Maintenance Of Phenotype Research Articles

OR07-6 Disrupting DNA-binding Domain Dimerization In The Androgen Receptor In Mice Results In a Partial Androgen Insensitivity Model

Abstract The androgen receptor (AR) plays a crucial role in the development and maintenance of the male phenotype, as shown in patients with androgen insensitivity syndrome (AIS). The AR is a nuclear receptor that needs to homodimerize to execute its role as transcription factor. Dimerization can occur through three different modes: via the DNA-binding domain (DBD), via the ligand-binding domain and via an interaction between the LBD and the aminoterminal domain. Dimerization via the DBD is very well known and occurs through the D-box located in the second zinc finger. DBD dimerization is generally accepted for all steroid receptors, but some of them show that D-box mutations do not inactivate them completely. However, for the AR, no monomeric transactivation capacity was described yet. To study the role of D-box independent activity of the AR, we introduced a double point mutation in the D-box that is predicted to disrupt DBD dimerization (ARDmon). These two point mutations were found in separate patients suffering from partial AIS. In vitro studies with this mutant suggest that the remaining activity depends on the androgen response element under investigation. We show that ligand binding was not affected by the D-box mutation. Furthermore, BioID assays indicated no changes in coregulatory interactions. When introducing the corresponding mutation in mice, this led to a phenotype that is intermediate between global ARKO and wild type (WT) males. Based on their anogenital distance (AGD) and the absence of nipples at early age, the ARDmon/Y mice were identified as males. Follow-up of their AGD and body weight, however, revealed clear differences with both WT males and females. Furthermore, hypospadias was observed in the ARDmon/Y mice. Testes were smaller in size compared to the WT males and first analysis showed almost no expression of well-known AR regulated genes. Reminiscent structures of the epididymis were found. In summary, the ARDmon mouse model shows an intermediate AIS phenotype. Via orchidectomy in combination with androgen replacement therapy, we will now investigate remaining androgen responses in the ARDmon/Y mice and elucidate response elements on which the AR can act in a D-box independent way. Additionally, we generated the first rodent PAIS model with a genetic cause of hypospadias. Presentation: Saturday, June 11, 2022 12:45 p.m. - 1:00 p.m.

Crosstalk between metabolic reprogramming and epigenetics in cancer: updates on mechanisms and therapeutic opportunities

Reversible, spatial, and temporal regulation of metabolic reprogramming and epigenetic homeostasis are prominent hallmarks of carcinogenesis. Cancer cells reprogram their metabolism to meet the high bioenergetic and biosynthetic demands for vigorous proliferation. Epigenetic dysregulation is a common feature of human cancers, which contributes to tumorigenesis and maintenance of the malignant phenotypes by regulating gene expression. The epigenome is sensitive to metabolic changes. Metabolism produces various metabolites that are substrates, cofactors, or inhibitors of epigenetic enzymes. Alterations in metabolic pathways and fluctuations in intermediate metabolites convey information regarding the intracellular metabolic status into the nucleus by modulating the activity of epigenetic enzymes and thus remodeling the epigenetic landscape, inducing transcriptional responses to heterogeneous metabolic requirements. Cancer metabolism is regulated by epigenetic machinery at both transcriptional and post-transcriptional levels. Epigenetic modifiers, chromatin remodelers and non-coding RNAs are integral contributors to the regulatory networks involved in cancer metabolism, facilitating malignant transformation. However, the significance of the close connection between metabolism and epigenetics in the context of cancer has not been fully deciphered. Thus, it will be constructive to summarize and update the emerging new evidence supporting this bidirectional crosstalk and deeply assess how the crosstalk between metabolic reprogramming and epigenetic abnormalities could be exploited to optimize treatment paradigms and establish new therapeutic options. In this review, we summarize the central mechanisms by which epigenetics and metabolism reciprocally modulate each other in cancer and elaborate upon and update the major contributions of the interplays between epigenetic aberrations and metabolic rewiring to cancer initiation and development. Finally, we highlight the potential therapeutic opportunities for hematological malignancies and solid tumors by targeting this epigenetic-metabolic circuit. In summary, we endeavored to depict the current understanding of the coordination between these fundamental abnormalities more comprehensively and provide new perspectives for utilizing metabolic and epigenetic targets for cancer treatment.

