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.

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.

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.

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.

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.

[Reflections on the inner limiting membrane peeling and its derivatives in macular hole surgery].

Internal limiting membrane (ILM) peeling is a critical step in the process of macular hole surgery, giving rise to various modified techniques such as ILM flip-over coverage, ILM and other tissue tamponade procedures, and foveal-sparing ILM peeling. All these approaches aim to improve the postoperative closure rate of macular holes. The goal of macular hole surgery is to better preserve the integrity of the foveal center structure, with the aim of achieving functional recovery on the basis of anatomical restoration. However, in clinical practice, there is a tendency to excessively choose certain surgical methods solely to pursue the closure rate of the hole, which may not be beneficial for the visual function recovery of the patients. This article discusses how to correctly select the internal limiting membrane and its derivative procedures in macular hole surgery, combining clinical practice and relevant domestic and international research literature. It aims to provide insights for colleagues performing macular hole surgery as a reference regarding this clinical focus issue.

[Analysis of the therapeutic efficacy of pars plana vitrectomy without intraocular tamponade in the treatment of high myopic eyes with myopic foveoschisis and central foveal detachment].

Objective: To investigate the efficacy of pars plana vitrectomy (PPV) without intraocular tamponade in the treatment of high myopic eyes with myopic foveoschisis (MF) accompanied by foveal detachment (FD). Methods: A retrospective case series study was conducted. The medical records of patients diagnosed with unilateral MF accompanied by FD at the Eye & ENT Hospital of Fudan University between May 2018 and December 2021 were collected. All patients underwent 23-gauge PPV with posterior vitreous cortex clearance, and no intraocular tamponade was applied. The cases were divided into groups based on whether the internal limiting membrane was peeled during surgery or retained. Follow-up was conducted for at least 12 months. The main outcome measures included postoperative best-corrected visual acuity (BCVA, converted to logarithm of the minimum angle of resolution), central foveal thickness (CFT), MF resolution, and complications. Statistical analyses were performed using t-tests, chi-square tests, Fisher's exact tests, and univariate and multivariate linear regression. Results: A total of 40 patients (40 eyes) with MF and FD were included in the study, with 30.0% being male and 70.0% female. The mean age was (56.9±11.7) years, and the axial length of the eyes was (29.1±1.9) mm. At 12 months postoperatively, BCVA improved from baseline 1.15±0.58 to 0.73±0.39 (t=6.11, P<0.001), and CFT decreased from baseline (610.1±207.2) µm to (155.9±104.1) µm (t=13.47, P<0.001). Complete resolution of MF with foveal reattachment was observed in 80.0% of eyes, with a median time of 6 (5, 8) months. There was no significant difference in BCVA and CFT between the internal limiting membrane peeled group and retained group [0.68±0.39 vs. 0.79±0.40, t=0.85, P=0.403; (148.3±63.8)vs.(164.3±137.2)um,t=0.48, P=0.634]. One eye experienced macular hole and another eye developed retinal detachment postoperatively. Correlation analysis showed a positive correlation between BCVA at 12 months postoperatively and baseline BCVA (ß=0.433, P<0.001). Conclusions: Pars plana vitrectomy without intraocular tamponade is effective in treating MF accompanied by FD. The choice between internal limiting membrane peeling and retention does not significantly affect visual prognosis.

[Clinical observation of the intraocular distribution characteristics of indocyanine green after epiretinal membrane peeling using a fluorescence detection system developed in Python].

