Collagen Fibril Organization Research Articles

Phlpp1 alters the murine chondrocyte phospho-proteome during endochondral bone formation

Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, and maturation via Akt, PKC, Raf1, S6k, and other intracellular signaling cascades. Germline deletion of Phlpp1 suppresses bone lengthening in growth plate chondrocytes. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification, and we define the molecular differences between Phlpp1 and Phlpp2 in chondrocytes. Phlpp2−/− mice were phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. Deletion of Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT cells. By contrast, Phlpp1/2−/− (double knockout) mice resembled Phlpp1−/− mice phenotypically and molecularly, as the chondrocyte phospho-proteomes of Phlpp1−/− and Phlpp1/2−/− chondrocytes had similarities and were significantly different from WT and Phlpp2−/− chondrocyte phospho-proteomes. Data integration via multiparametric analysis showed that the transcriptome explained less variation in the data as a result of Phlpp1 or Phlpp2 deletion than proteome or phospho-proteome. Alterations in cell proliferation, collagen fibril organization, and Pdpk1 and Pak1/2 signaling pathways were identified in chondrocytes lacking Phlpp1, while cell cycle processes and Akt1 and Aurka signaling pathways were altered in chondrocytes lacking Phlpp2. These data demonstrate that Phlpp1, and to a lesser extent Phlpp2, regulate multiple and complex signaling cascades across the chondrocyte transcriptome, proteome, and phospho-proteome and that multi-omic data integration can reveal novel putative kinase targets that regulate endochondral ossification.

Tissue-engineered Bicipital Autologous Tendon Patch Enhances Massive Rotator Cuff Defect Repair in a Rabbit Infraspinatus Tendon Defect Model.

Massive rotator cuff defects represent an important source of shoulder pain and functional debilitation, substantially diminishing patients' quality of life. The primary treatment of massive rotator cuff defects includes complete or partial repair and patch augmentation. However, because of the tendon's limited regenerative ability, the tendon retear risk after rotator cuff defect repair is still high. Thus, a new therapy is needed to promote tendon regeneration for repair of massive rotator cuff defects. Using an in vitro analysis, we first asked: (1) What is the biocompatibility and collagen synthesis ability of fibrin glue, and what is the cell growth of tissue-engineered bicipital tendon patches, which is comprised of fibrin glue and biceps tendon tissue particles? Then, using an in vivo animal model of full-thickness defects in the infraspinatus tendon in New Zealand White rabbits, we asked: (2) What is the potential of the tissue-engineered bicipital autologous tendon patch to promote tendon regeneration? In vitro experiments were conducted to assess the survival, proliferation, and collagen synthesis ability of tendon stem/progenitor cells cultured in fibrin glue. This was achieved through an assay of live/dead cell viability, cell counting kit-8 (CCK-8) assay, and Sirius red staining, respectively. The in vivo animal study was conducted using 8- to 12-week-old New Zealand White rabbits. The left shoulder of each animal was operated on, with equal numbers of males and females. There were 12 rabbits in the control group and 15 rabbits each in the gel and patch groups. Six rabbits were allocated to each of the three groups at the 1- and 3-month time points and three rabbits each were in the gel and patch groups at 2-month time point. Through an infraspinatus tendon defect model, the effectiveness of tissue-engineered bicipital autologous tendon patches (patch group) in tendon repair was assessed compared with untreated (control group) and fibrin glue (gel group) treatments in vivo. This assessment included histological evaluation of repaired tissue morphology, transmission electron microscopy (TEM) evaluation of regenerated collagen fibrils, and RNA sequencing to explore the potential mechanisms of tissue-engineered bicipital autologous tendon patches in tendon regeneration. In vitro experiments demonstrated that fibrin glue enhanced the collagen synthesis ability of tendon stem/progenitor cells (0.38 ± 0.02) compared with standard cell culture alone (0.27 ± 0.02, mean difference 0.11 [95% CI 0.07 to 0.14]; p < 0.001). With prolonged cultivation, the cell growth area of tissue-engineered bicipital tendon patches showed a notable increase after culturing for 14 days (78.13% ± 3.68%) compared with 11 days (13.05% ± 8.78%, mean difference -65.08% [95% CI -77.99% to -52.15%]; p＜0.001), 7 days (2.67% ± 2.62%, mean difference -75.46% [95% CI -88.37% to -62.53%]; p＜0.001), and 1 day (0.33% ± 0.30%, mean difference -77.80% [95% CI -90.71% to -64.87%]; p＜0.001). At 3 months after transplantation, in vivo experiments revealed that compared with the control and gel groups, the patch group displayed improved repair outcomes. This was evidenced by better histological scores in the patch group (3.83 ± 2.01) compared with the gel group (10.67 ± 0.58, mean difference 6.84 [95% CI 3.67 to 10.00]; p = 0.001) and control group (10.75 ± 0.66, mean difference 6.92 [95% CI 3.75 to 10.08]; p = 0.001), and by regular alignment and larger diameters of newly formed collagen fibrils in the patch group (77.52 ± 44.41 nm) compared with the control group (53.34 ± 6.64 nm, mean difference 24.18 [95% CI 22.24 to 26.11]; p < 0.001). RNA sequencing analysis revealed that a tissue-engineered bicipital autologous tendon patch facilitated tendon regeneration by modulating the immune response, promoting collagen fibril organization, and alleviating vasoconstriction. This animal study demonstrates that the tissue-engineered bicipital autologous tendon patch effectively modulates an immune response and collagen fibril organization, leading to the promotion of tendon regeneration. The tissue-engineered bicipital autologous tendon patch represents a promising strategy for tendon regeneration, offering potential in the repair of massive rotator cuff defects during clinical rotator cuff surgery. Subsequent research could focus on large animal experiments using a tissue-engineered bicipital autologous tendon patch to explore their feasibility for clinical translation.