Designer injectable matrices of photocrosslinkable carboxymethyl cellulose methacrylate based hydrogels as cell carriers for gel type autologous chondrocyte implantation (GACI)

Gel-type autologous chondrocyte implantation (GACI) is the fourth-generation therapeutic strategy that has been introduced to address the limitations of earlier generation strategies, especially problems related to cell leakage upon implantation and loss of chondrocyte functionality owing to the dedifferentiation of cells in culture and fibrocartilage formation. In GACI, an injectable gel system is used, which acts as the cell carrier. However, the maintenance of the morphology and redifferentiation of chondrocytes with appropriate biofunctionality are major challenges in this technique. In this study, we prepared a photocrosslinkable injectable hydrogel based on carboxymethyl cellulose-methacrylate (CMC-MA) and polyethylene glycol diacrylate (PEGDA) and evaluated chondrocyte–matrix interactions and biofunctionality on different blend ratios of the gels with varying stiffness. Cell–matrix interaction was evaluated by immunostaining for actin filaments via phalloidin and cell adhesion markers such as focal adhesion kinase (FAK), integrin β1, and integrin αV. This study indicates that the stiffness of the substrates, along with the material chemistry, is a crucial factor when selecting an injectable gel-based system. Stiffer gels (2:8 CMC-MA/PEGDA) showed good chondrocyte cell attachment and growth with maintenance of the redifferentiated phenotype; therefore, they can be considered as an ideal matrix for GACI technique.

A Comprehensive Analysis and Anti-Cancer Activities of Quercetin in ROS-Mediated Cancer and Cancer Stem Cells.

Reactive oxygen species (ROS) induce carcinogenesis by causing genetic mutations, activating oncogenes, and increasing oxidative stress, all of which affect cell proliferation, survival, and apoptosis. When compared to normal cells, cancer cells have higher levels of ROS, and they are responsible for the maintenance of the cancer phenotype; this unique feature in cancer cells may, therefore, be exploited for targeted therapy. Quercetin (QC), a plant-derived bioflavonoid, is known for its ROS scavenging properties and was recently discovered to have various antitumor properties in a variety of solid tumors. Adaptive stress responses may be induced by persistent ROS stress, allowing cancer cells to survive with high levels of ROS while maintaining cellular viability. However, large amounts of ROS make cancer cells extremely susceptible to quercetin, one of the most available dietary flavonoids. Because of the molecular and metabolic distinctions between malignant and normal cells, targeting ROS metabolism might help overcome medication resistance and achieve therapeutic selectivity while having little or no effect on normal cells. The powerful bioactivity and modulatory role of quercetin has prompted extensive research into the chemical, which has identified a number of pathways that potentially work together to prevent cancer, alongside, QC has a great number of evidences to use as a therapeutic agent in cancer stem cells. This current study has broadly demonstrated the function-mechanistic relationship of quercetin and how it regulates ROS generation to kill cancer and cancer stem cells. Here, we have revealed the regulation and production of ROS in normal cells and cancer cells with a certain signaling mechanism. We demonstrated the specific molecular mechanisms of quercetin including MAPK/ERK1/2, p53, JAK/STAT and TRAIL, AMPKα1/ASK1/p38, RAGE/PI3K/AKT/mTOR axis, HMGB1 and NF-κB, Nrf2-induced signaling pathways and certain cell cycle arrest in cancer cell death, and how they regulate the specific cancer signaling pathways as long-searched cancer therapeutics.

