for regeneration, but how SCs regenerate muscle architecture is largely unknown. We address this problem using genetic labeling of mouse SCs and whole-mount imaging to reconstruct, in three dimensions, muscle regeneration. Unexpectedly, we found that myofibers form via two distinct phases of fusion and the residual basement membrane of necrotic myofibers is critical for promoting fusion and orienting regenerated myofibers. Furthermore, the centralized myonuclei characteristic of regenerated myofibers are associated with myofibrillogenesis and endure months post injury. Finally, we elucidate two cellular mechanisms for the formation of branched myofibers, a pathology characteristic of diseased muscle. We provide a synthesis of the cellular events of regeneration and show that these differ from those used during development.

Mapping functional to morphological variation reveals the basis of regional extracellular matrix subversion and nerve invasion in pancreatic cancer.

Intratumor morphological heterogeneity of pancreatic ductal adenocarcinoma (PDAC) predicts clinical outcomes but is only partially understood at the molecular level. To elucidate the gene expression programs underpinning intratumor morphological variation in PDAC, we investigated and deconvoluted at single cell level the molecular profiles of histologically distinct clusters of PDAC cells. We identified three major morphological and functional variants that co-exist in varying proportions in all PDACs, display limited genetic diversity, and are associated with a distinct organization of the extracellular matrix: a glandular variant with classical ductal features; a transitional variant displaying abortive ductal structures and mixed endodermal and myofibroblast-like gene expression; and a poorly differentiated variant lacking ductal features and basement membrane, and showing neuronal lineage priming. Ex vivo and in vitro evidence supports the occurrence of dynamic transitions among these variants in part influenced by extracellular matrix composition and stiffness and associated with local, specifically neural, invasion.

Internal limiting membrane peeling in rhegmatogenous retinal detachment: A meta-analysis.

PURPOSE: To determine whether pars plana vitrectomy (PPV) with internal limiting membrane (ILM) peeling for rhegmatogenous retinal detachment (RRD) could get better functional and anatomical outcomes. METHODS: A comprehensive literature search was performed to find relevant studies. A meta-analysis was conducted by comparing the weighted mean differences (WMD) in the mean change of best corrected visual acuity (BCVA) from baseline and calculating the odd ratios (OR) for rates of epiretinal membrane (ERM) formation and recurrence of retinal detachment (RD). RESULTS: Fourteen studies were selected, including 2259 eyes (825 eyes in the ILM peeling group and 1434 eyes in the non-ILM peeling group). There was no significant difference in terms of mean change in BCVA from baseline and the rate of RD recurrence (WMD = 0.02, 95% CI, -0.20 to 0.24, P = 0.86, and OR = 0.55, 95% CI, 0.24 to 1.26, P = 0.16), but ILM peeling was associated with a significantly lower frequency of postoperative ERM formation (OR = 0.13, 95% CI, 0.06 to 0.26, P<0.00001). Similar results were obtained in a sub-analysis based on macula-off RRD. CONCLUSION: ILM peeling results in similar BCVA, with same rate of RD recurrence, but lower rate of postoperative ERM development. ILM peeling could be considered in selected cases with risk factors that are likely to develop an ERM.

Basement membrane diversification relies on two competitive secretory routes defined by Rab10 and Rab8 and modulated by dystrophin and the exocyst complex.

The basement membrane (BM) is an essential structural element of tissues, and its diversification participates in organ morphogenesis. However, the traffic routes associated with BM formation and the mechanistic modulations explaining its diversification are still poorly understood. Drosophila melanogaster follicular epithelium relies on a BM composed of oriented BM fibrils and a more homogenous matrix. Here, we determined the specific molecular identity and cell exit sites of BM protein secretory routes. First, we found that Rab10 and Rab8 define two parallel routes for BM protein secretion. When both routes were abolished, BM production was fully blocked; however, genetic interactions revealed that these two routes competed. Rab10 promoted lateral and planar-polarized secretion, whereas Rab8 promoted basal secretion, leading to the formation of BM fibrils and homogenous BM, respectively. We also found that the dystrophin-associated protein complex (DAPC) and Rab10 were both present in a planar-polarized tubular compartment containing BM proteins. DAPC was essential for fibril formation and sufficient to reorient secretion towards the Rab10 route. Moreover, we identified a dual function for the exocyst complex in this context. First, the Exo70 subunit directly interacted with dystrophin to limit its planar polarization. Second, the exocyst complex was also required for the Rab8 route. Altogether, these results highlight important mechanistic aspects of BM protein secretion and illustrate how BM diversity can emerge from the spatial control of distinct traffic routes.