Transcriptomic Differences by RNA Sequencing for Evaluation of New Method for Long-Time In Vitro Culture of Cryopreserved Testicular Tissue for Oncologic Patients.

Earlier studies have established that culturing human ovarian tissue in a 3D system with a small amount of soluble Matrigel (a basement membrane protein) for 7 days in vitro increased gene fusion and alternative splicing events, cellular functions, and potentially impacted gene expression. However, this method was not suitable for in vitro culture of human testicular tissue. To test a new method for long-time in vitro culture of testicular fragments, thawed with two different regimes, with evaluation of transcriptomic differences by RNA sequencing. Testicular tissue samples were collected, cryopreserved (frozen and thawed), and evaluated immediately after thawing and following one week of in vitro culture. Before in vitro culture, tissue fragments were encapsulated in fibrin. Four experimental groups were formed. Group 1: tissue quickly thawed (in boiling water at 100 °C) and immediately evaluated. Group 2: tissue quickly thawed (in boiling water at 100 °C) and evaluated after one week of in vitro culture. Group 3: tissue slowly thawed (by a physiological temperature 37 °C) and immediately evaluated. Group 4: tissue slowly thawed (by a physiological temperature 37 °C) and evaluated after one week of in vitro culture. There are the fewest differentially expressed genes in the comparison between Group 2 and Group 4. In this comparison, significantly up-regulated genes included C4B_2, LOC107987373, and GJA4, while significantly down-regulated genes included SULT1A4, FBLN2, and CCN2. Differential genes in cells of Group 2 were mainly enriched in KEGG: regulation of actin cytoskeleton, lysosome, proteoglycans in cancer, TGF-beta signaling pathway, focal adhesion, and endocytosis. These Group 2- genes were mainly enriched in GO: spermatogenesis, cilium movement, collagen fibril organization, cell differentiation, meiotic cell cycle, and flagellated spermatozoa motility. Encapsulation of testicular tissue in fibrin and long-time in vitro culture with constant stirring in a large volume of culture medium can reduce the impact of thawing methods on cryopreserved testicular tissue.

Alternative splicing and intron retention: Their profiles and roles in cutaneous fibrosis of systemic sclerosis

BackgroundAlternative splicing (AS) and intron retention (IR) implicated in multiple pathophysiological processes, have rarely been reported in systemic sclerosis (SSc). MethodsWe integrated bulk RNA-seq and 4D label-free mass spectrometry to perform a multi-omics analysis of AS and IR in SSc skin tissue and fibroblasts. RMATS and iREAD were used to identify AS and IR, which were validated by real-time PCR. Spearman correlation and the LASSO method were employed to assess correlations among clinical features, introns, splicing factors (regulators of AS) and proteins. FindingsAS profiles showed distinct alterations in SSc skin tissue, with the most pronounced changes occurring in IR. AS and IR were associated with total modified Rodnan skin score (mRSS) and local skin score. Upon TGF-β stimulation, fibroblasts exhibited significant alterations in IR profiles, affecting genes related to fibroblast proliferation and collagen fibril organization. A comprehensive integrated analysis of introns, exons, and proteome profiles revealed that IR exerted a negative impact on protein expression, with certain changes being under intronic control. RT-PCR confirmed the presence of intron and exon-derived sequences of CTTN, OGA, MED16 and PHYKPL. Additionally, notable changes were observed in the regulatory network of splicing factors in SSc skin tissues. These factors are also involved in fibrosis pathways and correlated with clinical features. ConclusionTotally, abnormal AS, IR profiles and splicing factors were identified in SSc, altered IRs and splicing factors participated in fibrosis-related pathways. IR exerted a negative impact on protein expression in TGF-β-stimulated fibroblasts. Clarification of the IR mechanisms will provide new insights into the pathophysiology of SSc.

