Basement Membrane Research Articles

A comprehensive map of proteoglycan expression and deposition in the pulmonary arterial wall in health and pulmonary hypertension.

Changes in the extracellular matrix of pulmonary arteries (PAs) are a key aspect of vascular remodelling in pulmonary hypertension (PH). Yet, our understanding of the alterations affecting the proteoglycan (PG) family remains limited. We sought to investigate the expression and spatial distribution of major vascular PGs in PAs from healthy individuals and various PH groups (chronic obstructive pulmonary disease: PH-COPD, pulmonary fibrosis: PH-PF, idiopathic: IPAH). PG regulation, deposition, and synthesis were notably heightened in IPAH, followed by PH-PF, with minor alterations in PH-COPD. Single-cell analysis unveiled cell-type and disease-specific PG regulation. Agrin expression, a basement membrane PG, was increased in IPAH, with PA endothelial cells (PAECs) identified as a major source. PA smooth muscle cells (PASMCs) mainly produced large-PGs, aggrecan and versican, and small-leucine-like proteoglycan (SLRP) biglycan, while the major PGs produced by adventitial fibroblasts were SLRP decorin and lumican. In IPAH and PF-PH, the neointima-forming PASMC population increased the expression of all investigated large-PGs and SLRPs, except fibroblast-predominant DCN. Expression of lumican, versican, and biglycan also positively correlated with collagen 1α1/1α2 expression in PASMCs of IPAH and PH-PF patients. We demonstrated that TGF-β regulates versican and biglycan expression, indicating their contribution to vessel fibrosis in IPAH and PF-PH. We furthermore show that certain circulating PG levels display a disease-dependent pattern, with increased decorin and lumican across all patient groups, while versican was elevated in PH-COPD and IPAH and biglycan reduced in IPAH. These findings suggest unique compartment-specific PG regulation in different forms of PH, indicating distinct pathological processes.

Nerve Epidermal Growth Factor-Like 1 Protein (NELL-1) Expression in Mercury-Related Membranous Nephropathy: Is It a True Association or a Chance Occurrence?

Background: Neural epidermal-like growth factor-like 1 (NELL-1) is a protein kinase C binding protein expressed in osteoblasts and renal tubules. It is expressed in 5%–25% glomerular cells at the mRNA level. Membranous Nephropathy (MN) is characterized by the presence of antibodies against certain types of antigens on the glomerular basement membrane. The most common one implicated in primary MN is an antibody against PLA2R. Many newer antigens have been discovered in the recent past, which are proven to cause secondary MN, one of which is NELL-1. NELL-1 has been associated with malignancy-associated MN and also recently associated with traditional indigenous medications containing mercury. In this study, we study the expression of NELL-1 in mercury-associated MN. Materials and Methods: Records of ten cases of Mercury -associated MN were retrieved from the Institute medical archives and NELL-1 Immunohistochemistry was performed in all ten cases. Results: NELL-1 was found to be positive in 50% of the cases of Mercury associated MN. In addition, mass spectrometric studies was performed, which revealed the common Mercuric compound associated to be ‘Swaskalpa’, Sudarshana Melugu’ and Rasagandhi Mezhugu’. Conclusion: This study highlights why it is important to diagnose mercury-associated MN by a pathologist by picking up the finer histopathological clues and by using NELL-1 immunohistochemistry, especially in PLA2R-negative patients. The former is true as most of the time a history of mercuric compound intake is missed out.

Autoimmunity and epithelial dysplasia in patients with oral lichenoid diseases.