Profiling native pulmonary basement membrane stiffness using atomic force microscopy.

Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) substructure measuring only ∼100-400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the precise assignment of the Young's modulus E of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young's modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young's modulus E using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of Young's moduli and generation of Young's modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1-2 d.

Comprehensive analysis of basement membrane-related gene based on single-cell and bulk RNA sequencing data to predict prognosis and evaluate immune characteristics in colorectal cancer.

AIMS: Basement membrane-related genes (BMs) participate in regulating cell polarity, invasion, metastasis, and survival across different tumor types. Nevertheless, the specific functions of BMs in colorectal cancer (CRC) remain uncertain. METHODS: To investigate the clinical relevance of BMs in CRC, we retrieved both gene expression and clinical data from The Cancer Genome Atlas (TCGA) datasets for subsequent analysis. The Kaplan-Meier (K-M) survival curve was employed to evaluate prognosis in high- and low-risk groups. Furthermore, additional analyses, including nomogram construction, functional enrichment, examination of the tumor immune microenvironment, prediction of small-molecule drugs, and more, were conducted to delve into the significance of BM-related signatures in CRC. Single-cell data from seven CRC patients were obtained from the TISCH2 database, and expression validation and cell source exploration of BM-related signatures were performed. Lastly, the expression and function of TIMP1, a key gene in BMs that may play a role in the progression of CRC, was validated in vitro through a series of basic experiments. RESULTS: We constructed a seven BMs-based model to categorize CRC patients into high-risk and low-risk groups. K-M survival analysis indicated a poorer prognosis for high-risk CRC patients. Cox regression analysis further identified the risk score as an independent prognostic factor for CRC patients. The nomogram model exhibited superior discrimination and calibration abilities of CRC patients. Based on the results from GO/KEGG and GSEA, genes in the high-risk subgroup were implicated in immune-related pathways and exhibited a positive correlation with immune checkpoints. In single-cell data, we found that TIMP1 is highly expressed in many cells, especially in malignant tumor cells. We also observed up-regulation of TIMP1 in CRC cell lines, promoting cancer invasion and migration in vitro. CONCLUSIONS: Our study has discovered a novel prognostic index derived from BM-related genes in CRC patients. Specifically, the new model enables patient stratification, improving the selection of individuals likely to benefit from immunotherapy.

Low pH condition impairs BP-IgG binding to the basement membrane zone.

Bullous pemphigoid (BP), an autoimmune subepidermal blistering disease, shows tense blisters associated with urticarial erythema. Tissue-bound Immunoglobulin G (IgG) at the basement membrane zone (BMZ) detected by direct immunofluorescence (DIF) is strong evidence for a diagnosis of BP. The sensitivity of DIF is higher in complement component 3 (C3) than in IgG, but the reason for this different sensitivity is not fully understood. In this study, we performed several ex vivo studies to investigate the possible mechanism of IgG negativity and C3 positivity at the BMZ by DIF in some BP cases. First, sera from BP patients showing IgG negativity by DIF were found to clearly react to the BMZ in their own DIF skin samples. Next, indirect immunofluorescence (IIF) was performed using sera diluted with different pH phosphate-buffered saline (PBS), pH 7.4, 6.0, and 3.0. Patients' sera diluted with pH 7.4 PBS showed linear staining at the BMZ, but sera diluted with pH 6.0 PBS and pH 3.0 PBS showed lower fluorescence intensities. Finally, sections of skin from BP patients were pre-incubated with different pH PBS (pH 3.0, 6.0, and 7.4), followed by staining with anti-human IgG and C3. The fluorescence intensities were notably lower for IgG and C3 that had been pre-incubated with pH 3.0 PBS and pH 6.0 PBS than for IgG and C3 that had been pre-incubated with pH 7.4 PBS. These results suggest that a low pH condition hinders the binding of autoantibodies to the BMZ, that is, the drop in tissue pH induced by inflammation inhibits autoantibodies from depositing at the BMZ. Furthermore, the drop in tissue pH causes tissue-bound autoantibodies to detach from the BMZ. Complement fragments are activated not only on IgG but also on the cell surface of cells close to IgG during complement activation. IgG may detach from the BMZ under low pH condition induced by inflammation, but some complement fragments remain at the BMZ. These phenomena may help to explain why C3 is more sensitive than IgG when DIF is used to diagnose BP.

