Pax6 Expression Research Articles

Effects of electroacupuncture on gait and proliferation and differentiation of muscle satellite cell in rats with acute blunt trauma of gastrocnemius muscle

To observe the effects of electroacupuncture on threshold of pain, gait, proliferation and differentiation of muscle satellite cell in rats with acute blunt trauma of gastrocnemius muscle, and to explore the possible mechanism of electroacupuncture in promoting the repair of acute injury of skeletal muscle. A total of 48 SD rats were randomly divided into a blank group (6 rats), a model group (24 rats) and an electroacupuncture group (18 rats). In the model group and the electroacupuncture group, the model of acute blunt trauma of gastrocnemius muscle was established by self-made impactor. In the electroacupuncture group, electroacupuncture was applied at "Chengshan" (BL 57) and "Yanglingquan" (GB 34) on the right side, with disperse-dense wave, in frequency of 2 Hz/100 Hz, once a day, 30 min each time. Electroacupuncture intervention was performed for 3, 7 and 14 days according to the sampling time. On the 1st, 3rd, 7th and 14th days after modeling, the mechanical withdrawal pain threshold of hindfoot was detected by Von Frey method; the standing time and the maximum contact area of the right hindfoot were recorded by Cat Walk XTTM animal gait analysis instrument; the morphology of the right gastrocnemius muscle and the number of inflammatory cells were observed by HE staining; the positive expression of paired box gene 7 (Pax7) and myogenic differentiation (MyoD) of the right gastrocnemius muscle was detected by immunofluorescence. After modeling, the muscle fiber rupture and massive infiltration of red blood cells and inflammatory cells were observed in the right gastrocnemius muscle; after electroacupuncture intervention, the morphology of muscle fiber was intact and the infiltration of inflammatory cells was improved. Compared with the blank group, in the model group, the differences of mechanical withdrawal pain threshold between the left and right foot were increased (P<0.05), the standing time was shortened and the maximum contact area of the right hindfoot was decreased (P<0.05), the number of inflammatory cells and the positive expression of Pax7 and MyoD of the right gastrocnemius muscle were increased (P<0.05) on the 1st, 3rd, 7th and 14th days after modeling. Compared with the model group, in the electroacupuncture group, the differences of mechanical withdrawal pain threshold were decreased (P<0.05), the standing time was prolonged (P<0.05), the number of inflammatory cells of right gastrocnemius muscle was decreased (P<0.05) on the 7th and 14th days after modeling; the maximum contact area of the right hindfoot was increased (P<0.05), the positive expression of MyoD of the right gastrocnemius muscle was increased (P<0.05) on the 3rd, 7th and 14th days after modeling; the positive expression of Pax7 of the right gastrocnemius muscle was increased (P<0.05) on the 3rd day after modeling. Electroacupuncture can effectively improve the pain threshold and gait in rats with acute blunt trauma of gastrocnemius muscle, and promote the repair of skeletal muscle injury, the mechanism may be related to the up-regulation of Pax7 and MyoD, so as to promoting the proliferation and differentiation of muscle satellite cell.

Dipyridamole activates adenosine A2B receptor and AMPK/cAMP signaling and promotes myogenic differentiation of myoblastic C2C12 cells.

Introduction: Sarcopenia is defined as a loss of muscle mass and strength. ATP homeostasis is crucial during myogenesis. We determined how the purinergic system modulates myogenesis using dipyridamole (blocks adenosine taken up by the cells) and tenofovir (inhibits ATP release) in a myoblast cell line. Methods: C2C12 cells were differentiated in the presence/absence of tenofovir/dipyridamole, with/without the A2B selective inhibitor PSB-603. Extra-/intracellular nucleotides were examined via HPLC. The expression of muscle differentiation proteins (Pax7, Mif5, MyoD, MyoG, and MHC), PKA/CREB, adenosine receptors (A1, A2A, A2B, and A3), ATP-channel pannexin-1 and the P2X7 receptor was analyzed via WB and RT-PCR. cAMP and AMPK activation was measured. Results: Tenofovir increased intracellular ATP and reduced extracellular adenosine, decreasing Pax7 expression and increasing MHC expression prematurely. Dipyridamole increased intracellular AMP and extracellular adenosine, counteracting the premature myogenesis promoted by tenofovir. All adenosine receptors were expressed during differentiation with dipyridamole, increasing A2B expression. Tenofovir maintained inactive AMPK and decreased cAMP levels, as well as PKAα and pCREB expression, which were recovered with dipyridamole. Discussion: Adenosine and ATP act as mediators in muscle myogenesis. The blockade of ATP release by tenofovir promotes premature myogenesis, with dipyridamole counteracting the premature differentiation promoted by tenofovir via the adenosine A2B receptor and cAMP/AMPK pathways. Therefore, dipyridamole might be of interest as a therapeutic approach in sarcopenia.