The primary objective of this study was to explore relationship between autoimmunity and epithelial dysplasia in patients with oral lichenoid diseases. A total of 66 patients with oral lichen planus (OLP), 35 with oral lichenoid lesion (OLL), and 85 with oral lichenoid drug reaction (OLDR) were enrolled. OLP, OLL, and OLDR were diagnosed following the definitions of the modified World Health Organization criteria, except for the absence of epithelial dysplasia. All patients underwent diagnostic incisional biopsy and adjunctive direct immunofluorescence assays. An indirect immunofluorescence assay was conducted to determine the antinuclear antibody (ANA) positivity. OLP and OLDR patients with epithelial dysplasia demonstrated higher prevalence of serum ANA positivity compared to those without epithelial dysplasia. Elevated serum levels of high sensitivity-C reactive proteins were observed in the OLP, OLL, and OLDR patients with epithelial dysplasia. In the DIF analysis, patients with epithelial dysplasia in the OLP exhibited a higher prevalence of C3 deposition in the basement membrane zone. This study proposed that autoimmunity may contribute to elevating levels of focal and chronic systemic inflammation, potentially influencing abnormal wound healing and development of dysplastic changes in the oral epithelium among patients with oral lichenoid disease.

O20 Dissecting junctional epidermolysis bullosa heterogeneity

Abstract Introduction and aims Epidermolysis bullosa (EB) is a family of rare, incurable, inherited disorders that are characterized by extreme skin and mucosal fragility. One of the most severe forms is junctional EB (JEB). JEB is caused by mutations in genes encoding the skin basement membrane proteins laminin 332 (α3, β3, γ2), type XVII collagen or integrin α6β4. Despite similarities between the disease pathology of JEB and other forms of EB there is a dramatic reduction in life expectancy for patients with JEB. Patients with JEB rarely survive beyond the first year of life. To investigate this disparity, we have optimized a previously published conditional tamoxifen induced knockout JEB mouse model (Lama3flx/flxK14CreERT). This mouse model recapitulates all key features of JEB, such as thickening of the epidermis and visible blistering owing to the loss of laminin α3 at the basement membrane. Methods Single-cell (sc)RNA-Seq analysis was conducted on pooled skin samples from a healthy (control) and JEB mouse model (n = 3 per genotype). Immunofluorescent staining was conducted to confirm scRNA-Seq analysis including markers for skin differentiation (K14, transglutaminase 1, loricrin), proliferation (Ki67), basement membrane (laminin α3, laminin β3, laminin γ2, collagen VII) and immune markers (F4/80, Ly6-G/C, CD3, CD79). Results We can see large differences in cellular populations across the two conditions. With increased T- and B-immune-cell subpopulations in the JEB model compared with the healthy control. Utilizing CellChatDB we identified disease-specific signalling pathways that identify activated T- and B-cell populations interacting with JEB macrophage populations via proinflammatory pathways. Conclusions We have utilized an in vivo mouse model to gain insight into the molecular and cellular differences in a JEB diseased phenotype. Identifying changes in cellular pathways could be used as a targeted approach to identify a beneficial therapeutic treatment for JEB.

P16 Fibroblast signalling crosstalk drives junctional epidermolysis bullosa disease progression

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is a devastating hereditary skin disorder characterized by severe fragility of skin and mucus membrane, emerges as blistering with little or no trauma. The major classification of JEB includes JEB intermediate and JEB severe; the latter is the most common subtype, which is the result of loss of function mutations in the skin basement membrane protein laminin-332. Lacking this protein impairs basement membrane adhesion of epidermal keratinocytes and leads to deregulated epidermal–dermal crosstalk. The skin dermis is composed of multiple fibroblast subpopulations with different functions in skin homeostasis and repair; however, their specific involvement in skin diseases is largely unknown. Here, we investigate how fibroblast subpopulations, namely papillary and reticular fibroblasts, impact JEB disease progression and skin regeneration. Methods Three-dimensional (3D) organotypic (OT) combining healthy (control) and diseased (JEB) keratinocytes cells with distinct fibroblast subpopulations from different anatomical sites were developed and analysed by immunohistochemistry and RNA-Seq. Results JEB OTs with papillary fibroblasts showed increased epidermal growth, invagination of basal layer and deregulated differentiation compared with reticular fibroblasts. RNA-Seq analysis revealed altered activity in key signalling including immune, cytokine, gap junction, mitogen-activated protein kinase and remodelling of keratinization and ECM proteins, particularly in JEB OT cultured with papillary fibroblast. JEB OTs and cells also demonstrated variation in cell proliferation and activation of transforming growth factor-β1 and immune cell signalling. Currently, we are targeting identified signalling pathways in our two-dimensional and 3D cell culture models and identifying druggable targets, before testing selected candidates in our JEB mouse disease model for further preclinical validation. Conclusions By dissecting the pathological signalling interactions between keratinocytes and distinct fibroblast subpopulations, our goal is to identify new therapeutic target to improve the skin health and regeneration in patients with JEB.