The involvement of Slc7a11/xCT related iron accumulation on acupuncture intervention in 6-OHDA-induced Parkinson rat

BackgroundAcupuncture plays a critical role for the treatment of patients with Parkinson's disease (PD), which is characterized by ferroptosis of dopaminergic neurons. It has been found that acupuncture can reduce iron accumulation. However, the mechanism of acupuncture therapy against PD by inhibiting ferroptosis remains unclear. ObjectiveTo investigate the potential mechanism of acupuncture on alleviating ferroptosis in 6-OHDA-induced PD rats. MethodsMale Sprague-Dawley rats were randomly divided into sham group, acupuncture group, and PD model group, with the PD model induced by 6-OHDA. The motor function of the rats was tested by the rotation test and suspension test, respectively. Immunohistochemistry and iron staining were performed to analyze nerve damage, and the effect of acupuncture on the accumulation of iron in the substantia nigra (SN). RNA-seq analysis in the SN of rats were performed to discover ferroptosis-related genes. Differentially expressed genes (DEGs) resulted from the acupuncture treatment were identified and were used for Gene Ontology (GO) function, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A protein–protein interaction (PPI) network was constructed, and alternative splicing (AS) analysis of the transcriptome was performed. Western blot was used to detect the protein level change of Slc7a11/xCT. ResultsRotation test and suspension test indicated a positive effect of acupuncture on body weight gain and limb function. Perl's iron staining and ELISA test detected 6-OHDA induced iron accumulation in the SN and nerve damage, which was mitigated by acupuncture treatment. Acupuncture treatment brought about the differential expression of a total of 1088 genes in the SN of rats. GO and KEGG enrichment revealed that such DEGs were mainly associated with several key biological process (BP), cellular component (CC) and molecular function (MF) terms. These terms included collagen fibril organization, extracellular matrix organization, collagen trimer, extracellular matrix components, chemokine receptor binding and cargo receptor activity, etc. Additionally, the DEGs were found to be involved in pathways such as complement and coagulation. The PPI network of DEGs suggested a critical role of Slc7a11, which was further supported by an up-regulated Slc7a11/xCT protein level in the substantia nigra (SN) tissue of rats treated with acupuncture compared to their PD model counterparts (P = 0.045). ConclusionAcupuncture may prevent ferroptosis through Slc7a11/xCT as one of the targets in 6-OHDA-induced PD model and thereby could be promising for PD treatment.

Whole-exome sequencing screening for candidate genes and variants associated with primary sporadic keratoconus in Chinese patients

The pathogenesis of keratoconus (KC) is complex, and genetic factors play an important role. The purpose of this study was to screen and analyse candidate genes and variants in Chinese patients with primary sporadic KC. Whole-exome sequencing (WES) was performed to identify candidate genes and variants in 105 unrelated Chinese patients with primary sporadic KC. Through a series of screening processes, 54 candidate variants in 26 KC candidate genes were identified in 53 KC patients (53/105, 50.5%). These 54 candidate variants included 10 previously identified variants in 9 KC candidate genes and 44 novel variants in 20 KC candidate genes. The previously identified variants occurred in 25.7% (27/105) of patients. Of these, 4 variants (COL6A5, c.5014T > G; CAST, c.1814G > A; ZNF469, c.946G > A; and MPDZ, c.3836A > G) were identified for the first time in Chinese KC patients. The novel variants occurred in 33.3% (35/105) of patients. Of the 26 screened KC candidate genes, 11 KC candidate genes (CAT, COL12A1, FLG, HKDC1, HSPG2, PLOD1, ITGA2, TFAP2B, USH2A, WNT10A, and COL6A5) were found to be potentially pathogenic in Chinese KC patients for the first time. Gene Ontology (GO) biological process (BP) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the 26 KC candidate genes using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The results showed that the KC candidate genes were significantly enriched in biological processes such as collagen fibril organization and extracellular matrix (ECM) organization and in ECM-receptor interaction and protein digestion and absorption pathways. The results further expand the spectrum of KC candidate variants and provide a basis for further KC gene studies.

Effects of mineralization on the hierarchical organization of collagen-a synchrotron X-ray scattering and polarized second harmonic generation study.

The process of mineralization fundamentally alters collagenous tissue biomechanics. While the structure and organization of mineral particles have been widely studied, the impact of mineralization on collagen matrix structure, particularly at the molecular scale, requires further investigation. In this study, synchrotron X-ray scattering (XRD) and polarization-resolved second harmonic generation microscopy (pSHG) were used to study normally mineralizing turkey leg tendon in tissue zones representing different stages of mineralization. XRD data demonstrated statistically significant differences in collagen D-period, intermolecular spacing, fibril and molecular dispersion and relative supramolecular twists between non-mineralizing, early mineralizing and late mineralizing zones. pSHG analysis of the same tendon zones showed the degree of collagen fibril organization was significantly greater in early and late mineralizing zones compared to non-mineralizing zones. The combination of XRD and pSHG data provide new insights into hierarchical collagen-mineral interactions, notably concerning possible cleavage of intra- or interfibrillar bonds, occlusion and reorganization of collagen by mineral with time. The complementary application of XRD and fast, label-free and non-destructive pSHG optical measurements presents a pathway for future investigations into the dynamics of molecular scale changes in collagen in the presence of increasing mineral deposition.

