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Purpose: The deubiquitinating enzymes (DUBs) have been linked to cancer initiation and progression. Although ubiquitin-specific protease 33 (USP33) represents a significant factor in regulating various tumor cell behaviors, its specific biological functions and precise mechanisms in esophageal squamous cell carcinoma (ESCC) progression remain unclear. Methods: The expressions of USP33 mRNA in GEO databases, clinical ESCC samples, and USP33 protein were analyzed using bioinformatics, RT-PCR, and immunohistochemistry, respectively. Using Kaplan–Meier survival curves, the log-rank test was used to determine the cumulative survival rate. Western blotting was used to determine indicated protein expression. The cell biological functions were evaluated by cell growth assay, transwell, cell adhesion, and cell spreading assay, respectively. The interaction between USP33 and integrins was detected by immunoprecipitation, and the deubiquitination was performed by deubiquitination assay. The metastatic ability was checked by tail vein injection. Results: A significant positive correlation was found between USP33 expression and clinical TNM stage, T classification, and poor prognosis in patients with ESCC. USP33 promoted laminin-dependent adhesion, spreading, and migration of ESCC cells but not their proliferation. Mechanistically, USP33 mediates cell migration through binding, deubiquinating, and stabilizing integrin α6. USP33 knockdown could inhibit ESCC cell migration and metastasis majorly through integrin α6. Conclusion: This study reveals a novel mechanism of USP33 in promoting laminin-dependent ESCC cell migration and metastasis through integrin α6, suggesting that USP33 may be a promising target for treating ESCC. [ABSTRACT FROM AUTHOR]

Mutations in IDH1 and TP53 have a significant impact on glioma prognosis and progression; however, their roles in tumor cell invasion in terms of interactions with particular components of the extracellular matrix (ECM) are still unclear. Using gene editing protocol based on CRISPR-Cas 9 with cytidine deaminase, we introduced point mutations into U87MG glioblastoma cells to establish modified cell lines with heterozygous IDH1 R132H, homozygous TP53 R248Q and heterozygous IDH1 R132H, homozygous TP53 R248Q genotypes. A comparative study of cell migration on major ECM components was carried out by high-content microscopy. IDH1 R132H mutation introduced to U87MG glioblastoma cells was shown to decrease the migration speed on Matrigel and collagen IV substrates compared to the wild-type. This data were supported by cell adhesion quantification via the lateral shift assay performed by atomic force microscopy (AFM). TP53 R248Q mutation increased cell adhesion to various substrates and significantly promoted cell migration on hyaluronic acid and chondroitin sulfate but did not change the migration rates on laminin and collagens IV and I. A double-mutant genotype produced by consequently introducing IDH1 R132H and TP53 R248Q to parental glioblastoma cells was characterized by the highest migration among all the cell lines, with particularly faster motility on chondroitin sulfate. These findings underscore the complex interactions between glioma cells, with the most important driver mutations and specific ECM components regulating cancer cell migration, offering valuable insights for potential therapeutic targets in glioma treatment. [ABSTRACT FROM AUTHOR]

Abstract Bone marrow-derived mesenchymal stem cells (BMSCs) exhibit multi-lineage differentiation potential and robust proliferative capacity. The late stage of differentiation signifies the functional maturation and characterization of specific cell lineages, which is crucial for studying lineage-specific differentiation mechanisms. However, the molecular processes governing late-stage BMSC differentiation remain poorly understood. This study aimed to elucidate the key biological processes involved in late-stage BMSC differentiation. Publicly available transcriptomic data from human BMSCs were analyzed after approximately 14 days of osteogenic, adipogenic, and chondrogenic differentiation. Thirty-one differentially expressed genes (DEGs) associated with differentiation were identified. Pathway enrichment analysis indicated that the DEGs were involved in extracellular matrix (ECM)-receptor interactions, focal adhesion, and glycolipid biosynthesis, a ganglion series process. Subsequently, the target genes were validated using publicly available single-cell RNA-seq data from mouse BMSCs. Lamc1 exhibited predominant distribution in adipocytes and osteoblasts, primarily during the G2/M phase. Tln2 and Hexb were expressed in chondroblasts, osteoblasts, and adipocytes, while St3gal5 was abundantly distributed in stem cells. Cell communication analysis identified two receptors that interact with LAMCI. q-PCR results confirmed the upregulation of Lamc1, Tln2, Hexb, and St3gal5 during osteogenic differentiation and their downregulation during adipogenic differentiation. Knockdown of Lamc1 inhibited adipogenic and osteogenic differentiation. In conclusion, this study identified four genes, Lamc1, Tln2, Hexb, and St3gal5, that may play important roles in the late-stage differentiation of BMSCs. It elucidated their interactions and the pathways they influence, providing a foundation for further research on BMSC differentiation.

Introduction: Gingival recession is the exposure of the root surface of the tooth, characterised by the displacement of the gingival margin apically from the Cementoenamel Junction (CEJ). Various techniques have been suggested for treating recession, such as Free Gingival Graft (FGG), Connective tissue grafts, laterally or Coronally Advanced Flaps (CAF), and Guided Tissue Regeneration (GTR)-based procedures. Different membranes have been used for GTR, one of which is the amnion membrane. Aim: To evaluate root coverage using Coronally Advanced Flap (CAF) with and without amniotic membrane in the treatment of localised gingival recession. Materials and Methods: A randomised controlled trial was conducted at Pandit Deendayal Upadhyay Dental College and Hospital, Solapur, Maharashtra, India from March 2017 to February 2018. In this study, 30 teeth were treated, with 15 teeth undergoing CAF with amniotic membrane (Group II-Test Group) and 15 teeth treated with CAF without amniotic membrane (Group I-Control Group). Probing Depth (PD), Clinical Attachment Level (CAL), Relative Attachment Level (RAL), Recession Width (RW), Recession Depth (RD), Width of Keratinised Gingiva (WKG), Width of Attached Gingiva (WAG), and gingival biotype were assessed between baseline and after three months. Statistical analysis was performed using paired t-test, independent t-test, and Mann-Whitney U-Test. Statistical Package for Social Sciences (SPSS) software version 20.0 was used for analysis. The α error of 5% (p-value=0.05) and β error of 20% were taken into consideration. Results: The mean age of the study population was 30.36±4.55 years, with 18 males and 12 females. There was a decrease in RD, RW, PD, and a gain in CAL and RAL in both groups after three months compared to the baseline values. Additionally, there was an increase in WKG, WAG, and a significant change in the thickness of the gingival biotype from thin to thick in both groups at the end of three months. On inter group comparison, the changes in PD (p-value=1.00), RD (p-value=1.00), RW (0.176), CAL (0.664), RAL (1.00), WKG (p-value=0.313) were not statistically significant at the end of three months. WAG was higher in Group II compared to Group I (p-value=0.014*), and the mean root coverage was greater in Group II (15.62%) than in the control group (11.642%) (p-value=0.005) at the end of three months. There was a statistically significant difference (p-value=0.005*) in Group II (15.62%) compared to the control group (11.642%). Conclusion: Amniotic membrane in combination with CAF proves to be a successful option for root coverage in localised recession defects. However, histological evidence and a greater number of future longitudinal studies are necessary to study the efficacy of this approach.