O19 Improvement of a junctional epidermolysis bullosa mouse model

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is the most severe of the epidermolysis bullosa (EB) disorders and is caused by mutations in genes encoding the skin basement membrane proteins laminin 332, type XVII collagen or integrin α6β4. Affected individuals experience blistering from birth leading to scarring, granulation tissue and susceptibility to infection. In JEB, generalized severe, there is 50% mortality in the first 2 years of life, often owing to failure to thrive or overwhelming infection. A number of animal models for JEB exist; however, because of severe blistering on the entire surface of the skin, these mice die soon after birth, reducing their experimental utility. In 2017, the first adult inducible knockout mouse model of JEB was published. In this mouse model, the disruption of the Lama3 gene is specifically induced in epidermal basal keratinocytes after birth. These mice remain viable, which allows us to study the longer-term consequences of the loss of laminin α3. However, the mice did not develop the severe phenotype observed in patients with JEB. Methods We have optimized this previously published methodology of a conditional knockout JEB mouse model (Lama3flx/flxK14CreERT) to now include more regular tamoxifen injections, which Results in a more controlled and severe phenotype of JEB. Results Widespread skin blistering develops after 12 weeks of tamoxifen treatment with an impaired skin barrier (transepidermal water loss) and a loss of epidermal lipids. Immunofluorescent staining was conducted to assess skin differentiation (K14, transglutaminase 1, loricrin), proliferation (Ki67), basement membrane proteins (laminin α3, laminin β3, laminin γ2, collagen VII) and immune markers (F4/80, Ly6-G/C, CD3, CD79). Conclusions These mice recapitulate all key features of human JEB with loss of laminin α3 at the basement membrane, epidermal thickening and visible blisters. This allows us to delve deeper into the functional and downstream pathways affected by the loss of laminin α3 and discover why the prognosis of patients with JEB is so severe.

O08 Exploring the effect of skin ageing-related basement membrane collagen loss on nuclear mechanics

Abstract Introduction and aims Skin ageing is a universal process that can increase susceptibility to inflammation and disease. Basement membrane collagens (BMCs) are reduced during ageing and the consequences are poorly understood. BMC sensing of mechanical stress, vital for skin homeostasis, elicits signalling sensed via cytoskeletal actin filaments to the linker of nucleoskeleton and cytoskeleton (LINC) complex (composed of nesprin and sun proteins), nuclear lamina and lamina-associated chromatin. Our hypothesis is that BMC loss during ageing leads to a deformation of the nucleocytoskeleton and disrupts chromatin organization in keratinocytes, leading to impairment of epidermal differentiation and inability to withstand mechanical stress. Methods To explore the role of BMC in mechanical stress response, stable lentiviral short hairpin (sh)RNA knockdowns of Col7 (shCol7) and Col17 (shCol17) were generated in immortalized N/TERT keratinocytes. Western blot (WB) validated knockdowns were compared with shC nontargeting control. IncuCyte assays showed shCol17 had a hyperproliferative phenotype compared with shC. Sun2 was reduced in shCol17 (P < 0.001) compared with shC (n = 5 experimental replicates). The mechanical stretch effect on cells on a flexible membrane was studied using immunofluorescence and WB analysis. Images taken from the IN Cell Analyzer confocal microscope were analysed to detect changes in protein expression and localization. Nuclear and cytoplasmic fractions made before and after stretch were analysed by WB. Results We observed collagen-specific responses to stretch-induced stress with changes to actin, components of the nuclear membrane, and LINC complex proteins. Additionally, work on investigating chromatin changes with BMC knockdown using Cleavage Under Targets & Tagmentation and Cleavage Under Targeted Accessible Chromatin assays is ongoing. Conclusions Our data show an altered nucleocytoskeleton in the context of BMC loss and mechanotransduction in skin. These data could provide novel insights into epidermal regulation by BMC in ageing.