In vitro-generated human muscle reserve cells are heterogeneous for Pax7 with distinct molecular states and metabolic profiles

BackgroundThe capacity of skeletal muscles to regenerate relies on Pax7+ muscle stem cells (MuSC). While in vitro-amplified MuSC are activated and lose part of their regenerative capacity, in vitro-generated human muscle reserve cells (MuRC) are very similar to quiescent MuSC with properties required for their use in cell-based therapies.MethodsIn the present study, we investigated the heterogeneity of human MuRC and characterized their molecular signature and metabolic profile.ResultsWe observed that Notch signaling is active and essential for the generation of quiescent human Pax7+ MuRC in vitro. We also revealed, by immunofluorescence and flow cytometry, two distinct subpopulations of MuRC distinguished by their relative Pax7 expression. After 48 h in differentiation medium (DM), the Pax7High subpopulation represented 35% of the total MuRC pool and this percentage increased to 61% after 96 h in DM. Transcriptomic analysis revealed that Pax7High MuRC were less primed for myogenic differentiation as compared to Pax7Low MuRC and displayed a metabolic shift from glycolysis toward fatty acid oxidation. The bioenergetic profile of human MuRC displayed a 1.5-fold decrease in glycolysis, basal respiration and ATP-linked respiration as compared to myoblasts. We also observed that AMPKα1 expression was significantly upregulated in human MuRC that correlated with an increased phosphorylation of acetyl-CoA carboxylase (ACC). Finally, we showed that fatty acid uptake was increased in MuRC as compared to myoblasts, whereas no changes were observed for glucose uptake.ConclusionsOverall, these data reveal that the quiescent MuRC pool is heterogeneous for Pax7 with a Pax7High subpopulation being in a deeper quiescent state, less committed to differentiation and displaying a reduced metabolic activity. Altogether, our data suggest that human Pax7High MuRC may constitute an appropriate stem cell source for potential therapeutic applications in skeletal muscle diseases.

Vitamin A injection at birth improves muscle growth in lambs.

Vitamin A and its metabolite, retinoic acid (RA) play important roles in regulating skeletal muscle development. This study was conducted to investigate the effects of early intramuscular vitamin A injection on the muscle growth of lambs. A total of 16 newborn lambs were given weekly intramuscular injections of corn oil (control group, n=8) or 7,500 IU vitamin A palmitate (vitamin A group, n=8) from birth to 3wk of age (4 shots in total). At 3wk of age and weaning, biceps femoris muscle samples were taken to analyze the effects of vitamin A on the myogenic capacity of skeletal muscle cells. All lambs were slaughtered at 8 months of age. The results suggest that vitamin A treatment accelerated the growth rate of lambs and increased the loin eye area (P<0.05). Consistently, vitamin A increased the diameter of myofibers in longissimus thoracis muscle (P<0.01) and increased the final body weight of lambs (P<0.05). Vitamin A injection did not change the protein kinase B/mammalian target of rapamycin and myostatin signaling (P>0.05). Moreover, vitamin A upregulated the expression of PAX7 (P<0.05) and the myogenic marker genes including MYOD and MYOG (P<0.01). The skeletal muscle-derived mononuclear cells from vitamin A-treated lambs showed higher expression of myogenic genes (P<0.05) and formed more myotubes (P<0.01) when myogenic differentiation was induced invitro. In addition, invitro analysis showed that RA promoted myogenic differentiation of the skeletal muscle-derived mononuclear cells in the first 3d(P<0.05) but not at the later stage (P>0.05) as evidenced by myogenic gene expression and fusion index. Taken together, neonatal intramuscular vitamin A injection promotes lamb muscle growth by promoting the myogenic potential of satellite cells.

BET inhibition targets ABC-DLBCL constitutive B-cell receptor signaling through PAX5.