Bone marrow-derived mesenchymal stem cells (BMSCs) exhibit multi-lineage differentiation potential and robust proliferative capacity. The late stage of differentiation signifies the functional maturation and characterization of specific cell lineages, which is crucial for studying lineage-specific differentiation mechanisms. However, the molecular processes governing late-stage BMSC differentiation remain poorly understood. This study aimed to elucidate the key biological processes involved in late-stage BMSC differentiation. Publicly available transcriptomic data from human BMSCs were analyzed after approximately 14 days of osteogenic, adipogenic, and chondrogenic differentiation. Thirty-one differentially expressed genes (DEGs) associated with differentiation were identified. Pathway enrichment analysis indicated that the DEGs were involved in extracellular matrix (ECM)-receptor interactions, focal adhesion, and glycolipid biosynthesis, a ganglion series process. Subsequently, the target genes were validated using publicly available single-cell RNA-seq data from mouse BMSCs. Lamc1 exhibited predominant distribution in adipocytes and osteoblasts, primarily during the G2/M phase. Tln2 and Hexb were expressed in chondroblasts, osteoblasts, and adipocytes, while St3gal5 was abundantly distributed in stem cells. Cell communication analysis identified two receptors that interact with LAMCI. q-PCR results confirmed the upregulation of Lamc1, Tln2, Hexb, and St3gal5 during osteogenic differentiation and their downregulation during adipogenic differentiation. Knockdown of Lamc1 inhibited adipogenic and osteogenic differentiation. In conclusion, this study identified four genes, Lamc1, Tln2, Hexb, and St3gal5, that may play important roles in the late-stage differentiation of BMSCs. It elucidated their interactions and the pathways they influence, providing a foundation for further research on BMSC differentiation. [ABSTRACT FROM AUTHOR]

37/67 kDa laminin receptor (LamR)/ribosomal protein SA exhibits dual function as both a ribosomal protein and cell surface receptor for laminin. LamR influences critical cellular processes such as invasion, adhesion, and migration when acting as a receptor. Despite the acknowledged importance of LamR/67LR in various cellular processes, its contribution to the peripheral nervous system development is obscure. Thus, this study investigated the biological activity of LamR in peripheral axonal outgrowth in the presence of laminin-1 or Ile-Lys-Val-Ala- Val (IKVAV) peptide, whose important role in dorsal root ganglia (DRG) axonal outgrowth we recently showed. Unexpectedly, we did not observe LamR on the surface of DRG cells or in a conditioned medium, suggesting its intracellular action in the negative regulation of DRG axonal outgrowth. Using C-terminus LamR-targeting IgG, we demonstrated the role of LamR in that process, which is independent of the presence of Schwann cell precursors (SCPs) and is mediated by extracellular signal-regulated kinase (Erk) and Protein kinase B (Akt1/2/3) signaling pathways. Additionally, we show that the action of LamR towards laminin-1-dependent axonal outgrowth is unmasked only when the activity of integrin β1 is perturbed. We believe that modulation of LamR activity provides the basis for its use for inhibiting axon growth as a potential therapeutic agent for regulating abnormal or excessive neurite growth during neurodevelopmental diseases or pathological nerve regeneration. [ABSTRACT FROM AUTHOR]

Introduction: Enteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of wellestablished in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed. Methods: We examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3' bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice. Results: We identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1b treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation. Discussion: Together, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation. [ABSTRACT FROM AUTHOR]

COVID-19 patients often suffer from post-COVID-19 acute sequelae (PASC). Pulmonary fibrosis has the most significant long-term impact on the respiratory health of patients, known as post-COVID-19 pulmonary fibrosis (PC19-PF). PC19-PF can be caused by acute respiratory distress syndrome (ARDS) or COVID-19-induced pneumonia. Individuals who experience COVID-19 pneumonia symptoms (including cough, shortness of breath, dyspnea on exertion, and desaturation) for at least 12 weeks after diagnosis, almost all develop PC19-PF. Extracellular matrix molecules: laminin (LN), type IV collagen (IV Col), procollagen III N-terminal peptide (PIIINP), and hyaluronic acid (HA) are involved in the development and progression of PC19-PF. This study aimed to investigate the relationship between the progression of PC19-PF and serum levels of laminin, IV COL, PIIINP, and hyaluronic acid. This retrospective study included 162 PC19-PF patients treated and 160 healthy controls who received treatment at Shenzhen Longgang District Third People's Hospital, Hebei PetroChina Central Hospital and Changzhi People's Hospital from January 2021 to December 2023. Serum levels of LN, IV COL, PIIINP, and HA were detected by chemiluminescence immunoassay using commercial kits. Predicted forced vital capacity percentage (FVC% pred), predicted carbon monoxide lung diffusion capacity percentage (DLCO% pred), high-resolution computed tomography (HRCT) scores were assessed, and patient mortality was compared with healthy controls. Serum levels of LN, IV Col, PIIINP, and HA were significantly higher in PC19-PF or CTD-ILD patients than in healthy controls (all p < 0.05), and they were further elevated in acute exacerbation cases (all p < 0.01). In patients, HA was positively associated with HRCT scores and negatively associated with FVC% pred and DLCO% pred (all p < 0.05). Serum levels of LN, IV COL, PIIINP, and HA were significantly lower in surviving patients than in those who deceased (all p > 0.05). Serum levels of LN, IV C, PIIINP, and HA may affect the progression of PC19-PF and may serve as indicators of PC19-PF severity. [ABSTRACT FROM AUTHOR]