Role of smooth muscle-derived S100A4 in an atherosclerotic mouse model

Abstract Background S100A4, a small calcium binding protein, plays an important role in vascular smooth muscle cell (SMC) phenotypic switch but its implication in atherosclerotic plaque development and particularly in SMC phenotypic plasticity is not clear yet. By neutralizing S100A4 using a monoclonal antibody, we have previously shown a reduction in overall atherosclerotic burden. However, this strategy did not distinguish the different contribution between SMCs or other S100A4-expressing cells (e.g. macrophages). Methods Herein, we used a lineage tracing mouse model in which we induced a SMC specific deletion of S100A4 (SMC-S100A4Δ/Δ) in an ApoE−/− background. High cholesterol diet was maintained for 12 weeks, after which, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed and aortas processed for staining and single cell RNA sequencing (scRNA-seq). Results We showed that S100A4 deletion modulated plaque composition rather than plaque size. ScRNA-seq analysis from SMC-S100A4Δ/Δ and SMC-S100A4wt/wt showed a reduction in macrophages and dendritic cells and an increase in total SMCs, likely participating in fibrous cap stabilization. SMC populations showed a decrease in macrophage-like inflammatory phenotype and an increase in fibroblast-like repair phenotype, as well as retention of specific SMC markers, namely Acta2 and Myh11. Gene expression analysis revealed a reduction in the expression of inflammation markers, fatty acid metabolism, and extracellular matrix-related genes, and an increase in classical SMC markers and maintenance of the contractile phenotype. Conclusion We report an intrinsic function of S100A4 in the establishment of SMC fate within the plaque and, surprisingly, a global impact on the atherosclerotic plaque microenvironnement and inflammation status. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation

Suramin enhances chondrogenic properties by regulating the p67phox/PI3K/AKT/SOX9 signalling pathway.

Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism. Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN); COL1A1; COL10A1; SRY-box transcription factor 9 (SOX9); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); interleukin (IL)-1β; tumour necrosis factor alpha (TNFα); IL-8; and matrix metallopeptidase 13 (MMP-13) in chondrocytes at both messenger RNA (mRNA) and protein levels was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. In addition, the supplementation of suramin to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by biochemical analyses and immunofluorescence, as well as by immunohistochemistry. The expression of reactive oxygen species (ROS) and NOX activity were assessed by luciferase reporter gene assay, immunofluorescence analysis, and flow cytometry. Mutagenesis analysis, Alcian blue staining, reverse transcriptase polymerase chain reaction (RT-PCR), and western blot assay were used to determine whether p67phox was involved in suramin-enhanced chondrocyte phenotype maintenance. Suramin enhanced the COL2A1 and ACAN expression and lowered COL1A1 synthesis. Also, in 3D pellet culture GAG and COL2A1 production was significantly higher in pellets consisting of chondrocytes expanded with suramin compared to controls. Surprisingly, suramin also increased ROS generation, which is largely caused by enhanced NOX (p67phox) activity and membrane translocation. Overexpression of p67phox but not p67phoxAD (deleting amino acid (a.a) 199 to 212) mutant, which does not support ROS production in chondrocytes, significantly enhanced chondrocyte phenotype maintenance, SOX9 expression, and AKT (S473) phosphorylation. Knockdown of p67phox with its specific short hairpin (sh) RNA (shRNA) abolished the suramin-induced effects. Moreover, when these cells were treated with the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor LY294002 or shRNA of AKT1, p67phox-induced COL2A1 and ACAN expression was significantly inhibited. Suramin could redifferentiate dedifferentiated chondrocytes dependent on p67phox activation, which is mediated by the PI3K/AKT/SOX9 signalling pathway.Cite this article: Bone Joint Res2022;11(10):693-708.

Semaglutide (GLP-1 receptor agonist) stimulates browning on subcutaneous fat adipocytes and mitigates inflammation and endoplasmic reticulum stress in visceral fat adipocytes of obese mice.