O10 Tuneable Caf1 protein polymers as an innovative and scalable solution for regenerative wound care

Abstract Introduction and aims Wound healing is a complex process essential for tissue restoration, where the extracellular matrix (ECM) plays a crucial role by providing structural and biochemical support to cells. Laminin 332, a key ECM component, is a promising target for protein-based therapeutics in view of its pathogenic role in junctional epidermolysis bullosa (JEB). Bioengineered capsular antigen F1 protein (Caf1) has emerged as a potential candidate molecule. We have developed the technology to enable bacteria to produce Caf1 polymers with multiple tailored bioactive motifs. Additionally, the unique properties of Caf1 minimize the potential for a host immune response. In this study, we engineered Caf1 proteins to express specific synthetic basement membrane components related to laminin 332 with the aim of restoring ECM equilibrium and achieving effective wound healing. Methods To evaluate Caf1 construct effects on human epidermal keratinocytes (HEKs) (CCD 1106) and human dermal fibroblasts (HDFs), scratch assays were performed under different treatment with Caf constructs. The Incucyte® ZOOM system (Sartorius AG, Göttingen, Germany) automated scratch wound and Live-Cell Analysis System screened constructs enhancing wound healing, capturing images every 2 h. Analysis involved quantifying the area under the curve of wound confluence (%) over 72 h to assess wound closure. Results The migration of HDFs was enhanced by 10.4% ± 0.95% (P < 0.05 Anova, n = 9 independent experiments) when there was interaction with Caf1-CTB4 (PMQKMRGDVFSP). The migration of CCDs was enhanced by 12.7% ± 1.03% when interacting with Caf1-J3 (KNSFMALYLSKGR) and by 14.4% ± 1.11% when interacting with Caf1-J4 (TALIRATYGEYSTGYI) (P < 0.05 Anova; n = 3 independent experiments). Combining Caf1-J3 and Caf1-J4 synergistically boosts CCD migration by 19.1% ± 1.42% (P < 0.05 Anova; n = 3 independent experiments), yielding better Results compared with individual treatments. Conclusions Caf1 exhibits promise as a platform for motif delivery, and the incorporation of cell migration motifs into Caf1 holds the potential to enhance wound healing.

The NM_033380.2 transcript of the COL4A5 gene contains a variable splice site c.4822–10T>C, which has been identified as a causative factor for Alport syndrome

Alport Syndrome (AS) is a genetic kidney disorder characterized by progressive hearing loss and atypical eye symptoms, resulting in a poor prognosis and lack of effective targeted therapy. The primary mode of inheritance is X-linked dominant (XLAS) due to variants in the COL4A5 gene. This study revealed a previously unidentified alternative form of the COL4A5 gene, namely, the c.4822–10T>C variant, which was confirmed through in vitro experiments. To investigate the impact of a splicing variant on COL4A5 mRNA production, an in vitro minigene splicing assay was utilized. Additionally, molecular dynamics was employed to predict the ability of α5(IV) to form a triple helix. Results from the experiment revealed that the wild-type (WT) plasmid produced two distinct mRNA products simultaneously. Sequence analysis using the BLAST database revealed a 173-bp deletion in the mRNA sequence of the first product, indicating a potential similarity to the XM_016942897.2 transcript of Pan troglodytes. The second mRNA product of the WT plasmid contained the full sequence of exons 51, 52, and 53, as anticipated. Conversely, the mutant (MT) plasmid generated a single mRNA product with a 173-bp deletion in exon 52, leading to the identification of the mature mRNA expression as NM_033380.2: COL4A5: c.4822_4994del. In the context of nonsense-mediated mRNA decay (NMD), the deletion c.4822_4994 results in the production of a truncated protein, p.His1608*, that terminates prematurely. This truncated protein may disrupt the secondary structure of α5(IV) and potentially cause an abnormal conformation of α345(IV). This study examines the relationship between the variable splicing pattern in the NM_033380.2 transcript of the COL4A5 gene in XLAS patients and the presence of the COL4A5 gene splice variant c.4822–10T>C. Our findings indicate that the c.4822–10T>C splice variant leads to activation of nonsense-mediated mRNA degradation (NMD) and reduced COL4A5 mRNA expression, resulting in inadequate synthesis of the corresponding proteins. This aligns with the patient’s immunofluorescence results showing negative α5(IV) chain presence at the glomerular basement membrane, bursa, and tubular basement membrane, confirming the pathogenic nature of c.4822–10T>C.