Introduction: Gingival recession is the exposure of the root surface of the tooth, characterised by the displacement of the gingival margin apically from the Cementoenamel Junction (CEJ). Various techniques have been suggested for treating recession, such as Free Gingival Graft (FGG), Connective tissue grafts, laterally or Coronally Advanced Flaps (CAF), and Guided Tissue Regeneration (GTR)-based procedures. Different membranes have been used for GTR, one of which is the amnion membrane. Aim: To evaluate root coverage using Coronally Advanced Flap (CAF) with and without amniotic membrane in the treatment of localised gingival recession. Materials and Methods: A randomised controlled trial was conducted at Pandit Deendayal Upadhyay Dental College and Hospital, Solapur, Maharashtra, India from March 2017 to February 2018. In this study, 30 teeth were treated, with 15 teeth undergoing CAF with amniotic membrane (Group IITest Group) and 15 teeth treated with CAF without amniotic membrane (Group I-Control Group). Probing Depth (PD), Clinical Attachment Level (CAL), Relative Attachment Level (RAL), Recession Width (RW), Recession Depth (RD), Width of Keratinised Gingiva (WKG), Width of Attached Gingiva (WAG), and gingival biotype were assessed between baseline and after three months. Statistical analysis was performed using paired t-test, independent t-test, and Mann-Whitney U-Test. Statistical Package for Social Sciences (SPSS) software version 20.0 was used for analysis. The α error of 5% (p-value=0.05) and β error of 20% were taken into consideration. Results: The mean age of the study population was 30.36±4.55 years, with 18 males and 12 females. There was a decrease in RD, RW, PD, and a gain in CAL and RAL in both groups after three months compared to the baseline values. Additionally, there was an increase in WKG, WAG, and a significant change in the thickness of the gingival biotype from thin to thick in both groups at the end of three months. On inter group comparison, the changes in PD (p-value=1.00), RD (p-value=1.00), RW (0.176), CAL (0.664), RAL (1.00), WKG (p-value=0.313) were not statistically significant at the end of three months. WAG was higher in Group II compared to Group I (p-value=0.014*), and the mean root coverage was greater in Group II (15.62%) than in the control group (11.642%) (p-value=0.005) at the end of three months. There was a statistically significant difference (p-value=0.005*) in Group II (15.62%) compared to the control group (11.642%). Conclusion: Amniotic membrane in combination with CAF proves to be a successful option for root coverage in localised recession defects. However, histological evidence and a greater number of future longitudinal studies are necessary to study the efficacy of this approach. [ABSTRACT FROM AUTHOR]

The spectrum of canine muscular dystrophies has rapidly grown with the recent identification of several more affected breeds and associated mutations. Defects include those in genes and protein products associated with the sarcolemma (dystrophin deficient X-linked muscular dystrophy and sarcoglycan-deficient limb-girdle muscular dystrophy) and with the extracellular matrix (collagen 6, laminin α2, and α-dystroglycan-deficient congenital muscular dystrophies). With the increasing application of whole genome sequencing and whole exome sequencing, the clinical and pathological spectra associated with specific neuromuscular genetic defects are constantly evolving. In this report, we provide a brief overview of the current status of gene defects reported in canine muscular dystrophies. We also report the causative mutations for novel forms of X-linked muscular dystrophy in Brittany spaniels and in a French bulldog.

Oogenesis involves transduction of mechanical forces from the cytoskeleton to the nuclear envelope (NE). In Caenorhabditis elegans, oocyte nuclei lacking the single lamin protein LMN-1 are vulnerable to collapse under forces mediated through LINC (linker of nucleoskeleton and cytoskeleton) complexes. Here, we use cytological analysis and in vivo imaging to investigate the balance of forces that drive this collapse and protect oocyte nuclei. We also use a mechano-node-pore sensing device to directly measure the effect of genetic mutations on oocyte nuclear stiffness. We find that nuclear collapse is not a consequence of apoptosis. It is promoted by dynein, which induces polarization of a LINC complex composed of Sad1 and UNC-84 homology 1 (SUN-1) and ZYGote defective 12 (ZYG-12). Lamins contribute to oocyte nuclear stiffness and cooperate with other inner nuclear membrane proteins to distribute LINC complexes and protect nuclei from collapse. We speculate that a similar network may protect oocyte integrity during extended oocyte arrest in mammals.

Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (∼30-150 nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3β1 and α6β1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 μm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3β1 and α6β1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design.

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.

The notochord is a defining feature of all chordates. The transcription factors Zic and ETS regulate enhancer activity within the notochord. We conduct high-throughput screens of genomic elements within developing Ciona embryos to understand how Zic and ETS sites encode notochord activity. Our screen discovers an enhancer located near Lama, a gene critical for notochord development. Reversing the orientation of an ETS site within this enhancer abolishes expression, indicating that enhancer grammar is critical for notochord activity. Similarly organized clusters of Zic and ETS sites occur within mouse and human Lama1 introns. Within a Brachyury (Bra) enhancer, FoxA and Bra, in combination with Zic and ETS binding sites, are necessary and sufficient for notochord expression. This binding site logic also occurs within other Ciona and vertebrate Bra enhancers. Collectively, this study uncovers the importance of grammar within notochord enhancers and discovers signatures of enhancer logic and grammar conserved across chordates.