Identification of Potential lncRNA-miRNA-mRNA Regulatory Network Contributing to Arrhythmogenic Right Ventricular Cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) can lead to sudden cardiac death and life-threatening heart failure. Due to its high fatality rate and limited therapies, the pathogenesis and diagnosis biomarker of ARVC needs to be explored urgently. This study aimed to explore the lncRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) network in ARVC. The mRNA and lncRNA expression datasets obtained from the Gene Expression Omnibus (GEO) database were used to analyze differentially expressed mRNA (DEM) and lncRNA (DElnc) between ARVC and non-failing controls. Differentially expressed miRNAs (DEmiRs) were obtained from the previous profiling work. Using starBase to predict targets of DEmiRs and intersecting with DEM and DElnc, a ceRNA network of lncRNA-miRNA-mRNA was constructed. The DEM and DElnc were validated by real-time quantitative PCR in human heart tissue. Protein-protein interaction network and weighted gene co-expression network analyses were used to identify hub genes. A logistic regression model for ARVC diagnostic prediction was established with the hub genes and their ceRNA pairs in the network. A total of 448 DEMs (282 upregulated and 166 downregulated) were identified, mainly enriched in extracellular matrix and fibrosis-related GO terms and KEGG pathways, such as extracellular matrix organization and collagen fibril organization. Four mRNAs and two lncRNAs, including COL1A1, COL5A1, FBN1, BGN, XIST, and LINC00173 identified through the ceRNA network, were validated by real-time quantitative PCR in human heart tissue and used to construct a logistic regression model. Good ARVC diagnostic prediction performance for the model was shown in both the training set and the validation set. The potential lncRNA-miRNA-mRNA regulatory network and logistic regression model established in our study may provide promising diagnostic methods for ARVC.

Transcriptome and proteome revealed the differences in 3 colors of earlobe in Jiangshan Black-bone chicken

The earlobe is a featherless, exposed thickening located beneath the ear canal of chickens, which plays a visual signaling role in age, performance, mental vitality, reproduction, and other aspects. However, despite its importance, there have been few studies on the color differences and formation mechanisms of chicken earlobes, particularly the structurally blue earlobes characteristic of the Jiangshan black-bone chicken. In this study, we explored the physiological mechanisms that may influence the formation of differently colored earlobes using 3 types of earlobes from Jiangshan black-bone chickens: light peacock green (Green group), dark peacock green (Blue group), and dark reddish purple (Black group). All 3 earlobe colors exhibited positive melanin Masson-Fontana staining, and the thickness of collagen fibers in the dermis decreased in the order of Green, Blue, and Black groups. A total of 1,953 differentially expressed genes (DEGs) were detected in the 3 earlobes through mRNA sequencing, among which the GO term "collagen trimer" was significantly enriched in DEGs between groups. Additionally, 716 differentially expressed proteins (DEPs) were identified in the 3 earlobes using 4D-DIA proteomics, with the term "collagen fibril organization" being significantly enriched in DEPs between the Green and Black groups. Integrated analysis of transcriptome and proteome data revealed that 12 DEGs and DEPs were commonly differentially expressed between the Green and Black groups, including the gene LUM (corneal keratan sulfate proteoglycan), which was significantly enriched in the "collagen fibril organization" GO term. In conclusion, our study suggests that LUM plays a crucial role in the formation of peacock green earlobes in Jiangshan black-bone chickens. The high level of LUM in peacock green (Green and Blue groups) may affect collagen nanostructures, leading to a stronger effect of melanin-supported dermal collagen on the production of non-iridescent structural colors through coherent scattering, resulting in a bright structural blue color in Jiangshan black-bone chickens. In contrast, the low expression of LUM in dark reddish purple (Black group) reduces the reflection of non-iridescent structural colors, making the earlobe color appear almost black, similar to melanin.

Abstract PO5-21-05: An Endocrine Resistant-Related Gene Signature Revealing the Tumor Microenvironment to Predict the Prognosis of Hormone Receptor-Positive Breast Cancer Patients