Objective: To utilize a Python-based fluorescence area detection system to observe and quantitatively analyze the intraocular distribution characteristics and metabolic patterns of Indocyanine Green (ICG) following epiretinal membrane peeling. Methods: A prospective case series study was conducted on patients with idiopathic epiretinal membrane undergoing vitrectomy at West China Hospital of Sichuan University from March 2019 to March 2021. ICG staining was applied during surgery for peeling the epiretinal membrane and internal limiting membrane. Patients were followed up at 1 week, 1 month, 3 months, 6 months, and 12 months postoperatively, with assessments including best-corrected visual acuity, intraocular pressure, fundus photography, near-infrared fundus fluorescence imaging (NIR-FF), and optical coherence tomography (OCT). A Python-based ICG intraocular metabolism detection system was developed to measure the residual area of ICG fluorescence on NIR-FF, predict the ICG metabolic pattern equation, and correlate it with postoperative visual acuity and peripapillary retinal nerve fiber layer thickness. Results: A total of 64 patients (64 eyes) were included, with an average age of 64.6±8.4 years, including 25 males (39.1%) and 39 females (60.9%). Preoperative NIR-FF images showed no ICG strong fluorescence. At 1 week postoperatively, diffuse ICG strong fluorescence appeared in the posterior pole, and the internal limiting membrane removal area exhibited a ring-like weak fluorescence. Over time, ICG strong fluorescence was observed along the vascular arch and nerve fiber trajectory, gradually diminishing toward the optic disc, with residual ICG fluorescence still visible at the optic disc at 1 year. The Python-based ICG fluorescence area detection system effectively measured intraocular residual ICG area. A predictive equation for the 12-month residual ICG area was constructed through linear regression analysis (Residual ICG area=0.22 × Residual ICG area at 6 months, R2=16%, P=0.002). Except for a negative correlation between the ICG residual area at 1 month and postoperative visual acuity (P=0.017, r=-0.195), no correlation was found between intraocular ICG fluorescence residual area and postoperative visual acuity or peripapillary retinal nerve fiber layer thickness at other follow-up times (all P>0.05). Conclusions: In patients with idiopathic epiretinal membrane undergoing ICG staining for internal limiting membrane peeling, ICG exhibits characteristic metabolic processes in the eye, with strong fluorescence along the vascular arch and nerve fiber trajectory, gradually converging toward the optic disc over time. The Python-based ICG fluorescence area detection system provides a clear display of the intraocular distribution characteristics of ICG after epiretinal membrane peeling and serves as a tool for predicting the metabolic patterns of ICG in the eye.

Comparison of cognitive workload and surgical outcomes between a three-dimensional and conventional microscope macular hole surgery.

BACKGROUND: Performing a surgical task subjects the surgeon to multitudinal stressors, especially with the newer 3D technology. The quantum of cognitive workload using this modern surgical system in comparison to the Conventional microscope system remains unexplored. We evaluate the surgeon's cognitive workload and the surgical outcomes of macular hole(MH) surgery performed on a 3D versus a Conventional microscope operating system. METHODS: 50 eyes of 50 patients with MH undergoing surgery using the 3D or Conventional microscope visualization system. Cognitive workload assessment was done by real-time tools(Surgeons' heart rate [HR] and oxygen saturation[SPO2]) and self-report tool(Surgery Task Load Index[SURG-TLX] questionnaire) of three Vitreoretinal surgeons. Based on the SURG-TLX questionnaire, an assessment of the workload was performed. RESULTS: Of the 50 eyes, 30 eyes and 20 eyes underwent surgery with the Conventional microscope and the 3D system, respectively. No difference was noted in the MH basal-diameter(p = 0.128), total surgical-duration(p = 0.299), internal-limiting membrane(ILM) peel time(p = 0.682), and the final visual acuity (VA; p = 0.515) between the two groups. Both groups showed significant improvement in VA(p < 0.001) with a 90% closure rate at one-month post-surgery. Cognitive workload comparison, the intraoperative HR(p = 0.024), total workload score(P = 0.005), and temporal-demand dimension(p = 0.004) were significantly more in Conventional microscope group as compared to 3D group. In both the groups, the HR increased significantly from the baseline while performing ILM peeling and at the end. CONCLUSION: The surgeon's cognitive workload is markedly reduced while performing macular hole surgery with a 3D viewing system. Moreover, duration of surgery including ILM peel time, MH closure rates, and visual outcomes remains unaffected irrespective of the operating microscope system.

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.

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.

ITGAM-mediated macrophages contribute to basement membrane damage in diabetic nephropathy and atherosclerosis.

BACKGROUND: Diabetic nephropathy (DN) and atherosclerosis (AS) are prevalent and severe complications associated with diabetes, exhibiting lesions in the basement membrane, an essential component found within the glomerulus, tubules, and arteries. These lesions contribute significantly to the progression of both diseases, however, the precise underlying mechanisms, as well as any potential shared pathogenic processes between them, remain elusive. METHODS: Our study analyzed transcriptomic profiles from DN and AS patients, sourced from the Gene Expression Omnibus database. A combination of integrated bioinformatics approaches and machine learning models were deployed to identify crucial genes connected to basement membrane lesions in both conditions. The role of integrin subunit alpha M (ITGAM) was further explored using immune infiltration analysis and genetic correlation studies. Single-cell sequencing analysis was employed to delineate the expression of ITGAM across different cell types within DN and AS tissues. RESULTS: Our analyses identified ITGAM as a key gene involved in basement membrane alterations and revealed its primary expression within macrophages in both DN and AS. ITGAM was significantly correlated with tissue immune infiltration within these diseases. Furthermore, the expression of genes encoding core components of the basement membrane was influenced by the expression level of ITGAM. CONCLUSION: Our findings suggest that macrophages may contribute to basement membrane lesions in DN and AS through the action of ITGAM. Moreover, therapeutic strategies that target ITGAM may offer potential avenues to mitigate basement membrane lesions in these two diabetes-related complications.