Patients with squamous cell carcinoma (SCC) have significantly lower survival upon the development of distant metastases. The extracellular matrix (ECM) is a consistent yet dynamic influence on the metastatic capacity of SCCs. The ECM encompasses a milieu of structural proteins, signaling molecules, and enzymes. Just over 40 years ago, the fibrous ECM glycoprotein laminin was identified. Roughly four decades of research have revealed a pivotal role of laminins in metastasis. However, trends in ECM alterations in some cancers have been applied broadly to all metastatic diseases, despite evidence that these characteristics vary by tumor type. We will summarize how laminins influence the SCC metastatic process exclusively. Enhanced laminin protein deposition occurs at the invasive edge of SCC tumors, which correlates with elevated levels of laminin-binding β1 integrins on SCC cells, increased MMP-3 presence, worse prognosis, and lymphatic dissemination. Although these findings are significant, gaps in knowledge of the formation of a premetastatic niche, the processes of intra- and extravasation, and the contributions of the ECM to SCC metastatic cell dormancy persist. Bridging these gaps requires novel in vitro systems and animal models that reproduce tumor-stromal interactions and spontaneous metastasis seen in the clinic. These advances will allow accurate assessment of laminins to predict responders to transforming growth factor-β inhibitors and immunotherapy, as well as potential combinatorial therapies with the standard of care. Such clinical interventions may drastically improve quality of life and patient survival by explicitly targeting SCC metastasis.

Benign and malignant nerve sheath tumors (NST) pose a major challenge in routine diagnostic anatomic pathology because of shared histomorphological features with other soft-tissue tumors (STT). As a result, NST are often diagnosed as STT, a broad category that encompasses various entities including perivascular wall tumors (PWT) and that represents approximately 15% of all skin tumors in dogs. Immunohistochemistry (IHC) can assist the identification of histologic subtypes of STT. This IHC pilot study applies various markers largely expressed by peripheral nerves to twelve benign and six malignant NST and determines the intratumoral protein expression of laminin, periaxin-1, Sox-10 and S-100 in the NST subtypes. Furthermore, this study assesses the usefulness of peripheral nerve markers applied to diagnostic work cases and demonstrates the relevance of laminin expression patterns, periaxin-1 and Sox-10 in assisting the differentiation of NST from other STT, in particular from PWT.

Prostate cancer is one of the most common neoplasm in the male population. It is not known why some tumors become more aggressive than others. Although most studies show changes in the expression of cell adhesion molecules and the extracellular matrix correlated with the Gleason score, no study has objectively measured the tissue content of these molecules. This study aims to measure the content and tissue expression of collagen type I and IV and laminin in the extracellular matrix of patients with prostate adenocarcinoma and correlate these findings with the Gleason score and clinical characteristics. Forty-one patients who underwent radical prostate surgery at the Urology Department of a reference Hospital in Brazil between January 2015 and December 2020 were studied. The tissue protein content was estimated under light microscopy at a final magnification of 200 ×. The mean collagen I score in prostate adenocarcinoma tissue samples was 7.16 ± 1.03 pixels/field. The mean type IV collagen score was 3.44 ± 0.61 pixels/field. The mean laminin score was 5.19 ± 0.79 pixels/field. The total Gleason score was correlated with both collagen and laminin. All the correlations were negative, which shows that the higher the collagen/laminin expression was, the lower the total Gleason score (p-value < 0,05). According to the Pearson correlation analysis, age has no statistical relationship with collagen and laminin content. PSA, in turn, showed a correlation only with laminin, but r = -0.378 (p = 0.015). Among the associated diseases and lifestyle habits, there is only statistical significance in the comparison of alcoholism for collagen I. For collagen IV and laminin, no statistical significance was obtained with the clinical variables analyzed. [ABSTRACT FROM AUTHOR]

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) stands as a prominent global cause of disability, with motor deficits being a common consequence. Despite its widespread impact, the precise pathological mechanisms underlying motor deficits after TBI remain elusive. In this study, hindlimb postural asymmetry (HL-PA) development in rats subjected to focal TBI was investigated to explore the potential roles of collagen IV and laminin within the extracellular matrix (ECM) of selected hindlimb muscles in the emergence of motor deficits following TBI. A focal TBI was induced by ablating the left sensorimotor cortex in rats and motor deficits were assessed by measuring HL-PA. The expression of laminin and collagen IV in eight selected muscles on each side of the hindlimbs from both TBI- and sham-operated rats were studied using immunohistochemistry and semi-quantitatively analyzed. The results indicated that the TBI rats exhibited HL-PA, characterized by flexion of the contralateral (right) hindlimb. In the sham-operated rats, the immunoreactive components of laminin and collagen IV were evenly and smoothly distributed along the border of the muscle fibers in all the investigated muscles. In contrast, in the TBI rats, the pattern was broken into aggregated, granule-like, immunoreactive components. Such a labeling pattern was detected in all the investigated muscles both from the contra- and ipsilateral sides of the TBI rats. However, in TBI rats, most of the muscles from the contralateral hindlimb showed a significantly increased expression of these two proteins in comparison with those from the ipsilateral hindlimb. In comparison to sham-operated rats, there was a significant increase in laminin and collagen IV expression in various contralateral hindlimb muscles in the TBI rats. These findings suggest potential implications of laminin and collagen IV in the development of motor deficits following a focal TBI. [ABSTRACT FROM AUTHOR]

37/67 kDa laminin receptor (LamR)/ribosomal protein SA exhibits dual function as both a ribosomal protein and cell surface receptor for laminin. LamR influences critical cellular processes such as invasion, adhesion, and migration when acting as a receptor. Despite the acknowledged importance of LamR/67LR in various cellular processes, its contribution to the peripheral nervous system development is obscure. Thus, this study investigated the biological activity of LamR in peripheral axonal outgrowth in the presence of laminin-1 or Ile-Lys-Val-Ala-Val (IKVAV) peptide, whose important role in dorsal root ganglia (DRG) axonal outgrowth we recently showed. Unexpectedly, we did not observe LamR on the surface of DRG cells or in a conditioned medium, suggesting its intracellular action in the negative regulation of DRG axonal outgrowth. Using C-terminus LamR-targeting IgG, we demonstrated the role of LamR in that process, which is independent of the presence of Schwann cell precursors (SCPs) and is mediated by extracellular signal-regulated kinase (Erk) and Protein kinase B (Akt1/2/3) signaling pathways. Additionally, we show that the action of LamR towards laminin-1-dependent axonal outgrowth is unmasked only when the activity of integrin β1 is perturbed. We believe that modulation of LamR activity provides the basis for its use for inhibiting axon growth as a potential therapeutic agent for regulating abnormal or excessive neurite growth during neurodevelopmental diseases or pathological nerve regeneration.

IntroductionEnteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of well-established in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed.MethodsWe examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3’ bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice.ResultsWe identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1β treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation.DiscussionTogether, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation.