Abstract Key words: breast cancer; hormone receptor-positive; predictive model; prognosis; endocrine therapy; drug resistance Hormone receptor positive (HR+) breast cancer is the most common type of breast cancer, accounting for approximately 70% of all breast cancer, in whom endocrine therapy plays a vital role. However, the limited effectiveness of endocrine therapy due to intricate mechanisms of drug resistance poses a significant challenge in treating certain patients. Therefore, it is imperative to identify novel indicators of endocrine resistance and develop new models for predicting recurrence-free survival (RFS) in patients with HR+ breast cancer. In this study, we successfully established an endocrine resistance-related risk model based on multicenter RNA sequencing data. This model could represent tumor microenvironment characteristics and provide valuable insights into predicting the prognosis of HR+ breast cancer patients. A seven gene risk model reflecting endocrine therapy resistance in 208 early HR+ breast cancer patients from two centers was constructed by LASSO analysis and multivariate cox regression. Based on the median value of risk score, patients were divided into high-risk group and low-risk group. Compared with the low-risk group, the high-risk group exhibited a significantly worse prognosis, which showed consistent across two external validation cohorts (TCGA-BRCA and METABRIC cohort). Time dependent ROC curve showed excellent capacity of gene model in predicting 1,3 and 5-year RFS. The IC50 analysis revealed that patients in low-risk group exhibited greater sensitivity to endocrine drugs, such as tamoxifen, fulvestrant and palbociclib, which strongly suggested that the identified gene signature can effectively differentiate between endocrine-resistant and -sensitive status. Gene-set enrichment analysis revealed regulation of cell killing, positive regulation of inflammatory response and T cell mediated cytotoxicity were enriched in low risk group, suggesting immune activation and a higher immune infiltration status. Conversely, the high-risk group demonstrated associations with collagen metabolic process, extracellular matrix structural constituent and collagen fibril organization, which were the indicative of the location and function of cancer-associated fibroblasts (CAFs). As expected, ssGSEA results revealed elevated expression levels of immune cells (e.g., natural killer cells, tumor-infiltrating lymphocytes, CD8+ T cells) and immune functions (e.g., T cell co-stimulation, inflammation-promoting, cytolytic activity) in the low-risk group. However, three diverse acknowledged methods (TIDE, EPIC and MCP-counter) consistently identified CAFs as predominantly content in the high-risk groups. Furthermore, considering that CD8+ T cell is the primary cell type responsible for exerting cytotoxic effects, we observed a negative correlation between CD8+ T cells and CAFs. This suggests that CAFs may inhibit the infiltration of CD8+ T cells, thereby weakening their cytotoxic capabilities, leading to a shorter RFS in high-risk patient groups. In conclusion, a novel gene signature constructed with 7 endocrine resistance response-related genes can be used for prognostic prediction and reflecting the tumor microenvironment status in HR+ early breast cancer. The low-risk group exhibited enhanced anti-tumor immunity, resulting in a favorable prognosis. In contrast, the high-risk group displayed pronounced infiltration of CAFs, indicating their potential to impede immune cell infiltration and impact the efficacy of endocrine drugs through the formation of physical barriers and recruitment of immunosuppressive factors. Consequently, patients in the high-risk group experienced a worse prognosis. Therefore, inhibiting these genes may be a therapeutic option for overcoming endocrine therapy resistance in HR+ breast cancer patients. Citation Format: Xiyu Kang, Jiaxiang Liu, Xin Wang. An Endocrine Resistant-Related Gene Signature Revealing the Tumor Microenvironment to Predict the Prognosis of Hormone Receptor-Positive Breast Cancer Patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-21-05.

Investigating cartilage-related diseases by polarization-resolved second harmonic generation (P-SHG) imaging.

Establishing quantitative parameters for differentiating between healthy and diseased cartilage tissues by examining collagen fibril degradation patterns facilitates the understanding of tissue characteristics during disease progression. These findings could also complement existing clinical methods used to diagnose cartilage-related diseases. In this study, cartilage samples from normal, osteoarthritis (OA), and rheumatoid arthritis (RA) tissues were prepared and analyzed using polarization-resolved second harmonic generation (P-SHG) imaging and quantitative image texture analysis. The enhanced molecular contrast obtained from this approach is expected to aid in distinguishing between healthy and diseased cartilage tissues. P-SHG image analysis revealed distinct parameters in the cartilage samples, reflecting variations in collagen fibril arrangement and organization across different pathological states. Normal tissues exhibited distinct χ33/χ31 values compared with those of OA and RA, indicating collagen type transition and cartilage erosion with chondrocyte swelling, respectively. Compared with those of normal tissues, OA samples demonstrated a higher degree of linear polarization, suggesting increased tissue birefringence due to the deposition of type-I collagen in the extracellular matrix. The distribution of the planar orientation of collagen fibrils revealed a more directional orientation in the OA samples, associated with increased type-I collagen, while the RA samples exhibited a heterogeneous molecular orientation. This study revealed that the imaging technique, the quantitative analysis of the images, and the derived parameters presented in this study could be used as a reference for disease diagnostics, providing a clear understanding of collagen fibril degradation in cartilage.

Tissue Interactions in Dense Tissues of the Orofacial Region in Patients with Connective Tissue Dysplasia.

This study aimed to investigate the rate and quality of maturation of the mineral component of impacted teeth 38 and 48 and a fragment of the human lower jaw with connective tissue dysplasia (CTD) in different periods of postnatal ontogenesis. The study involved 102 males (76 with CTD and 26 without CTD), divided into age groups: 31-40, 41-50 and 51-60 years. For medical reasons, teeth 38 and 48 were removed from each patient, as well as a fragment of the alveolar part of the lower jaw in the projection of teeth 38 and 48 measuring 0.5 × 0.5 cm. The odontological parameters, the mineral density of the enamel and the lower jaw, the length and width of the enamel prisms, the spatial organisation of collagen fibrils and the dimensions of the bone plates of the lower jaw were determined. A decrease in optical density was observed at the age of 41-50 and 51-60 years with dysplasia, which indicated a decrease in mineral density and the presence of total areas of hypomineralisation relative to the age of 31-40 years. In the age groups of 41-50 and 51-60 years, pronounced sclerosis and deformation of the delimiting elements were observed at the border of the connective tissue structures and the periosteum. At the age of 31-40 years, the level of stratification of the bone plates was local; after 40 years, it was generalised. Progressive osteoporosis of the lower jaw and incomplete amelogenesis are obstacles to the correct and harmonious eruption of the lower 'wisdom' teeth after 30 years.