Development of a novel zebrafish model of diabetic nephropathy.

Diabetic nephropathy (DN) is a substantial healthcare challenge as a complication of diabetes, owing to the high risk of morbidity and mortality involved. Although significant progress has been made in understanding the pathogenesis of DN, more efficient models are required to develop new therapeutics. Here, we created a DN model in zebrafish by crossing diabetic Tg(acta1:dnIGF1R-EGFP) and proteinuria-tracing Tg(l-fabp::VDBP-GFP) lines, named zMIR/VDBP. Overfed adult zMIR/VDBP fish developed severe hyperglycemia and proteinuria, which were not observed in wild-type zebrafish. Renal histopathology revealed human DN-like characteristics, such as glomerular basement membrane thickening, foot process effacement, and glomerular sclerosis. Glomerular dysfunction is restored upon calorie restriction. RNA sequencing (RNA-seq) analysis demonstrated that zebrafish DN kidneys exhibited transcriptional patterns similar to human DN pathogenesis. Notably, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway was activated, a phenomenon observed in the early phase of human DN. In addition, metformin improved hyperglycemia and proteinuria in DN zebrafish by modulating AKT phosphorylation. Our results indicated that zMIR/VDBP fish are suitable for elucidating the mechanisms underlying human DN and could be a powerful tool for therapeutic discovery.

Comparison of serum SDMA and creatinine as a biomarker for the detection of meloxicam-induced kidney injury in cats

IntroductionSerum symmetric dimethylarginine (SDMA) and creatinine are commonly used biomarkers of renal function in cats. We hypothesize that the serum analytes creatinine and SDMA are equally effective at detecting impaired renal function caused by meloxicam-induced renal injury in cats. Our primary objective was to compare serum concentrations of SDMA and creatinine in cats before, during, and after induction of renal injury from repeated dosages of meloxicam in the context of a small pilot study.MethodsThis follow-up study results from data collected in a well-controlled study that included 12 healthy female adult purpose-bred cats. Cats in the treatment group received meloxicam 0.3 mg/kg subcutaneously (SC) every 24 h for 31 days. Cats in the control group received saline (0.1 mL SC). Renal injury was defined as the presence of tubular damage, basement membrane damage, and/or interstitial inflammation in histological sections of kidney tissue. Serum creatinine and SDMA concentration were measured every 4 days.ResultsIn the control group, no cats developed renal azotemia. In the treatment group, four out of six cats developed elevated serum creatinine and histopathological evidence of renal injury. Three of these cats developed an elevation in serum SDMA. The time to the development of renal azotemia using serum creatinine or SDMA was not significantly different (p > 0.05).DiscussionIn this pilot study, there was no evidence that serum SDMA was superior to serum creatinine at detecting impaired renal function caused by meloxicam-induced renal injury in cats.

Identification of seven variants in the col4a1 gene that alter RNA splicing by minigene assay.

Type IV collagen is an integral component of basement membranes. Mutations in COL4A1, one of the key genes encoding Type IV collagen, can result in a variety of diseases. It is clear that a significant proportion of mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. Here, we analyzed exonic mutations and intronic mutations described in the COL4A1 gene using bioinformatics programs and identified candidate mutations that may alter the normal splicing pattern through a minigene system. We identified seven variants that induce splicing alterations by disrupting normal splice sites, creating new ones, or altering splice regulatory elements. These mutations are predicted to impact protein function. Our results help in the correct molecular characterization of variants in COL4A1 and may help develop more personalized treatment options.