It is generally recognized that the main function of α-tocopherol (αToc), which is the most active form of vitamin E, is its antioxidant effect, while non-antioxidant effects have also been reported. We previously found that αToc ameliorates diabetic nephropathy via diacylglycerol kinase alpha (DGKα) activation in vivo, and the activation was not related to the antioxidant effect. However, the underlying mechanism of how αToc activates DGKα have been enigmatic. We report that the membrane-bound 67 kDa laminin receptor (67LR), which has previously been shown to serve as a receptor for epigallocatechin gallate (EGCG), also contains a novel binding site for vitamin E, and its association with Vitamin E mediates DGKα activation by αToc. We employed hydrogen-deuterium exchange mass spectrometry (HDX/MS) and molecular dynamics (MD) simulations to identify the specific binding site of αToc on the 67LR and discovered the conformation of the specific hydrophobic pocket that accommodates αToc. Also, HDX/MS and MD simulations demonstrated the detailed binding of EGCG to a water-exposed hydrophilic site on 67LR, while in contrast αToc binds to a distinct hydrophobic site. We demonstrated that 67LR triggers an important signaling pathway mediating non-antioxidant effects of αToc, such as DGKα activation. This is the first evidence demonstrating a membrane receptor for αToc and one of the underlying mechanisms of a non-antioxidant function for αToc.

Objective(s): Bone tissue engineering is considered a new method in the treatment of bone defects and can be an effective alternative to surgery and bone grafting. The use of adipose tissue mesenchymal stem cells (ADMSCs) on synthetic polymer scaffolds is one of the new approaches in bone tissue engineering. In this study, we aimed to investigate the effect of laminin coating on biocompatibility and osteogenic differentiation of ADMSCs seeded on polycaprolactone (PCL) scaffolds.Materials and Methods: The morphology of the electrospun scaffold was evaluated using a scanning electron microscope (SEM). Cell proliferation and cytotoxicity were determined by MTT assay. The adipogenic and osteogenic differentiation potential of the cells was evaluated. The osteogenic differentiation of ADMSCs cultured on the PCL scaffold coated with laminin was assessed by evaluating the level of alkaline phosphatase (ALP) activity, intracellular calcium content, and expression of bone-specific genes.Results: The results showed that the ADMSCs cultured on PCL/laminin showed enhanced osteogenic differentiation compared to those cultured on non-coated PCL or control medium (P

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Nidogen, also known as entactin, is a multifunctional glycoprotein that plays a crucial role in the maintenance of the basement membrane (BM), morphogenesis and neuronal plasticity. This review aims to provide an overview of the structural features, molecular interactions and diverse functions associated with Nidogen. As a bridging molecule within the BM, Nidogen acts as a linchpin connecting various extracellular matrix (ECM) components. Its involvement in tissue development, homeostasis, and pathological conditions underscores its biological and medical significance. We discuss the current state of knowledge regarding Nidogen's role in tissue maintenance, cell adhesion, migration, and signaling, shedding light on its intricate contributions to physiological and pathological processes. [ABSTRACT FROM AUTHOR]

Mutations of PKD1 coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these are the leucine-rich repeats (LRRs) in the far N-terminus of PC1. Using zebrafish (Danio rerio) as an in vivo model system, we explored the role of LRRs in the function of PC1. Zebrafish expresses two human PKD1 paralogs, pkd1a and pkd1b. Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal-axis curvature and pronephric cyst formation. We found that overexpression of LRRs suppressed both phenotypes in pkd1-morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5β1γ1) in vitro. Mutations of amino acid residues in LRRs structurally predicted to bind laminin-511 disrupted LRR–laminin interaction in vitro and neutralized the ability of LRRs to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1 deficiency. [ABSTRACT FROM AUTHOR]

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected. [ABSTRACT FROM AUTHOR]

Objective(s): Bone tissue engineering is considered a new method in the treatment of bone defects and can be an effective alternative to surgery and bone grafting. The use of adipose tissue mesenchymal stem cells (ADMSCs) on synthetic polymer scaffolds is one of the new approaches in bone tissue engineering. In this study, we aimed to investigate the effect of laminin coating on biocompatibility and osteogenic differentiation of ADMSCs seeded on polycaprolactone (PCL) scaffolds. Materials and Methods: The morphology of the electrospun scaffold was evaluated using a scanning electron microscope (SEM). Cell proliferation and cytotoxicity were determined by MTT assay. The adipogenic and osteogenic differentiation potential of the cells was evaluated. The osteogenic differentiation of ADMSCs cultured on the PCL scaffold coated with laminin was assessed by evaluating the level of alkaline phosphatase (ALP) activity, intracellular calcium content, and expression of bone-specific genes. Results: The results showed that the ADMSCs cultured on PCL/laminin showed enhanced osteogenic differentiation compared to those cultured on non-coated PCL or control medium (P<0.05). Conclusion: It seems that laminin enhances the physicochemical properties and biocompatibility of PCL nanofiber scaffolds; and by modifying the surface of the scaffold, improves the differentiation of ADMSCs into osteogenic cells. [ABSTRACT FROM AUTHOR]

Basement membranes are among the most widespread, non‐cellular functional materials in metazoan organisms. Despite this ubiquity, the links between their compositional and biophysical properties are often difficult to establish due to their thin and delicate nature. In this article, we examine these features on a molecular level by combining results from proteomics, elastic, and nanomechanical analyses across a selection of human basement membranes. Comparing results between these different membranes connects certain compositional attributes to distinct nanomechanical signatures and further demonstrates to what extent water defines these properties. In all, these data underline BMs as stiff yet highly elastic connective tissue layers and highlight how the interplay between composition, mechanics and hydration yields such exceptionally adaptable materials. [ABSTRACT FROM AUTHOR]