A Novel Ligamentous Complex in the Anteromedial Region of the Knee: A Cadaveric Study.

The medial knee structures have a primary role in stabilizing valgus and rotational stress, which makes them important in assessing the ligament-injured knee globally and choosing the most adequate treatment. To conduct a layer-by-layer dissection of the knee's anteromedial side and provide a qualitative and quantitative description of the anatomy and histology of a ligament in the anteromedial region of the knee, which we have termed the anterior oblique ligament (AOL). Also, to describe the AOL relationship with what we have termed the medial cross-a ligament complex that stabilizes the medial pivot. Descriptive laboratory study. A total of 35 fresh-frozen knees from transfemoral amputations that were exclusively performed for vascular reasons were dissected. Structures were identified after meticulous dissection, respecting the same protocol, measured with a digital caliper rule, and histologically studied for data. The AOL was found in all dissected knees, with a mean length of 31.47 ± 5.06 mm. This structure presented a ligament histology with densely organized collagen fibrils. The medial cross was represented by the superficial medial collateral ligament, AOL (anterior region), and posterior oblique ligament. This study demonstrated the presence of a ligament in the anteromedial region of the knee, termed the AOL. This structure was in the anterior part of a ligament complex-the medial cross. Studying and revisiting the medial compartment can provide important information for understanding joint instability and promoting better results in ligament reconstructions.

Transcriptomic and proteomic profiles of fetal versus adult mesenchymal stromal cells and mesenchymal stromal cell-derived extracellular vesicles

BackgroundMesenchymal stem/stromal cells (MSCs) can regenerate tissues through engraftment and differentiation but also via paracrine signalling via extracellular vesicles (EVs). Fetal-derived MSCs (fMSCs) have been shown, both in vitro and in animal studies, to be more efficient than adult MSC (aMSCs) in generating bone and muscle but the underlying reason for this difference has not yet been clearly elucidated. In this study, we aimed to systematically investigate the differences between fetal and adult MSCs and MSC-derived EVs at the phenotypic, RNA, and protein levels.MethodsWe carried out a detailed and comparative characterization of culture-expanded fetal liver derived MSCs (fMSCs) and adult bone marrow derived MSCs (aMSCs) phenotypically, and the MSCs and MSC-derived EVs were analysed using transcriptomics and proteomics approaches with RNA Sequencing and Mass Spectrometry.ResultsFetal MSCs were smaller, exhibited increased proliferation and colony-forming capacity, delayed onset of senescence, and demonstrated superior osteoblast differentiation capability compared to their adult counterparts. Gene Ontology analysis revealed that fMSCs displayed upregulated gene sets such as “Positive regulation of stem cell populations”, “Maintenance of stemness” and “Muscle cell development/contraction/Myogenesis” in comparison to aMSCs. Conversely, aMSCs displayed upregulated gene sets such as “Complement cascade”, “Adipogenesis”, “Extracellular matrix glycoproteins” and “Cellular metabolism”, and on the protein level, “Epithelial cell differentiation” pathways. Signalling entropy analysis suggested that fMSCs exhibit higher signalling promiscuity and hence, higher potency than aMSCs. Gene ontology comparisons revealed that fetal MSC-derived EVs (fEVs) were enriched for “Collagen fibril organization”, “Protein folding”, and “Response to transforming growth factor beta” compared to adult MSC-derived EVs (aEVs), whereas no significant difference in protein expression in aEVs compared to fEVs could be detected.ConclusionsThis study provides detailed and systematic insight into the differences between fMSCs and aMSCs, and MSC-derived EVs. The key finding across phenotypic, transcriptomic and proteomic levels is that fMSCs exhibit higher potency than aMSCs, meaning they are in a more undifferentiated state. Additionally, fMSCs and fMSC-derived EVs may possess greater bone forming capacity compared to aMSCs. Therefore, using fMSCs may lead to better treatment efficacy, especially in musculoskeletal diseases.