Semaglutide (GLP-1 agonist) was approved for treating obesity. Although the effects on weight loss and metabolism are known, the responses of adipocytes to semaglutide are yet limited. C57BL/6 male mice (n = 20/group) were fed a control diet (C) or a high-fat(HF)diet for 16 weeks and then separated into four groups (n = 10/group) for an additional four weeks: C, C diet and semaglutide, HF, and HF diet and semaglutide. Epididymal white adipose tissue(eWAT) and subcutaneous white adipose tissue(sWAT) fat pads were studied with biochemistry, immunohistochemistry/fluorescence, stereology, and reverse transcription-quantitative polymerase chain reaction. In obese mice, semaglutide reduced the fat pad masses (eWAT, -55%; sWAT, -40%), plasmatic cytokines, and proinflammatory gene expressions: tumor necrosis factor-alpha (-60%); interleukin (IL)-6 (-55%); IL-1beta (-40%); monocyte chemoattractant protein-1 (-90%); andleptin (-80%). Semaglutide also lessened endoplasmic reticulum (ER) stress genes of activating transcription factor-4 (-85%), CCAAT enhancer-binding protein homologous protein (-55%), and growth arrest and DNA damage-inducible gene 45 (-45%). The obese mice's adipocyte hypertrophy and macrophage infiltration were equally reduced by semaglutide. Semaglutide enhanced multiloculation and uncoupled protein 1 (UCP1) labeling in obese mice: peroxisome proliferator-activated receptor-alpha (+560%) and gamma (+150%), fibronectin type III domain-containing protein 5 (+215%), peroxisome proliferator-activated receptor-alpha coactivator (+110%), nuclear respiratory factor 1 (+260%), and mitochondrial transcription factor A (+120%). Semaglutide also increased thermogenetic gene expressions for the browning phenotype maintenance: beta-3 adrenergic receptor (+520%), PR domain containing 16 (+90%), and Ucp1 (+110%). In conclusion, semaglutide showed significant beneficial effects beyond weight loss, directly on fat pads and adipocytes of obese mice, remarkably anti-inflammatory, and reduced adipocyte size and ER stress. Besides, semaglutide activated adipocyte browning, improving UCP1, mitochondrial biogenesis, and thermogenic marker expressions help weight loss.

Shining Light on Autophagy in Skin Pigmentation and Pigmentary Disorders.

Autophagy is a vital process for cell survival and it preserves homeostasis by recycling or disassembling unnecessary or dysfunctional cellular constituents. Autophagy ameliorates skin integrity, regulating epidermal differentiation and constitutive pigmentation. It induces melanogenesis and contributes to skin color through melanosome turnover. Autophagy activity is involved in skin phenotypic plasticity and cell function maintenance and, if altered, it concurs to the onset and/or progression of hypopigmentary and hyperpigmentary disorders. Overexpression of autophagy exerts a protective role against the intrinsic metabolic stress occurring in vitiligo skin, while its dysfunction has been linked to the tuberous sclerosis complex hypopigmentation. Again, autophagy impairment reduces melanosome degradation by concurring to pigment accumulation characterizing senile lentigo and melasma. Here we provide an updated review that describes recent findings on the crucial role of autophagy in skin pigmentation, thus revealing the complex interplay among melanocyte biology, skin environment and autophagy. Hence, targeting this process may also represent a promising strategy for treating pigmentary disorders.

Maintaining Myofibroblastic-Like Cancer-Associated Fibroblasts by Cancer Stemness Signal Transduction Feedback Loop.

Background: Myofibroblast-like cancer-associated fibroblasts (myCAF) in the tumor microenvironment (TME) promote cancer stemness, growth, and metastasis. Cancer cell-derived osteopontin (OPN) has been reported as a biomarker related to malignant cancer growth. In this study, we confirm that cancer cell stemness is required for the maintenance of an OPN-induced myCAF phenotype. Methods: MDA-MB-231 or HepG2 cells and Sox2 knockout variants were co-cultured with human mesenchymal stem cells (MSC). In selected instances, the OPN bioactivity inhibitor OPN-R3 aptamer (APT), OPN-R3 mutant aptamer (MuAPT), or cancer cell stemness inhibitor BBI-608 were added separately. MDA-MB-231 cancer stemness and myCAF markers were quantified by real-time PCR. Stemness-lacking cancer cell mice models were created to confirm that stemness is required for the maintenance of the OPN-induced myCAF phenotype in vivo. Results: In an MDA-MB-231 co-culture system, myCAF and stemness markers increased. Osteopontin and stemness blockade in this co-culture system decreased both myCAF and stemness marker expression, but OPN blockade after 72 hours had no effect. In contrast, when BBI608 was added at 72 hours, myCAF markers were abated after 36-hour treatment. Replacing wildtype with MDA-MB-231(-/-sox2) in co-cultures at 72 hours decreased myCAF marker expression to baseline despite the Western blot confirming the presence of OPN. Conversely, replacing MDA-MB-231(-/-sox2) cells with wildtype increased myCAF marker expression to a level equivalent to the MDA-MB-231+MSC co-culture system. In vivo osteopontin blockade diminished stemness and myCAF marker expression and stemness lacking cancer cell models, indicated by decreasing myCAF presence. Experiments were repeated in a HepG2 cell line with identical results. Conclusions: Cancer and myCAF crosstalk increases myCAF maintenance and cancer cell stemness. In this study using human breast and liver cancer cell lines, maintenance of the OPN-induced myCAF phenotype also requires cancer stemness. This indicates that the myCAF phenotype requires two distinct signaling pathways: initiation and maintenance.