Recent research suggests that T-cell receptor (TCR) sequences expanded during human immunodeficiency virus and SARS-CoV-2 infections unexpectedly mimic these viruses. The hypothesis tested here is that TCR sequences expanded in patients with type 1 diabetes mellitus (T1DM) and autoimmune myocarditis (AM) mimic the infectious triggers of these diseases. Indeed, TCR sequences mimicking coxsackieviruses, which are implicated as triggers of both diseases, are statistically significantly increased in both T1DM and AM patients. However, TCRs mimicking Clostridia antigens are significantly expanded in T1DM, whereas TCRs mimicking Streptococcal antigens are expanded in AM. Notably, Clostridia antigens mimic T1DM autoantigens, such as insulin and glutamic acid decarboxylase, whereas Streptococcal antigens mimic cardiac autoantigens, such as myosin and laminins. Thus, T1DM may be triggered by combined infections of coxsackieviruses with Clostridia bacteria, while AM may be triggered by coxsackieviruses with Streptococci. These TCR results are consistent with both epidemiological and clinical data and recent experimental studies of cross-reactivities of coxsackievirus, Clostridial, and Streptococcal antibodies with T1DM and AM antigens. These data provide the basis for developing novel animal models of AM and T1DM and may provide a generalizable method for revealing the etiologies of other autoimmune diseases. Theories to explain these results are explored. [ABSTRACT FROM AUTHOR]

In Alzheimer's disease, the blood–brain barrier breakdown, blood vessel damage and re-organization are early events. Deposits of the small toxic peptide beta-amyloid (Aβ) cause the formation of extracellular plaques and accumulate in vessels disrupting the blood flow but may also play a role in blood clotting. In the present study, we aim to explore the impact of Aβ on the migration of endothelial cells and subsequent vessel formation. We use organotypic brain slices of postnatal day 10 wildtype mice (C57BL/6) and connect them to small microcontact prints (µCPs) of collagen. Our data show that laminin-positive endothelial cells migrate onto collagen µCPs, but without any vessel formation after 4 weeks. When the µCPs are loaded with human Aβ40, (aggregated) human Aβ42 and mouse Aβ42 peptides, the number and migration distance of endothelial cells are significantly reduced, but with a more pronounced subsequent vessel formation. The vessel formation is verified by zonula occludens (ZO)-1 and -2 stainings and confocal microscopy. In addition, the vessel formation is accompanied by a stronger GFAP-positive astroglial formation. Finally, we show that vessels can grow towards convergence when two opposed slices are connected via microcontact-printed lanes. In conclusion, our data show that Aβ promotes vessel formation, and organotypic brain slices connected to collagen µCPs provide a potent tool to study vessel formation. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem cells (mESCs) have been widely used as a model system to study the basic biology of pluripotency and to develop cell-based therapies. Traditionally, mESCs have been cultured in a medium supplemented with fetal bovine serum (FBS). However, serum with its inconsistent chemical composition has been problematic for reproducibility and for studying the role of specific components. While some serum-free media have been reported, these media contain commercial additives whose detailed components have not been disclosed. Recently, we developed a serum-free medium, DA-X medium, which can maintain a wide variety of adherent cancer lines. In this study, we modified the DA-X medium and established a novel serum-free condition for both naïve mESCs in which all components are chemically defined and disclosed (DA-X-modified medium for robust growth of pluripotent stem cells: DARP medium). The DARP medium fully supports the normal transcriptome and differentiation potential in teratoma and the establishment of mESCs from blastocysts that retain the developmental potential in all three germ layers, including germ cells in chimeric embryos. Utility of chemically defined DA-X medium for primed mouse epiblast stem cells (mEpiSCs) revealed that an optimal amount of cholesterol is required for the robust growth of naïve-state mESCs, but is dispensable for the maintenance of primed-state mEpiSCs. Thus, this study provides reliable and reproducible culture methods to investigate the role of specific components regulating self-renewal and pluripotency in a wide range of pluripotent states.

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin β1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/β1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor β1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.

Visceral leiomyosarcoma is well described in dogs, but information about non-visceral locations and prevalence is lacking. The diagnosis of leiomyosarcoma is challenging without a gold standard, and often includes the use of immunohistochemical (IHC) stains. We used defined histopathologic patterns, histochemical staining, and IHC staining for smooth muscle actin (SMA), desmin, and laminin to characterize suspected non-visceral leiomyosarcoma in dogs at a single academic institution. In a retrospective search, we identified 24 dogs with a definitive or suspected histologic diagnosis of leiomyosarcoma in a non-visceral location. Histopathology results and clinical details were obtained. Biopsy sections were reviewed by a single pathologist using standardized histologic criteria, including light microscopic appearance, immunohistochemistry (more than two-thirds of neoplastic cells labeled with SMA and desmin or laminin), and histochemical staining (minimal-to-mild matrix deposition by Masson trichrome). Of the 24 cases of possible non-visceral leiomyosarcomas, 4 were consistent with a definitive diagnosis of non-visceral leiomyosarcoma (3) or leiomyoma (1) based on the established criteria. Only the leiomyoma had more than two-thirds of neoplastic cells label with all 3 markers; all 3 leiomyosarcomas had more than two-thirds of neoplastic cells label with SMA and laminin. Our data highlight the uncommon nature of non-visceral leiomyosarcoma and the importance of IHC for their diagnosis. A definitive diagnosis could not be made based on SMA alone, and desmin was not useful in this cohort. Further studies are needed to clarify the histopathologic, IHC, and clinical features of canine non-visceral SMA-positive mesenchymal tumors.

A 2-year-old female spayed dog was presented with a chronic history of short-strided gait and inability to completely open the jaw. Clinical signs were present since the dog was adopted from a humane society at a few months of age. Serum creatine kinase activity was abnormally high. Neurological examination, electromyography, muscle biopsies with immunofluorescent staining, and whole genome sequencing (WGS) were performed. A dystrophic phenotype was identified histologically in muscle biopsies, deficiency of laminin α2 protein was confirmed by immunofluorescent staining, and a deletion in the LAMA2 gene was identified by analysis of the WGS data. Congenital muscular dystrophy associated with a disease variant in LAMA2 was identified.