B-cell receptor (BCR) signaling is essential for the diffuse large B-cell lymphoma (DLBCL) subtype that originates from activated B-cells (ABCs). ABC-DLBCL cells are sensitive to Bruton tyrosine kinase intervention. However, patients with relapsed or refractory ABC-DLBCL had overall response rates from 33% to 37% for Bruton tyrosine kinase inhibitors, suggesting the evaluation of combination-based treatment for improved efficacy. We investigated the efficacy and mechanism of the bromodomain and extraterminal motif (BET) inhibitor AZD5153 combined with the Bruton tyrosine kinase inhibitor acalabrutinib in ABC-DLBCL preclinical models. AZD5153 is a bivalent BET inhibitor that simultaneously engages the 2 bromodomains of BRD4. Adding AZD5153 to acalabrutinib demonstrated combination benefits in ABC-DLBCL cell line and patient-derived xenograft models. Differential expression analyses revealed PAX5 transcriptional activity as a novel downstream effector of this drug combination. PAX5 is a transcription factor for BCR signaling genes and may be critical for perpetually active BCR signaling in ABC-DLBCL. Our analyses further indicated significant alterations in BCR, RELB/alternative NF-κB, and toll-like receptor/interferon signaling. Validation of these results mapped a positive-feedback signaling loop regulated by PAX5. We demonstrated that AZD5153 decreased PAX5 expression, whereas acalabrutinib disruption of BCR signaling inhibited PAX5 activation. Furthermore, several interferon levels were decreased by AZD5153 and acalabrutinib in tumors. Adding interferon-beta1 (IFNβ1) to cells treated with acalabrutinib partially rescued PAX5 activation. Our results demonstrate that AZD5153 enhances the efficacy of acalabrutinib through PAX5 and BCR mechanisms that are critical for ABC-DLBCL.

Limbal Stem Cell Dysfunction Induced by Severe Dry Eye via Activation of the p38 MAPK Signaling Pathway

Severe dry eye (SDE) can cause grievous damage of the ocular surface and result in vision impairment and even blindness. To investigate the fate of limbal stem cells in SDE and the underlying mechanism, we established an SDE rat model by removing the extraorbital and infraorbital lacrimal glands and maintaining them in a low-humidity environment. One month after the surgery, aqueous tear secretion was reduced dramatically, blood vessels invaded into the central cornea, and inflammatory cells infiltrated into the limbal stroma. The expression of K12 and Pax6 were down-regulated dramatically, while those of K10, Sprr1b, and MUC5AC were up-regulated in the corneal epithelium of the SDE rats. Cell proliferation in the limbal epithelium was up-regulated, while the stem/progenitor marker adenosine 5'-triphosphate-binding cassette member 2 and the limbal epithelial colony-forming efficiency were decreased in the SDE condition. Furthermore, the p38 mitogen-activated protein kinase signaling pathway was activated in the limbal corneal epithelium of SDE rats. The abnormal differentiation and stemness loss in the cornea epithelium could be reversed upon treatment with a p38 inhibitor in a SDE invivo model and invitro hyperosmolar corneal epithelial culture conditions. In conclusion, SDE can lead to limbal stem cell dysfunction, and p38 mitogen-activated protein kinase signaling pathway activation plays an essential role in this process.

Production and limbal lineage commitment of aniridia patient-derived induced pluripotent stem cells.

Congenital aniridia is caused by heterozygous mutations on the PAX6 gene leading to reduced amount of PAX6 protein (haploinsufficiency), abnormal eye development and aniridia-associated keratopathy (AAK). This progressive corneal opacification resembles late-onset limbal stem cell (LSC) deficiency, leading to disrupted corneal epithelial renewal. The factors leading to AAK are not known and defects in native LSC differentiation and/or features leading to ocular surface dysfunction like inflammation and loss of innervation could contribute to development of AAK. Here, we produced induced pluripotent stem cells (hiPSC) from three AAK patients and examined whether PAX6 haploinsufficiency affects LSC lineage commitment. During LSC differentiation, characterization of the AAK lines showed lowered PAX6 expression as compared to wild type (WT) controls and expression peak of PAX6 during early phase of differentiation was detected only in the WT hiPSC lines. Whether it reflects developmental regulation remains to be studied further. Nevertheless, the AAK-hiPSCs successfully differentiated towards LSC lineage, in line with the presence of LSCs in young patients before cell loss later in life. In addition, patient specific LSCs showed similar wound healing capacity as WT cells. However, extensive batch-related variation in the LSC marker expression and wound healing efficacy was detected without clear correlation to AAK. As development and maintenance of corneal epithelium involves an interplay between LSCs and their environment, the AAK-hiPSCs generated here can be further used to study the crosstalk between LSCs and limbal niche including e.g. corneal immune cells, stroma cells and neurons.