G-quadruplexes formation within the promoter of TEAD4 oncogene and their interaction with Vimentin.

G-quadruplexes (G4s) are nucleic acid secondary structures detected within human chromosomes, that cluster at gene promoters and enhancers. This suggests that G4s may play specific roles in the regulation of gene expression. Within a distinct subgroup of G-rich domains, the formation of two or more adjacent G4 units (G4-repeats) is feasible. Recently it was shown that Vimentin, a protein highly expressed within mesenchymal cells, selectively recognizes these arrangements. Putative G4-repeats have been searched within the human gene proximal promoters by the bioinformatics tool QPARSE and they resulted to be enriched at genes related to epithelial-to-mesenchymal transition (EMT). This suggested that Vimentin binding at these sites might be relevant for the maintenance of the mesenchymal phenotype. Among all the identified sequences, in the present study we selected the one located within the promoter of the TEAD4 oncogene. TEAD4 codifies for a transcriptional enhancer factor, TEAD4, that actively promotes EMT, supporting, cell proliferation and migration. Moreover, in colorectal cancer cells TEAD4 directly enhances the expression of Vimentin. Thus, the possible interaction of Vimentin with TEAD4 promoter could highlight a positive feedback loop between these two factors, associated to important tumor metastasis related events. Here, we exploited spectroscopic and electrophoretic measurements under different conditions to address the folding behavior of the selected sequence. This allowed us to validate the folding of TEAD4 promoter into a G4-repeat able to interact with Vimentin.

Targeting of MCL-1 in breast cancer-associated fibroblasts reverses their myofibroblastic phenotype and pro-invasive properties

Cancer-associated fibroblasts (CAF) are a major cellular component of epithelial tumors. In breast cancers in particular these stromal cells have numerous tumorigenic effects in part due to their acquisition of a myofibroblastic phenotype. Breast CAFs (bCAFs) typically express MCL-1. We show here that pharmacological inhibition or knock down of this regulator of mitochondrial integrity in primary bCAFs directly derived from human samples mitigates myofibroblastic features. This decreases expression of genes involved in actomyosin organization and contractility (associated with a cytoplasmic retention of the transcriptional regulator, yes-associated protein—YAP) and decreases bCAFs ability to promote cancer cells invasion in 3D coculture assays. Our findings underscore the usefulness of targeting MCL-1 in breast cancer ecosystems, not only to favor death of cancer cells but also to counteract the tumorigenic activation of fibroblasts with which they co-evolve. Mechanistically, pharmacological inhibition of MCL-1 with a specific BH3 mimetic promotes mitochondrial fragmentation in bCAFs. Inhibition of the mitochondrial fission activity of DRP-1, which interacts with MCL-1 upon BH3 mimetic treatment, allows the maintenance of the myofibroblastic phenotype of bCAFs.

Transgenic overexpression of α7 integrin in smooth muscle attenuates allergen-induced airway inflammation in a murine model of asthma.