Identifying hub genes and dysregulated pathways in Duchenne muscular dystrophy

Purpose The aim of this study was to identify the hub genes and dysregulated pathways in the progression of duchenne muscular dystrophy (DMD) and to unveil detailedly the cellular and molecular mechanisms associated with DMD for developing efficacious treatments in the future. Material and methods Three mRNA microarray datasets (GSE13608, GSE38417 and GSE109178) were downloaded from Gene Expression Omnibus (GEO). The differentially expressed genes (DEGs) between DMD and normal tissues were obtained via R package. Function enrichment analyses were implemented respectively using DAVID online database. The network analysis of protein-protein interaction network (PPI) was conducted using String. Cytoscape and String were used to analyse modules and screen hub genes. The expression of the identified hub genes was confirmed in mdx mice through using qRT-PCR. Results In total, 519 DEGs were identified, consisting of 393 upregulated genes and 126 downregulated genes. The enriched functions and pathways of the DEGs mainly involve extracellular matrix organization, collagen fibril organization, interferon-gamma-mediated signaling pathway, muscle contraction, endoplasmic reticulum lumen, MHC class II receptor activity, phagosome, graft-versus-host disease, cardiomyocytes, calcium signaling pathway. Twelve hub genes were discovered and biological process analysis proved that these genes were mainly enriched cell cycle, cell division. The result of qRT-PCR suggested that increase in expression of CD44, ECT2, TYMS, MAGEL2, HLA-DMA, SERPINH1, TNNT2 was confirmed in mdx mice and the downregulation of ASB2 and LEPREL1 was also observed. Conclusion In conclusion, DEGs and hub genes identified in the current research help us probe the molecular mechanisms underlying the pathogenesis and progression of DMD, and provide candidate targets for diagnosis and treatment of DMD.

Ultrasound Stimulation of Piezoelectric Nanocomposite Hydrogels Boosts Chondrogenic Differentiation in Vitro, in Both a Normal and Inflammatory Milieu.

The use of piezoelectric nanomaterials combined with ultrasound stimulation is emerging as a promising approach for wirelessly triggering the regeneration of different tissue types. However, it has never been explored for boosting chondrogenesis. Furthermore, the ultrasound stimulation parameters used are often not adequately controlled. In this study, we show that adipose-tissue-derived mesenchymal stromal cells embedded in a nanocomposite hydrogel containing piezoelectric barium titanate nanoparticles and graphene oxide nanoflakes and stimulated with ultrasound waves with precisely controlled parameters (1 MHz and 250 mW/cm2, for 5 min once every 2 days for 10 days) dramatically boost chondrogenic cell commitment in vitro. Moreover, fibrotic and catabolic factors are strongly down-modulated: proteomic analyses reveal that such stimulation influences biological processes involved in cytoskeleton and extracellular matrix organization, collagen fibril organization, and metabolic processes. The optimal stimulation regimen also has a considerable anti-inflammatory effect and keeps its ability to boost chondrogenesis in vitro, even in an inflammatory milieu. An analytical model to predict the voltage generated by piezoelectric nanoparticles invested by ultrasound waves is proposed, together with a computational tool that takes into consideration nanoparticle clustering within the cell vacuoles and predicts the electric field streamline distribution in the cell cytoplasm. The proposed nanocomposite hydrogel shows good injectability and adhesion to the cartilage tissue ex vivo, as well as excellent biocompatibility in vivo, according to ISO 10993. Future perspectives will involve preclinical testing of this paradigm for cartilage regeneration.

Identification of a novel lymphangiogenesis signature associated with immune cell infiltration in colorectal cancer based on bioinformatics analysis

BackgroundLymphangiogenesis plays an important role in tumor progression and is significantly associated with tumor immune infiltration. However, the role and mechanisms of lymphangiogenesis in colorectal cancer (CRC) are still unknown. Thus, the objective is to identify the lymphangiogenesis-related genes associated with immune infiltration and investigation of their prognosis value.MethodsmRNA expression profiles and corresponding clinical information of CRC samples were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The lymphangiogenesis-related genes (LymRGs) were collected from the Molecular Signatures database (MSigDB). Lymphangiogenesis score (LymScore) and immune cell infiltrating levels were quantified using ssGSEA. LymScore) and immune cell infiltrating levels-related hub genes were identified using weighted gene co-expression network analysis (WGCNA). Univariate Cox and LASSO regression analyses were performed to identify the prognostic gene signature and construct a risk model. Furthermore, a predictive nomogram was constructed based on the independent risk factor generated from a multivariate Cox model.ResultsA total of 1076 LymScore and immune cell infiltrating levels-related hub genes from three key modules were identified by WGCNA. Lymscore is positively associated with natural killer cells as well as regulator T cells infiltrating. These modular genes were enriched in extracellular matrix and structure, collagen fibril organization, cell-substrate adhesion, etc. NUMBL, TSPAN11, PHF21A, PDGFRA, ZNF385A, and RIMKLB were eventually identified as the prognostic gene signature in CRC. And patients were divided into high-risk and low-risk groups based on the median risk score, the patients in the high-risk group indicated poor survival and were predisposed to metastasis and advanced stages. NUMBL and PHF21A were upregulated but PDGFRA was downregulated in tumor samples compared with normal samples in the Human Protein Atlas (HPA) database.ConclusionOur finding highlights the critical role of lymphangiogenesis in CRC progression and metastasis and provides a novel gene signature for CRC and novel therapeutic strategies for anti-lymphangiogenic therapies in CRC.

Identification of ECM and EMT relevant genes involved in the progression of bladder cancer through bioinformatics analysis.