Single-nucleus RNA sequencing of developing superior colliculus identifies neuronal diversity and candidate mediators of circuit assembly

The superior colliculus (SC) is a sensorimotor structure in the midbrain that integrates input from multiple sensory modalities to initiate motor commands. It undergoes well-characterized steps of circuit assembly during development, rendering the mouse SC a popular model to study establishment of neural connectivity. Here we perform single-nucleus RNA-sequencing analysis of the mouse SC isolated at various developmental time points. Our study provides a transcriptomic landscape of the cell types that comprise the SC across murine development with particular emphasis on neuronal heterogeneity. We report a repertoire of genes differentially expressed across the different postnatal ages, many of which are known to regulate axon guidance and synapse formation. Using these data, we find that Pax7 expression is restricted to a subset of GABAergic neurons. Our data provide a valuable resource for interrogating the mechanisms of circuit development and identifying markers for manipulating specific SC neuronal populations and circuits.

The fibronectin concentration that optimally maintains porcine satellite cells.

'Cultured meat' has been suggested as means of solving the problems associated with overpopulation and gas emissions. Satellite cells are a major component in the production of cultured meat; however, these cells cannot be maintained in vitro over long periods. Fibronectin is a glycoprotein that affects biological processes such as cell adhesion, differentiation, and migration. Unfortunately, the characteristics of porcine satellite cells grown in a long-term culture when exposed to fibronectin-coated dishes are unknown. The objective of this study was to investigate the appropriate concentration of fibronectin coated dishes for proliferation and maintenance of porcine satellite cells at long-term culture. In this study, we isolated the satellite cells and fibroblast cells with pre-plating method. We next analyzed the cell doubling time, cell cycle, and rate of expressed paired box 7 (Pax7) and myogenic differentiation 1 (MyoD1) in porcine satellite cells cultured with 20 μg/mL of fibronectin-, gelatin-, and non-coated dishes at early and late passage. We then analyzed the proliferation of porcine satellite cells with various concentrations of mixed gelatin/fibronectin. We next determined the optimal concentration of fibronectin that would encourage proliferation and maintenance of porcine satellite cells in a long-term culture. Doubling time was lowest when 20 μg/mL of fibronectin was used (as tested during an early and late passage). Levels of expressed Pax7 and MyoD1, assessed using immunocytochemistry, were highest in cells grown using fibronectin-coated dishes. The proliferation of gelatin/fibronectin mixed coatings had no significant effect on porcine satellite cells. The concentration of 5 μg/mL fibronectin coated dishes showed the lowest doubling time and maintained expression of Pax7. Fibronectin with 5μg/mL effectively maintains porcine satellite cells, a discovery that will be of interest to those developing the next generation of artificial meats.

Traditional Chinese Medicine for Topical Treatment of Skeletal Muscle Injury.

Muscle injuries are common musculoskeletal problems, but the pharmaceutical agent for muscle repair and healing is insufficient. Traditional Chinese Medicine (TCM) frequently uses topical treatments to treat muscle injuries, although scientific evidence supporting their efficacy is scarce. In this study, an in vitro assay was used to test the cytotoxicity of a topical TCM formula containing Carthami Flos, Dipsaci Radix, and Rhei Rhizoma (CDR). Then, a muscle contusion rat model was developed to investigate the in vivo effect and basic mechanisms underlying CDR on muscle regeneration. The in vitro assay illustrated that CDR was non-cytotoxic to immortalized rat myoblast culture and increased cell viability. Histological results demonstrated that the CDR treatment facilitated muscle repair by increasing the number of new muscle fibers and promoting muscle integrity. The CDR treatment also upregulated the expression of Pax7, MyoD and myogenin, as evidenced by an immunohistochemical study. A gene expression analysis indicated that the CDR treatment accelerated the regeneration and remodeling phases during muscle repair. This study demonstrated that topical CDR treatment was effective at facilitating muscle injury repair.