Asthma is a chronic inflammatory disorder of the lower airways characterized by modulation of airway smooth muscle (ASM) function. Infiltration of smooth muscle by inflammatory mediators is partially regulated by transmembrane integrins and the major smooth muscle laminin receptor α7β1 integrin plays a critical role in the maintenance of ASM phenotype. The goal of the current study was to investigate the role of α7 integrin in asthma using smooth muscle-specific α7 integrin transgenic mice (TgSM-Itgα7) using both acute and chronic OVA sensitization and challenge protocols that mimic mild to severe asthmatic phenotypes. Transgenic over-expression of the α7 integrin in smooth muscle resulted in a significant decrease in airway resistance relative to controls, reduced the total number of inflammatory cells and substantially inhibited the production of crucial Th2 and Th17 cytokines in airways. This was accompanied by decreased secretion of various inflammatory chemokines such as eotaxin/CCL11, KC/CXCL3, MCP-1/CCL2, and MIP-1β/CCL4. Additionally, α7 integrin overexpression significantly decreased ERK1/2 phosphorylation in the lungs of TgSM-Itgα7 mice and affected proliferative, contractile, and inflammatory downstream effectors of ERK1/2 that drive smooth muscle phenotype in the lung. Taken together, these results support the hypothesis that enhanced expression of α7 integrin in vivo inhibits allergic inflammation and airway resistance. Moreover, we identify ERK1/2 as a potential target by which α7 integrin signals to regulate airway inflammation. We conclude that identification of therapeutics targeting an increase in smooth muscle α7 integrin expression could serve as a potential novel treatment for asthma.

Reprogramming—Evolving Path to Functional Surrogate β-Cells

Numerous cell sources are being explored to replenish functional β-cell mass since the proof-of -concept for cell therapy of diabetes was laid down by transplantation of islets. Many of these cell sources have been shown to possess a degree of plasticity permitting differentiation along new lineages into insulin-secreting β-cells. In this review, we explore emerging reprograming pathways that aim to generate bone fide insulin producing cells. We focus on small molecules and key transcriptional regulators that orchestrate phenotypic conversion and maintenance of engineered cells.

Synthetic Extracellular Matrices for 3D Culture of Schwann Cells, Hepatocytes, and HUVECs.

Synthetic hydrogels from polyisocyanides (PIC) are a type of novel thermoreversible biomaterials, which can covalently bind biomolecules such as adhesion peptides to provide a suitable extracellular matrix (ECM)-like microenvironment for different cells. Although we have demonstrated that PIC is suitable for three-dimensional (3D) culture of several cell types, it is unknown whether this hydrogel sustains the proliferation and passaging of cells originating from different germ layers. In the present study, we propose a 3D culture system for three representative cell sources: Schwann cells (ectoderm), hepatocytes (endoderm), and endothelial cells (mesoderm). Both Schwann cells and hepatocytes proliferated into multicellular spheroids and maintained their properties, regardless of the amount of cell-adhesive RGD motifs in long-term culture. Notably, Schwann cells grew into larger spheroids in RGD-free PIC than in PIC-RGD, while HL-7702 showed the opposite behavior. Endothelial cells (human umbilical vein endothelial cells, HUVECs) spread and formed an endothelial cell (EC) network only in PIC-RGD. Moreover, in a hepatocyte/HUVEC co-culture system, the characteristics of both cells were well kept for a long period in PIC-RGD. In all, our work highlights a simple ECM mimic that supports the growth and phenotype maintenance of cells from all germ layers in the long term. Our findings might contribute to research on biological development, organoid engineering, and in vitro drug screening.

Development of high resilience spiral wound suture-embedded gelatin/PCL/heparin nanofiber membrane scaffolds for tendon tissue engineering

This study develops a spiral wound scaffold based on gelatin/PCL/heparin (GPH) nanofiber membranes for tendon tissue engineering. By embedding sutures in dual layers of aligned GPH nanofiber membranes, prepared from mixed electrospinning of gelatin and PCL/heparin solutions, we fabricate a high resilience scaffold intended for the high loading environment experienced by tendons. The basic fibroblast growth factor (bFGF) was anchored to GPH scaffold through bioaffinity between heparin and bFGF, aim to provide biological cues for maintenance of tenogenic phenotype. In addition, the aligned nanofiber morphology is expected to provide physical cues toward seeded tenocytes. With sustained release of bFGF, GPH-bFGF can enhance proliferation, up-regulate tenogenic gene expression, and increase synthesis of tendon-specific proteins by tenocytes in vitro. Furthermore, by properly maintaining tendon phenotypes, GPH-bFGF/tenocytes constructs showed improved mechanical properties over GPH-bFGF. From in vivo study using GPH-bFGF/tenocytes constructs to repair rabbit Achilles tendon defects, neotendon tissue formation was confirmed from histological staining and biomechanical analysis. These findings collectively demonstrate that the newly designed GPH-bFGF scaffold could provide a niche for inducing tendon tissue regeneration by effectively restoring the tendon tissue structure and function.