A 4-month-old, male Italian Greyhound with clinical signs of a neuromuscular disease was investigated. The affected dog presented with an abnormal short-strided gait, generalized muscle atrophy, and poor growth since 2-months of age. Serum biochemistry revealed a marked elevation in creatine kinase activity. Electrodiagnostic testing supported a myopathy. Histopathology of muscle biopsies confirmed a dystrophic phenotype with excessive variability in myofiber size, degenerating fibers, and endomysial fibrosis. A heritable form of congenital muscular dystrophy (CMD) was suspected, and a genetic analysis initiated. We sequenced the genome of the affected dog and compared the data to that of 795 control genomes. This search revealed a private homozygous nonsense variant in LAMA2, XM_022419950.1:c.3285G>A, predicted to truncate 65% of the open reading frame of the wild type laminin α2 protein, XP_022275658.1:p.(Trp1095*). Immunofluorescent staining performed on muscle cryosections from the affected dog confirmed the complete absence of laminin α2 in skeletal muscle. LAMA2 loss of function variants were shown to cause severe laminin α2-related CMD in humans, mouse models, and in one previously described dog. Our data together with current knowledge on other species suggest the LAMA2 nonsense variant as cause for the CMD phenotype in the investigated dog.

Nidogen, also known as entactin, is a multifunctional glycoprotein that plays a crucial role in the maintenance of the basement membrane (BM), morphogenesis and neuronal plasticity. This review aims to provide an overview of the structural features, molecular interactions and diverse functions associated with Nidogen. As a bridging molecule within the BM, Nidogen acts as a linchpin connecting various extracellular matrix (ECM) components. Its involvement in tissue development, homeostasis, and pathological conditions underscores its biological and medical significance. We discuss the current state of knowledge regarding Nidogen’s role in tissue maintenance, cell adhesion, migration, and signaling, shedding light on its intricate contributions to physiological and pathological processes.

Regeneration of periodontal tissues requires an integrated approach to the restoration of the periodontal ligament, cementum, and alveolar bone surrounding the teeth. Current strategies in endogenous regenerative dentistry widely use biomaterials, in particular the decellularized extracellular matrix (dECM), to facilitate the recruitment of populations of resident cells into damaged tissues and stimulate their proliferation and differentiation. The purpose of our study was to evaluate the effect of the exogenous components of the extracellular matrix (hyaluronic acid, laminin, fibronectin) on the differentiation of periodontal ligament stem cells (PDLSCs) cultured with dECM (combinations of decellularized tooth matrices and periodontal ligament) in a 3D collagen I hydrogel. The immunohistochemical expression of various markers in PDLSCs was assessed quantitatively and semi-quantitatively on paraffin sections. The results showed that PDLSCs cultured under these conditions for 14 days exhibited phenotypic characteristics consistent with osteoblast-like and odontoblast-like cells. This potential has been demonstrated by the expression of osteogenic differentiation markers (OC, OPN, ALP) and odontogenic markers (DSPP). This phenomenon corresponds to the in vivo state of the periodontal ligament, in which cells at the interface between bone and cementum tend to differentiate into osteoblasts or cementoblasts. The addition of fibronectin to the dECM most effectively induces the differentiation of PDLSCs into osteoblast-like and odontoblast-like cells under 3D culture conditions. Therefore, this bioengineered construct has a high potential for future use in periodontal tissue regeneration. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) stands as a prominent global cause of disability, with motor deficits being a common consequence. Despite its widespread impact, the precise pathological mechanisms underlying motor deficits after TBI remain elusive. In this study, hindlimb postural asymmetry (HL-PA) development in rats subjected to focal TBI was investigated to explore the potential roles of collagen IV and laminin within the extracellular matrix (ECM) of selected hindlimb muscles in the emergence of motor deficits following TBI. A focal TBI was induced by ablating the left sensorimotor cortex in rats and motor deficits were assessed by measuring HL-PA. The expression of laminin and collagen IV in eight selected muscles on each side of the hindlimbs from both TBI- and sham-operated rats were studied using immunohistochemistry and semi-quantitatively analyzed. The results indicated that the TBI rats exhibited HL-PA, characterized by flexion of the contralateral (right) hindlimb. In the sham-operated rats, the immunoreactive components of laminin and collagen IV were evenly and smoothly distributed along the border of the muscle fibers in all the investigated muscles. In contrast, in the TBI rats, the pattern was broken into aggregated, granule-like, immunoreactive components. Such a labeling pattern was detected in all the investigated muscles both from the contra- and ipsilateral sides of the TBI rats. However, in TBI rats, most of the muscles from the contralateral hindlimb showed a significantly increased expression of these two proteins in comparison with those from the ipsilateral hindlimb. In comparison to sham-operated rats, there was a significant increase in laminin and collagen IV expression in various contralateral hindlimb muscles in the TBI rats. These findings suggest potential implications of laminin and collagen IV in the development of motor deficits following a focal TBI.

Simple Summary: MPN blood cancers are characterized by elevated blood cell counts. Chronic inflammation drives MPNs, which after 10–30 years, may lead to bone marrow failure due to replacement of bone marrow by connective tissue. Chronic inflammation also impacts other organs causing impaired vision, osteoporosis, fractures, heart failure and decreased lung and kidney function. MPNs are also associated with an increased risk of other cancers. Novel, noninvasive technologies are needed to improve the early detection of fibrosis development and MPN progression and to monitor the efficacy of treatment. Herein, we review current knowledge and focus on novel technologies for the measurement of circulating connective tissue biomarkers in MPNs. Future research directions are provided, suggesting studies on single markers or combinations before and during treatment with old and new agents, both targeting the diseased bone marrow cells and the inflammatory processes that drive the progressive fibrosis, which if left untreated, may ultimately lead to bone marrow failure or leukemic transformation. Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) arise due to acquired somatic driver mutations in stem cells and develop over 10–30 years from the earliest cancer stages (essential thrombocythemia, polycythemia vera) towards the advanced myelofibrosis stage with bone marrow failure. The JAK2V617F mutation is the most prevalent driver mutation. Chronic inflammation is considered to be a major pathogenetic player, both as a trigger of MPN development and as a driver of disease progression. Chronic inflammation in MPNs is characterized by persistent connective tissue remodeling, which leads to organ dysfunction and ultimately, organ failure, due to excessive accumulation of extracellular matrix (ECM). Considering that MPNs are acquired clonal stem cell diseases developing in an inflammatory microenvironment in which the hematopoietic cell populations are progressively replaced by stromal proliferation—"a wound that never heals"—we herein aim to provide a comprehensive review of previous promising research in the field of circulating ECM fragments in the diagnosis, treatment and monitoring of MPNs. We address the rationales and highlight new perspectives for the use of circulating ECM protein fragments as biologically plausible, noninvasive disease markers in the management of MPNs. [ABSTRACT FROM AUTHOR]