Associations of the Serum KL-6 with Severity and Prognosis in Patients with Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

As a biomarker of alveolar-capillary basement membrane injury, Krebs von den Lungen-6 (KL-6) is involved in the occurrence and development of pulmonary diseases. However, the role of the KL-6 in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) has yet to be elucidated. This prospective study was designed to clarify the associations of the serum KL-6 with the severity and prognosis in patients with AECOPD. This study enrolled 199 eligible AECOPD patients. Demographic data and clinical characteristics were recorded. Follow-up was tracked to evaluate acute exacerbation and death. The serum KL-6 concentration was measured via an enzyme-linked immunosorbent assay. Serum KL-6 level at admission was higher in AECOPD patients than in control subjects. The serum KL-6 concentration gradually elevated with increasing severity of AECOPD. Pearson and Spearman analyses revealed that the serum KL-6 concentration was positively correlated with the severity score, monocyte count and concentrations of C-reactive protein, interleukin-6, uric acid, and lactate dehydrogenase in AECOPD patients during hospitalization. A statistical analysis of long-term follow-up data showed that elevated KL-6 level at admission was associated with longer hospital stays, an increased risk of future frequent acute exacerbations, and increased severity of exacerbation in COPD patients. Serum KL-6 level at admission is positively correlated with increased disease severity, prolonged hospital stay and increased risk of future acute exacerbations in COPD patients. There are positive dose-response associations of elevated serum KL-6 with severity and poor prognosis in COPD patients. The serum KL-6 concentration could be a novel diagnostic and prognostic biomarker in AECOPD patients.

Elucidating the Functional Mechanism of PTK7 in Cancer Development through Spatial Assembly Analysis Using Super Resolution Imaging.

Protein tyrosine kinase-7 (PTK7) has been reported as a vital participant in the Wnt signaling pathway, influencing tumorigenesis and metastasis. However, their specific roles in the mechanisms underlying cancer development and progression remain elusive. Here, using direct stochastic optical reconstruction microscopy (dSTORM) with aptamer-probe labeling, we first revealed that a weakening clustering distribution of PTK7 on the basal membranes happened as cellular migration increased during cancer progression. This correspondence was further supported by a diminished aggregated state of PTK7 caused by direct enhancement of cell migration. By comparing the alterations in PTK7 distribution with activation or inhibition of specific Wnt signaling pathway, we speculated that PTK7 could modulate cell migration by participating in the interplay between canonical Wnt (in MCF7 cells) and noncanonical Wnt signals (in MDA-MB-231 cells). Furthermore, we discovered that the spatial distribution morphology of PTK7 was also subject to the hydrolysis ability and activation state of the related hydrolase Matrix metallopeptidase14 (MMP14). This function-related specific assembly of PTK7 reveals a clear relationship between PTK7 and cancer. Meanwhile, potential molecular interactions predicted by the apparent assembly morphology can promote a deep understanding of the functional mechanism of PTK7 in cancer progress.

Developing a gel-col heterogeneous network hydrogel scaffold for tissue-engineered skin with enhanced basement membrane formation

Tissue-engineered skin is constructed utilizing biocompatible matrix materials as scaffolds, and incorporating human epidermal keratinocytes (HEKs), dermal fibroblasts (HDFs), and bioactive molecules, addressing clinical issues such as skin graft rejection and donor scarcity. Conventional fabrication methods of tissue-engineered skin typically rely on homogeneous single-network scaffolds, which may not adequately address the distinct requirements of epidermal and dermal cells or the critical formation of the basement membrane, potentially impacting the development and functional integrity of the skin tissue. Cell culture studies employing GelMA hydrogels of varied concentrations have elucidated that epidermal cells exhibit increased viability on scaffolds with higher concentrations (15 %), whereas fibroblasts show increased proliferation within scaffolds of lower concentrations (5 %). Consequently, we have engineered a GelMA-collagen (Gel-Col) hydrogel scaffold with a heterogeneous network of dense and sparse matrices, conducive to the growth of both fibroblasts and epidermal cells. Culturing epidermal cells on this innovative scaffold, coupled with histological section staining, RT-PCR, and Western blot analyses, has demonstrated that the scaffold significantly enhances cellular adhesion and promotes the development of the epidermal basement membrane compared to single-network hydrogels. Mouse wound transplantation experiments have further confirmed the scaffold’s effectiveness in accelerating wound repair, supporting the development of the basement membrane, and enhancing epidermal regeneration. This research provides a reference for constructing tissue-engineered skin that more precisely emulates the intricate architecture and cellular dynamics of natural skin, propelling the advancement of tissue-engineered skin grafts in the clinical treatment of skin injuries.