Miat and interacting protein Metadherin maintain a stem-like niche to promote medulloblastoma tumorigenesis and treatment resistance

Long noncoding RNAs (lncRNAs) play essential roles in the development and progression of many cancers. However, the contributions of lncRNAs to medulloblastoma (MB) remain poorly understood. Here, we identify Miat as an lncRNA enriched in the sonic hedgehog group of MB that is required for maintenance of a treatment-resistant stem-like phenotype in the disease. Loss of Miat results in the differentiation of tumor-initiating, stem-like MB cells and enforces the differentiation of tumorigenic stem-like MB cells into a nontumorigenic state. Miat expression in stem-like MB cells also facilitates treatment resistance by down-regulating p53 signaling and impairing radiation-induced cell death, which can be reversed by therapeutic inhibition of Miat using antisense oligonucleotides. Mechanistically, the RNA binding protein Metadherin (Mtdh), previously linked to resistance to cytotoxic therapy in cancer, binds to Miat in stem-like MB cells. Like the loss of Miat, the loss of Mtdh reduces tumorigenicity and increases sensitivity to radiation-induced death in stem-like MB cells. Moreover, Miat and Mtdh function to regulate the biogenesis of several microRNAs and facilitate tumorigenesis and treatment resistance. Taken together, these data reveal an essential role for the lncRNA Miat in sustaining a treatment-resistant pool of tumorigenic stem-like MB cells.

Identification regulatory noncoding RNAs of human papilloma virus type 16 (Papillomaviridae: Alphapapillomavirus: Human papillomavirus) in cervical tumors

High carcinogenic-risk human papillomaviruses (hrHPVs) are recognized as etiological agents of cervical cancer. Constant expression of the viral oncoproteins, E6 and E7, is required for maintenance of the malignant phenotype of tumor cells. The exact mechanism of regulation of viral oncogenes expression in tumor cells is not fully elucidated. identification of viral noncoding RNAs (ncRNAs) in HPV16-positve cervical cancer. The reverse transcription polymerase chain reactions were used to detect viral ncRNAs in HPV16-positve primary cervical squamous cell carcinomas and SiHa and CasKi cell lines. The knockdown technique with oligonucleotides complementary to ncRNAs was used to elucidate their functions. We have identified ncRNAs transcribed in the upstream regulatory region of HPV16 in the cervical carcinoma cell lines and in 32 out 32 cervical squamous cell carcinomas with episomal or integrated forms of HPV16 DNA. Knockdown of sense or antisense strains of ncRNAs by oligonucleotides results in a decrease or increase of the E6 and E7 oncogenes mRNA levels in cells, respectively. These changes of oncogenes mRNA levels are accompanied by the modulation of the levels of the p53 protein, the main target of the E6 oncoprotein. The presence of regulatory ncRNAs in all examined tumors and cell lines revealed for the first time indicates their necessity for maintenance of constant expression of E6 and E7 oncogenes in them. The findings can be useful for understanding of the fundamental aspects of the viral expression regulation in HPV16-positive tumors.

Transcriptional Profile of the Developing Subthalamic Nucleus.