Porous Polymeric Nanofilms for Recreating the Basement Membrane in an Endothelial Barrier-on-Chip.

Organs-on-chips (OoCs) support an organotypic human cell culture in vitro. Precise representation of basement membranes (BMs) is critical for mimicking physiological functions of tissue interfaces. Artificial membranes in polyester (PES) and polycarbonate (PC) commonly used in in vitro models and OoCs do not replicate the characteristics of the natural BMs, such as submicrometric thickness, selective permeability, and elasticity. This study introduces porous poly(d,l-lactic acid) (PDLLA) nanofilms for replicating BMs in in vitro models and demonstrates their integration into microfluidic chips. Using roll-to-roll gravure coating and polymer phase separation, we fabricated transparent ∼200 nm thick PDLLA films. These nanofilms are 60 times thinner and 27 times more elastic than PES membranes and show uniformly distributed pores of controlled diameter (0.4 to 1.6 µm), which favor cell compartmentalization and exchange of large water-soluble molecules. Human umbilical vein endothelial cells (HUVECs) on PDLLA nanofilms stretched across microchannels exhibited 97% viability, enhanced adhesion, and a higher proliferation rate compared to their performance on PES membranes and glass substrates. After 5 days of culture, HUVECs formed a functional barrier on suspended PDLLA nanofilms, confirmed by a more than 10-fold increase in transendothelial electrical resistance and blocked 150 kDa dextran diffusion. When integrated between two microfluidic channels and exposed to physiological shear stress, despite their ultrathin thickness, PDLLA nanofilms upheld their integrity and efficiently maintained separation of the channels. The successful formation of an adherent endothelium and the coculture of HUVECs and human astrocytes on either side of the suspended nanofilm validate it as an artificial BM for OoCs. Its submicrometric thickness guarantees intimate contact, a key feature to mimic the blood-brain barrier and to study paracrine signaling between the two cell types. In summary, porous PDLLA nanofilms hold the potential for improving the accuracy and physiological relevance of the OoC as in vitro models and drug discovery tools.

Quantitative assessment of glomerular basement membrane collagen IV α chains in paraffin sections from patients with focal segmental glomerulosclerosis and Alport gene variants.

Focal segmental glomerulosclerosis (FSGS) lesions have been linked to variants in COL4A3/A4/A5 genes, which are also mutated in Alport syndrome. Although it could be useful for diagnosis, quantitative evaluation of glomerular basement membrane (GBM) type IV collagen (colIV) networks is not widely used to assess these patients. To do so, we developed immunofluorescence imaging for collagen α5(IV) and α1/2(IV) on kidney paraffin sections with Airyscan confocal microscopy that clearly distinguishes GBM collagen α3α4α5(IV) and α1α1α2(IV) as two distinct layers, allowing quantitative assessment of both colIV networks. The ratios of collagen α5(IV):α1/2(IV) mean fluorescence intensities (α5:α1/2 intensity ratios) and thicknesses (α5:α1/2 thickness ratios) were calculated to represent the levels of collagen α3α4α5(IV) relative to α1α1α2(IV). The α5:α1/2 intensity and thickness ratios were comparable across all 11 control samples, while both ratios were significantly and markedly decreased in all patients with pathogenic or likely pathogenic Alport COL4A variants, supporting validity of this approach. Thus, with further validation of this technique, quantitative measurement of GBM colIV subtype abundance by immunofluorescence, may potentially serve to identify the subgroup of patients with FSGS lesions likely to harbor pathogenic COL4A variants who could benefit from genetic testing.

Whole Mount Imaging to Visualize and Quantify Peripheral Lens Structure, Cell Morphology, and Organization.