Morphological evaluation of adult domestic cat testicular biopsy after vitrification.

Testicular biopsies (9 mm3) from domestic cats (n = 10) submitted to orchiectomy were submitted to equilibrium vitrification in the presence of ethylene glycol (EG) alone or combined with dimethylsulfoxide (DMSO) as intracellular cryoprotectants, and sucrose or trehalose as extracellular cryoprotectants. The samples were vitrified with 40% EG or 20% EG + 20% DMSO, plus 0.1 M or 0.5 M of sucrose or trehalose. The study was divided into Step 1 and Step 2. In Step 1, intratubular cells (spermatogonia, spermatids, spermatocytes, and Sertoli cells) were quantified and classified as intact or degenerated (pyknotic and/or vacuolated cells). Cryodamage of seminiferous cords was determined by spermatogonia and Sertoli cell scoring of nuclei alterations, tubular basement membrane detachment, epithelium shrinkage, and tubular measures (total area, epithelium area, larger and smaller diameter, and height of the epithelium). In Step 2, Hoechst 33342 stain and propidium iodide (PI) fluorescent stain were used to assess the cell viability of the four best experimental groups in Step 1. The effect of treatments on all analyses was accessed using analysis of variance (ANOVA), and Fisher's post hoc test at P < 0.05 significance was considered. In Step 1, the mean percentage of spermatogonia and Sertoli cells morphological integrity did not show a difference when using both sugars at different concentrations, but their morphology was more affected when DMSO was used. EG use associated with 0.1 M of sucrose or trehalose positively affected spermatocyte and spermatid morphology, respectively. The larger diameter and epithelium height of seminiferous tubules were increased using DMSO plus 0.5 M sucrose and DMSO plus 0.1 M trehalose. The changes in spermatogonial/Sertoli nucleoli visualization were best scored in the EG groups, while the nuclei condensation was lower with sucrose. The basement membrane was satisfactorily preserved with 0.1 M sucrose. In Step 2, the percentage of cell viability was higher when EG plus 0.1 M sucrose was used. Therefore, DMSO's negative effect on the vitrification of testicular biopsies of adult domestic cats was evident. The EG plus 0.1 M of sucrose or trehalose associations are the most suitable CPAs to preserve the testicular histology structure of adult domestic cats in vitrification.

The effect of antiresorptive therapy on the prevalence and severity of oral lichen planus: a retrospective study

BackgroundAntiresorptive therapy (AR) disrupts osseous homeostasis and can induce direct irritation over the gastrointestinal mucosa; however, its possible erosive effects on the oral epithelium have not been totally described. Among the most frequent oral erosive lesions, oral lichen planus (OLP) frequently presents as painful mucosal ulcerations, arising from basal membrane inflammatory damage. Thus, the aim of this retrospective study was to describe the association between AR and the incidence of OLP.MethodsThis case-control study included data from 148 patients (17 patients undergoing AR therapy (AR group) / 131 without AR therapy (Control group)). Each patient record was systematically processed and the association between AR drugs and OLP clinical characteristics within both groups was assessed.ResultsThe erosive form of OLP was significantly more frequent in the AR group than in the Control group (p = 0.029). Indeed, the AR treatment using alendronic acid (41.2%) was the most frequently reported. Additionally, the erosive form of OLP showed the strongest association with pain and burning sensation among the OLP types (p < 0.050). However, disease worsening and AR consumption were not significantly associated (p = 0.150).ConclusionsPatients under AR therapy show more clinical symptoms associated to the erosive type of OLP. Regardless of the AR therapy, the erosive type of OLP is associated with more severe symptoms.

Rediscovery of the implication of albuminuria in heart failure: emerging classic index for cardiorenal interaction.