The subthalamic nucleus (STN) is a small, excitatory nucleus that regulates the output of basal ganglia motor circuits. The functions of the STN and its role in the pathophysiology of Parkinson’s disease are now well established. However, some basic characteristics like the developmental origin and molecular phenotype of neuronal subpopulations are still being debated. The classical model of forebrain development attributed the origin of STN within the diencephalon. Recent studies of gene expression patterns exposed shortcomings of the classical model. To accommodate these findings, the prosomeric model was developed. In this concept, STN develops within the hypothalamic primordium, which is no longer a part of the diencephalic primordium. This concept is further supported by the expression patterns of many transcription factors. It is interesting to note that many transcription factors involved in the development of the STN are also involved in the pathogenesis of neurodevelopmental disorders. Thus, the study of neurodevelopmental disorders could provide us with valuable information on the roles of these transcription factors in the development and maintenance of STN phenotype. In this review, we summarize historical theories about the developmental origin of the STN and interpret the gene expression data within the prosomeric conceptual framework. Finally, we discuss the importance of neurodevelopmental disorders for the development of the STN and its potential role in the pathophysiology of neurodevelopmental disorders.

Tissue origin matters: Maintenance of tenogenic phenotype on the tendon and not skin collagen‐derived devices

AbstractRecent data suggest that collagen retains the memory of the tissue that it derives from. With this in mind, collagen (from bovine skin and tendon) sponges were fabricated with different crosslinking densities, and their physicochemical and biological properties were assessed. As the crosslinking density was increased, the resistance to collagenase digestion, denaturation temperature, compressive stress, and compressive modulus was significantly increased and the free amine content, % swelling, and human dermal fibroblast cytocompatibility were significantly reduced. The tendon‐collagen‐derived scaffolds exhibited significantly higher compressive stress and compressive modulus values and induced significantly higher human tenocyte DNA concentration and metabolic activity than the skin‐collagen‐derived scaffolds. In human tenocyte cultures on day 14, the 1 mM 4‐arm polyethylene‐glycol succinimidyl glutarate tendon‐collagen‐derived collagen sponges induced significantly higher collagen type III synthesis (as expected at early stages of physiological tendon healing) and downregulated actin alpha 2 (associated with myofibroblast differentiation) and the skin‐collagen‐derived collagen sponges induced significantly higher collagen type IV synthesis (found primarily at the dermal‐epidermal junction) and upregulated prolyl 4‐hydroxylase subunit alpha‐1 (associated with collagen biosynthesis and constitutes a target for antifibrotic compounds). Our data indicate that the tissue from which collagen is extracted should be considered in the development of medical devices.

Acute And Chronic NGF Responses During A Training Season In Young Swimmers

Nerve Growth Factor (NGF) is crucial for the functional accuracy of the cholinergic neurons in the central nervous system and for the growth and phenotypic maintenance of neurons in the peripheral nervous system. PURPOSE: We investigated the acute and chronic effects of a swimming training season of young athletes on serum NGF, both at rest and after a maximal exercise bout. METHODS: Eleven well-trained male swimmers (14.08 ± 1.0 yrs) participated in the study. Measurements were carried out at the beginning of the training season (T1) as well as pre- and post- taper of each of the two competitive periods (i.e., T2, T3 for the first macrocycle, and T4, T5 for the second macrocycle, respectively). Blood samples were drawn at pre- and 1 hour post a maximal, 400 m swimming testing. Serum NGF levels were measured by ELISA. Adjustment for exercise-induced plasma volume changes was performed before data analysis. Two-way ANOVA with repeated measures was used for statistics. RESULTS: Rest NGF values were not significantly different throughout the experimental period, although there was a tendency for them to increase from T1 to T5 (56%: T1 15.56 ± 1.4 vs T5 24.23 ± 5.6; p > 0.05). No significant differences were found among the post 400 m testing timepoints or between pre-post testing (p > 0.05) (T1: 15.56 ± 1.4 vs 19.31 ± 5.0; p > 0.05, T2: 15.16 ± 1.9 vs 14.35 ± 1.7; p > 0.05, T3: 16.34 ± 1.3 vs 13.99 ± 1.8; p > 0.05, T4: 16.81 ± 1.7 vs 17.18 ± 2.3; p > 0.05, T5: 24.23 ± 5.6 vs 18.27 ± 2.8; p > 0.05). CONCLUSIONS: This data suggest that long-term swimming training may affect the resting concentrations of circulating NGF in young swimmers. More studies are needed to further examine the NGF responses at different timepoints during the recovery period after acute bout(s) of swimming exercise.