Bladder cancer (BC) is very common among cancers of urinary system. It was usually categorized into two types: non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). NMIBC and MIBC groupings are heterogeneous and have different characteristics. The study was aimed to find some hub genes and related signal pathways which might be engaged in the progression of BC and to investigate the relationship with clinical stages and its prognostic significance. GSE37317 datasets were acquired from Gene Expression Omnibus (GEO) database. GEO2R on-line tool was selected to screen the differentially expressed genes (DEGs) of the two different types of BC. Then, Gene Ontology (GO) enrichment and KOBAS-Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these DEGs were conducted. A protein-protein interaction (PPI) network was employed to help us screen hub genes and find significant modules. Finally, we made analysis of gene expression and survival curve by GEPIA and Kaplan-Meier plotter database. 224 DEGs were screened in total, with 110 showing increased expression and 114 demonstrating decreased expression. GO and KEGG pathway enrichment analysis showed that DEGs were mostly involved in collagen fibril organization, extracellular matrix (ECM) structural constituent, bHLH transcription factor binding, AGE-RAGE signaling pathway and TGF-beta signaling pathway. Only 3 hub genes (DCN, JUN, THBS1) displayed significantly higher expression compared to those in the healthy controls. These hub genes were also strongly related to clinical stages as well as overall survival (OS) of BC patients. Taken together, most of hub genes involved in the progression of BC were related to ECM and EMT. In addition, 3 hub genes (DCN, JUN, THBS1) were strongly related with clinical stages and OS of BC patients. This study can enhance our comprehension of the progression of NMIBC and identify novel potential targets for MIBC.

Relevance Analysis of TPM2 and Clinicopathological Characteristics in Breast Cancer.

The function of tropomyosin 2 (TPM2) in breast cancer is still far understudied. In this study, we aim to explore the roles of TPM2 in breast cancer progression. This research included 155 breast cancer tissues. The expression of TPM2 was analyzed by immunohistochemical staining and grading. The mRNA expression of TPM2 in pan-cancer was analyzed with The Cancer Genome Atlas (TCGA) data plate form. The differential expression of TPM2 protein and the differential promoter methylation level of TPM2 between breast cancer tissues and normal breast tissues were analyzed by the UALCAN online database. The relationship between TPM2 and signaling pathways was interpreted by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) pathway enrichment analyses. The survival curve of TPM2 was analyzed across the Kaplan-Meier plotter online database. Furthermore, the relationship between TPM2 expression and infiltrating macrophages was validated through in vitro co-culture experiments. TPM2 expression was significantly down-regulated in breast cancer samples. In addition, TPM2 expression was correlated with lymph node metastasis and high-grade histopathological morphology. The receiver operating characteristic (ROC) curve indicated that TPM2 expression could well distinguish between normal breast tissue and breast cancer tissue. TPM2 may have potential value in breast cancer diagnosis. Bioinformatics analysis illustrated that TPM2 was mainly involved in extracellular matrix organization, collagen fibril organization, cell junction assembly, focal adhesion, cAMP signaling pathway, estrogen signaling pathway, Wnt signaling pathway, and adaptive immune system. TPM2 expression was correlated with immune infiltrating cells and immune checkpoint molecules. Our in vitro co-culture experiments showed that the M2 macrophages could upregulate the expression of TPM2. TPM2 may play key roles in breast cancer occurrence and development, especially in cancer metastasis. TPM2 may be a potential biomarker for breast cancer diagnosis.

Integrated analysis reveals ceRNA network of cardiac remodeling by SGLT2 inhibitor in middle-aged hypertensive rats

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) represent an innovative class of antidiabetic agents that have demonstrated promise in mitigating cardiac remodeling. However, the transcriptional regulatory mechanisms underpinning their impact on blood pressure and the reversal of hypertension-induced cardiac remodeling remain largely unexplored. Given this context, our study concentrated on comparing the cardiac expression profiles of lncRNAs and mRNAs between Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). To validate our results, we performed blood pressure measurements, tissue staining, and qRT-PCR. The treatment led to a significant reduction in systolic blood pressure and improved cardiac remodeling by reducing myocardial fibrosis and regulating the inflammatory response. Our examination disclosed that ventricular tissue mRNA, regulated by hypertension, was primarily concentrated in the complement and coagulation cascades and cytokine-cytokine receptor interactions. Compared with SHR, the SGLT2i treatment group was associated with myocardial contraction. Investigation into the lncRNA-mRNA regulatory network and competing endogenous RNA (ceRNA) network suggested that the potential roles of these differentially expressed (DE) lncRNAs and mRNAs were tied to processes such as collagen fibril organization, inflammatory response, and extracellular matrix (ECM) modifications. We found that the expression of Col3a1, C1qa, and lncRNA NONRATT007139.2 were altered in the SHR group and that SGLT2i treatment reversed these changes. Our results suggest that dapagliflozin effectively reverses hypertension-induced myocardial remodeling through a lncRNA-mRNA transcriptional regulatory network, with immune cell-mediated ECM deposition as a potential regulatory target. This underlines the potentiality of SGLT2i and genes related to immunity as promising targets for the treatment of hypertension.