The development of new drugs and device therapies has led to remarkable advancements in heart failure (HF) treatment in the past couple of decades. However, it becomes increasingly evident that guideline-directed medical therapy cannot be one-size-fits-all across a wide range of ejection fractions (EFs) and various aetiologies. Therefore, classifications solely relying on EF and natriuretic peptide make optimization of treatment challenging, and there is a growing exploration of new indicators that enable efficient risk stratification of HF patients. Particularly when considering HF as a multi-organ interaction syndrome, the cardiorenal interaction plays a central role in its pathophysiology, and albuminuria has gained great prominence as its biomarker, independent from glomerular filtration rate. Albuminuria has been shown to exhibit a linear correlation with cardiovascular disease and HF prognosis in multiple epidemiological studies, ranging from normal (<30mg/g) to high levels (>300mg/g). However, on the other hand, it is only recently that the details of the pathological mechanisms that give rise to albuminuria have begun to be elucidated, including the efficient compaction/tightening of the glomerular basement membrane by podocytes and mesangial cells. Interestingly, renal disease, diabetes, and HF damage these components associated with albuminuria, and experimental models have demonstrated that recently developed HF drugs reduce albuminuria by ameliorating these pathological phenotypes. In this review, facing the rapid expansion of horizons in HF treatment, we aim to clarify the current understanding of the pathophysiology of albuminuria and explore the comprehensive understanding of albuminuria by examining the clinically established evidence to date, the pathophysiological mechanisms leading to its occurrence, and the outcomes of clinical studies utilizing various drug classes committed to specific pathological mechanisms to put albuminuria as a novel axis to depict the pathophysiology of HF.

Insertions of the striated muscles in the skin and mucosa: a histological study of fetuses and cadavers.

Striated muscle insertions into the skin and mucosa are present in the head, neck, and pelvic floor. We reexamined the histology of these tissues to elucidate their role in transmission of the force. We examined histological sections of 25 human fetuses (gestational ages of ~11-19 weeks and ~26-40 weeks) and 6 cadavers of elderly individuals. Facial muscle insertion or terminal almost always formed as an interdigitation with another muscle or as a circular arrangement in which muscle fiber insertions were sandwiched and mechanically supported by other muscle fibers (like an in-series muscle). Our examination of the face revealed some limited exceptions in which muscle fibers that approached the dermis were always in the nasalis and mentalis muscles, and often in the levator labii superioris alaeque nasi muscle. The buccinator muscle was consistently inserted into the basement membrane of the oral mucosa. Parts of the uvulae muscle in the soft palate and of the intrinsic vertical muscle of the tongue were likely to direct toward the mucosa. In contrast, the pelvic floor did not contain striated muscle fibers that were directed toward the skin or mucosa. Although 'cutaneous muscle' is a common term, the actual insertion of a muscle into the skin or mucosa seemed to be very rare. Instead, superficial muscle insertion often consisted of interdigitated muscle bundles that had different functional vectors. In this case, the terminal of one muscle bundle was sandwiched and fixed mechanically by other bundles.

Impact of temporal resolution in single particle tracking analysis

Temporal resolution is a key parameter in the observation of dynamic processes, as in the case of single molecules motions visualized in real time in two-dimensions by wide field (fluorescence) microscopy, but a systematic investigation of its effects in all the single particle tracking analysis steps is still lacking. Here we present tools to quantify its impact on the estimation of diffusivity and of its distribution using one of the most popular tracking software for biological applications on simulated data and movies. We found important shifts and different widths for diffusivity distributions, depending on the interplay of temporal sampling conditions with various parameters, such as simulated diffusivity, density of spots, signal-to-noise ratio, lengths of trajectories, and kind of boundaries in the simulation. We examined conditions starting from the ones of experiments on the fluorescently labelled receptor p75NTR, a relatively fast-diffusing membrane receptor (diffusivity around 0.5–1 µm2/s), visualized by TIRF microscopy on the basal membrane of living cells. From the analysis of the simulations, we identified the best conditions in cases similar to these ones; considering also the experiments, we could confirm a range of values of temporal resolution suitable for obtaining reliable diffusivity results. The procedure we present can be exploited in different single particle/molecule tracking applications to find an optimal temporal resolution.