The ocular lens is a transparent flexible tissue that alters its shape to focus light from different distances onto the retina. Aside from a basement membrane surrounding the organ, called the capsule, the lens is entirely cellular consisting of a monolayer of epithelial cells on the anterior hemisphere and a bulk mass of lens fiber cells. Throughout life, epithelial cells proliferate in the germinative zone at the lens equator, and equatorial epithelial cells migrate, elongate, and differentiate into newly formed fiber cells. Equatorial epithelial cells substantially alter morphology from randomly packed cobble-stone-shaped cells into aligned hexagon-shaped cells forming meridional rows. Newly formed lens fiber cells retain the hexagonal cell shape and elongate toward the anterior and posterior poles, forming a new shell of cells that are overlaid onto previous generations of fibers. Little is known about the mechanisms that drive the remarkable morphogenesis of lens epithelial cells to fiber cells. To better understand lens structure, development, and function, new imaging protocols have been developed to image peripheral structures using whole mounts of ocular lenses. Here, methods to quantify capsule thickness, epithelial cell area, cell nuclear area and shape, meridional row cell order and packing, and fiber cell widths are shown. These measurements are essential for elucidating the cellular changes that occur during lifelong lens growth and understanding the changes that occur with age or pathology.

Comparison of stereopsis and foveal microstructure after internal limiting membrane peeling and inverted internal limiting membrane flap techniques in patients with macular hole.

PURPOSE: To compare stereopsis and foveal microstructure after internal limiting membrane peeling and inverted internal limiting membrane flap technique in patients with macular hole. DESIGN: Retrospective observational study. METHODS: Sixty-six patients with macular hole were included, of whom 41 underwent 25-gauge pars-plana vitrectomy with complete internal limiting membrane peeling (Peeling group) and 25 with the inverted flap technique (Inverted group). We evaluated stereopsis using the Titmus Stereo Test and the TNO stereo test, best-corrected visual acuity, macular hole closure rate, and foveal microstructure with optical coherence tomography before and at 3, 6, and 12 months after surgery. MAIN OUTCOME MEASURES: Stereopsis and foveal microstructure. RESULTS: Preoperatively, no difference was observed in the base and minimum diameters of macular hole, Titmus Stereo Test score, TNO stereo test score, and best-corrected visual acuity between the Peeling and Inverted groups. The macular hole closure rate in the Peeling and Inverted groups were 97.6% and 100%, respectively, with no significant difference between groups. At 12 months postoperatively, Titmus Stereo Test score (2.1 ± 0.4 in the peeling and 2.2 ± 0.4 in the inverted groups), TNO stereo test score (2.3 ± 0.4 and 2.2± 0.5), and best-corrected visual acuity (0.20 ± 0.18 and 0.24 ± 0.25) were not significantly different between groups (p = 0.596, 0.332, respectively). The defect of the external limiting membrane was more common in the Inverted group than in the Peeling group at 6 months after surgery (5.4 vs. 28.0%; p < 0.05). No statistically significant inter-group differences were noted in the ellipsoid zone defect ratio throughout the follow-up period. CONCLUSIONS: There was no difference in postoperative stereopsis nor foveal microstructure between the internal limiting membrane peeling group and the inverted group in patients with macular hole.

Complementary biomarkers of computed tomography for diagnostic grading of gastric cancer: DSCC1 and GINS1.

OBJECTIVE: Computed tomography (CT) is an important tool for grading gastric cancer. Gastric cancer typically originates from epithelial cells of gastric mucosa. However, complementary markers for gastric cancer, relationship between DSCC1, GINS1 and gastric cancer remain unclear. METHODS: Gastric cancer data were obtained from gene expression omnibus (GEO). Differentially expressed genes (DEGs) were identified, weighted gene co-expression network analysis (WGCNA) was conducted. Protein-protein interaction (PPI) network was constructed and analyzed. Functional enrichment analysis, gene set enrichment analysis (GSEA), gene expression heatmaps, immune infiltration analysis were performed. The most relevant diseases related to core genes were identified using Comparative Toxicogenomics Database (CTD). TargetScan was used to screen miRNAs. Validation was carried out using Western blotting (WB) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: 1243 DEGs were identified. Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) analyses revealed significant enrichment in cell cycle regulation, macrophage migration control, basement membrane, extracellular regions, growth factor binding, protein complex binding, P53 signaling pathway, protein digestion and absorption, metabolic pathways. Immune infiltration analysis indicated that high expression of activated Mast cells and Neutrophils, with a strong positive correlation between them, may influence progression of gastric cancer. CTD analysis revealed associations between DSCC1, GINS1 and gastric tumors, gastrointestinal diseases, tumors, gastritis, inflammation, necrosis. WB and RT-PCR results demonstrated high expression of DSCC1 and GINS1 in gastric cancer. CONCLUSION: The expressions of DSCC1 and GINS1 are up-regulated in gastric cancer, which can be used as supplementary markers for CT diagnostic grading of gastric cancer.