Stem cell quiescence, proliferation and differentiation are controlled by interactions with niche cells and a specialized extracellular matrix called basement membrane (BM). Direct interactions with adjacent BM are known to regulate stem cell quiescence; however, it is less clear how niche BM relays signals to stem cells that it does not contact. Here, we examine how niche BM regulates Caenorhabditis elegans primordial germ cells (PGCs). BM regulates PGC quiescence even though PGCs are enwrapped by somatic niche cells and do not contact the BM; this can be demonstrated by depleting laminin, which causes normally quiescent embryonic PGCs to proliferate. We show that following laminin depletion, niche cells relay proliferation-inducing signals from the gonadal BM to PGCs via integrin receptors. Disrupting the BM proteoglycan perlecan blocks PGC proliferation when laminin is depleted, indicating that laminin functions to inhibit a proliferation-inducing signal originating from perlecan. Reducing perlecan levels in fed larvae hampers germline growth, suggesting that BM signals regulate germ cell proliferation under physiological conditions. Our results reveal how BM signals can regulate stem cell quiescence indirectly, by activating niche cell integrin receptors. [ABSTRACT FROM AUTHOR]

Autoimmune cardiopathies (AC) following COVID-19 and vaccination against SARS-CoV-2 occur at significant rates but are of unknown etiology. This study investigated the possible roles of viral and bacterial mimicry, as well as viral-bacterial co-infections, as possible inducers of COVID-19 AC using proteomic methods and enzyme-linked immunoadsorption assays. BLAST and LALIGN results of this study demonstrate that SARS-CoV-2 shares a significantly greater number of high quality similarities to some cardiac protein compared with other viruses; that bacteria such as Streptococci, Staphylococci and Enterococci also display very significant similarities to cardiac proteins but to a different set than SARS-CoV-2; that the importance of these similarities is largely validated by ELISA experiments demonstrating that polyclonal antibodies against SARS-CoV-2 and COVID-19-associated bacteria recognize cardiac proteins with high affinity; that to account for the range of cardiac proteins targeted by autoantibodies in COVID-19-associated autoimmune myocarditis, both viral and bacterial triggers are probably required; that the targets of the viral and bacterial antibodies are often molecularly complementary antigens such as actin and myosin, laminin and collagen, or creatine kinase and pyruvate kinase, that are known to bind to each other; and that the corresponding viral and bacterial antibodies recognizing these complementary antigens also bind to each other with high affinity as if they have an idiotype-anti-idiotype relationship. These results suggest that AC results from SARS-CoV-2 infections or vaccination complicated by bacterial infections. Vaccination against some of these bacterial infections, such as Streptococci and Haemophilus, may therefore decrease AC risk, as may the appropriate and timely use of antibiotics among COVID-19 patients and careful screening of vaccinees for signs of infection such as fever, diarrhea, infected wounds, gum disease, etc. [ABSTRACT FROM AUTHOR]

Angiogenesis is a vital step in tissue regeneration. Hence, the current study aimed to prepare oxidized dextran (Odex)/collagen (Col)‐hydrogels with laminin (LMN), as an angiogenic extracellular matrix (ECM) component, for promoting human umbilical vein endothelial cell (HUVEC) proliferation and function. Odex/Col scaffolds were constructed at various concentrations and temperatures. Using oscillatory rheometry, scanning electron microscopy (SEM), and cell viability testing, the scaffolds were characterized, and then HUVEC proliferation and function was compared with or without LMN. The gelation time could be modified by altering the Odex/Col mass ratio as well as the temperature. SEM showed that Odex/Col hydrogels had a more regular three‐dimensional (3D) porous structure than the Col hydrogels. Moreover, HUVECs grew faster in the Col scaffold (12 mg/mL), whereas the Odex (30 mg/mL)/Col (6 mg/mL) scaffold exhibited the lowest apoptosis index. Furthermore, the expression level of vascular endothelial growth factor (VEGF) mRNA in the group without LMN was higher than that with LMN, and the Odex (30 mg/mL)/Col (6 mg/mL) scaffold without LMN had the highest VEGF protein secretion, allowing the cells to survive and function effectively. Odex/Col scaffolds, with or without LMN, are proposed as a tissue engineering construct to improve HUVEC survival and function for angiogenesis. [ABSTRACT FROM AUTHOR]

BackgroundBeyond its structural role in the skeleton, the extracellular matrix (ECM), particularly basement membrane proteins, facilitates communication with intracellular signaling pathways and cell to cell interactions to control differentiation, proliferation, migration and survival. Alterations in extracellular proteins cause a number of skeletal disorders, yet the consequences of an abnormal ECM on cellular communication remains less well understood METHODS: Clinical and radiographic examinations defined the phenotype in this unappreciated bent bone skeletal disorder. Exome analysis identified the genetic alteration, confirmed by Sanger sequencing. Quantitative PCR, western blot analyses, immunohistochemistry, luciferase assay for WNT signaling were employed to determine RNA, proteins levels and localization, and dissect out the underlying cell signaling abnormalities.  Migration and wound healing assays examined cell migration properties.FindingsThis bent bone dysplasia resulted from biallelic mutations in LAMA5, the gene encoding the alpha-5 laminin basement membrane protein. This finding uncovered a mechanism of disease driven by ECM-cell interactions between alpha-5-containing laminins, and integrin-mediated focal adhesion signaling, particularly in cartilage. Loss of LAMA5 altered β1 integrin signaling through the non-canonical kinase PYK2 and the skeletal enriched SRC kinase, FYN. Loss of LAMA5 negatively impacted the actin cytoskeleton, vinculin localization, and WNT signaling.InterpretationThis newly described mechanism revealed a LAMA5-β1 Integrin-PYK2-FYN focal adhesion complex that regulates skeletogenesis, impacted WNT signaling and, when dysregulated, produced a distinct skeletal disorder.FundingSupported by NIH awards R01 AR066124, R01 DE019567, R01 HD070394, and U54HG006493, and Czech Republic grants INTER-ACTION LTAUSA19030, V18-08-00567 and GA19-20123S.