Endothelial cells regulate alveolar morphogenesis by constructing basement membranes acting as a scaffold for myofibroblasts.

Alveologenesis is a spatially coordinated morphogenetic event, during which alveolar myofibroblasts surround the terminal sacs constructed by epithelial cells and endothelial cells (ECs), then contract to form secondary septa to generate alveoli in the lungs. Recent studies have demonstrated the important role of alveolar ECs in this morphogenetic event. However, the mechanisms underlying EC-mediated alveologenesis remain unknown. Herein, we show that ECs regulate alveologenesis by constructing basement membranes (BMs) acting as a scaffold for myofibroblasts to induce septa formation through activating mechanical signaling. Rap1, a small GTPase of the Ras superfamily, is known to stimulate integrin-mediated cell adhesions. EC-specific Rap1-deficient (Rap1iECKO) mice exhibit impaired septa formation and hypo-alveolarization due to the decreased mechanical signaling in myofibroblasts. In Rap1iECKO mice, ECs fail to stimulate integrin ß1 to recruit Collagen type IV (Col-4) into BMs required for myofibroblast-mediated septa formation. Consistently, EC-specific integrin ß1-deficient mice show hypo-alveolarization, defective mechanical signaling in myofibroblasts, and disorganized BMs. These data demonstrate that alveolar ECs promote integrin ß1-mediated Col-4 recruitment in a Rap1-dependent manner, thereby constructing BMs acting as a scaffold for myofibroblasts to induce mechanical signal-mediated alveologenesis. Thus, this study unveils a mechanism of organ morphogenesis mediated by ECs through intrinsic functions.

Dermal stiffness governs the topography of the epidermis and the underlying basement membrane in young and old human skin.

The epidermis is a stratified epithelium that forms the outer layer of the skin. It is composed primarily of keratinocytes and is constantly renewed by the proliferation of stem cells and their progeny that undergo terminal differentiation as they leave the basal layer and migrate to the skin surface. Basal keratinocytes rest on a basement membrane composed of an extracellular matrix that controls their fate via integrin-mediated focal adhesions and hemidesmosomes which are critical elements of the epidermal barrier and promote its regenerative capabilities. The distribution of basal cells with optimal activity provides the basement membrane with its characteristic undulating shape; this configuration disappears with age, leading to epidermal weakness. In this study, we present an in-depth imaging analysis of basal keratinocyte anchorage in samples of human skin from participants across the age spectrum. Our findings reveal that skin aging is associated with the depletion of hemidesmosomes that provide crucial support for stem cell maintenance; their depletion correlates with the loss of the characteristic basement membrane structure. Atomic force microscopy studies of skin and in vitro experiments revealed that the increase in tissue stiffness observed with aging triggers mechanical signals that alter the basement membrane structure and reduce the extent of basal keratinocyte anchorage, forcing them to differentiate. Genomic analysis revealed that epidermal aging was associated with mechanical induction of the transcription factor Krüppel-like factor 4. The altered mechanical properties of tissue being a new hallmark of aging, our work opens new avenues for the development of skin rejuvenation strategies.

Ophthalmologists' assessment of the handling characteristics of the novel Finesse Reflex Handle in comparison to those of a conventional handle.

Internal limiting membrane (ILM) peeling requires a delicate handling technique. It is also important that ophthalmologists can use the ILM forceps handle of their preference. This study objectively and subjectively evaluated the handling of the novel Finesse Reflex Handle (Reflex) in comparison with that of a conventional handle. The force required to close the forceps tips, evaluated using a digital force gauge, was significantly lesser for Reflex than for the conventional handle (3.14 ± 0.09 N vs. 3.84 ± 0.06 N, P < 0.001). Twenty-one ophthalmologists with various levels of experience answered a questionnaire after using both handles, and the total questionnaire score for Reflex was higher than that for the conventional handle (35.0 ± 3.7 vs. 30.0 ± 6.9, P = 0.01). Furthermore, the duration of experience as an ophthalmologist was negatively correlated with the vertical motion, assessed by video analysis, for the conventional handle (P = 0.02, r = - 0.50) but not for Reflex (P = 0.26). In conclusion, objective and subjective analyses revealed that compared with the conventional handle, the novel Reflex handle had more favourable handling characteristics. Most ophthalmologists preferred the handling of Reflex. Reflex may compensate for a lack of surgical experience.