Brain-specific Pd1 deficiency leads to cortical neurogenesis defects and depressive-like behaviors in mice.

Embryonic neurogenesis is tightly regulated by multiple factors to ensure the precise development of the cortex. Deficiency in neurogenesis may result in behavioral abnormalities. Pd1 is a well-known inhibitory immune molecule, but its function in brain development remains unknown. Here, we find brain specific deletion of Pd1 results in abnormal cortical neurogenesis, including enhanced proliferation of neural progenitors and reduced neuronal differentiation. In addition, neurons in Pd1 knockout mice exhibit abnormal morphology, both the total length and the number of primary dendrites were reduced. Moreover, Pd1cKO mice exhibit depressive-like behaviors, including immobility, despair, and anhedonia. Mechanistically, Pd1 regulates embryonic neurogenesis by targeting Pax3 through the β-catenin signaling pathway. The constitutive expression of Pax3 partly rescues the deficiency of neurogenesis in the Pd1 deleted embryonic brain. Besides, the administration of β-catenin inhibitor, XAV939, not only rescues abnormal brain development but also ameliorates depressive-like behaviors in Pd1cKO mice. Simultaneously, Pd1 plays a similar role in human neural progenitor cells (hNPCs) proliferation and differentiation. Taken together, our findings reveal the critical role and regulatory mechanism of Pd1 in embryonic neurogenesis and behavioral modulation, which could contribute to understanding immune molecules in brain development.

Expansion of bovine skeletal muscle stem cells from spinner flasks to benchtop stirred-tank bioreactors for up to 38 days.

Successful long-term expansion of skeletal muscle satellite cells (MuSCs) on a large scale is fundamental for cultivating animal cells for protein production. Prerequisites for efficient cell expansion include maintaining essential native cell activities such as cell adhesion, migration, proliferation, and differentiation while ensuring consistent reproducibility. This study investigated the growth of bovine MuSC culture using low-volume spinner flasks and a benchtop stirred-tank bioreactor (STR). Our results showed for the first time the expansion of primary MuSCs for 38 days in a bench-top STR run with low initial seeding density and FBS reduction, supported by increased expression of the satellite cell marker PAX7 and reduced expression of differentiation-inducing genes like MYOG, even without adding p38-MAPK inhibitors. Moreover, the cells retained their ability to proliferate, migrate, and differentiate after enzymatic dissociation from the microcarriers. We also showed reproducible results in a separate biological benchtop STR run.

Disease modeling and gene correction of LGMDR21 iPSCs elucidates the role of POGLUT1 in skeletal muscle maintenance, regeneration, and the satellite cell niche

Autosomal recessive limb-girdle muscular dystrophy 21 (LGMDR21) is caused by pathogenic variants in protein O-glucosyltransferase 1 (POGLUT1), which is responsible for O-glucosylation of specific epidermal growth factor (EGF) repeats found in ∼50 mammalian proteins, including Notch receptors. Previous data from patient biopsies indicated that impaired Notch signaling, reduction of muscle stem cells, and accelerated differentiation are probably involved in disease etiopathology. Using patient induced pluripotent stem cells (iPSCs), their corrected isotypes, and control iPSCs, gene expression profiling indicated dysregulation of POGLUT1, NOTCH, muscle development, extracellular matrix (ECM), cell adhesion, and migration as involved pathways. They also exhibited reduced invitro POGLUT1 enzymatic activity and NOTCH signaling as well as defective myogenesis, proliferation, migration and differentiation. Furthermore, invivo studies demonstrated significant reductions in engraftment, muscle stem cell formation, PAX7 expression, and maintenance, along with an increased percentage of mislocalized PAX7+ cells in the interstitial space. Gene correction in patient iPSCs using CRISPR-Cas9 nickase led to the rescue of the maininvitro and invivo phenotypes. These results demonstrate the efficacy of iPSCs and gene correction in disease modeling and rescue of the phenotypes and provide evidence of the involvement of muscle stem cell niche localization, PAX7 expression, and cell migration as possible mechanisms in LGMDR21.

Differentiation and traffic of IgM+ B cells between focal dark spots in skeletal muscle of Atlantic salmon, lymphoid and adipose tissues.

Focal dark spots (DS) in farmed Atlantic salmon fillets contain a significant number of B cells as revealed by the high abundance of immunoglobulin (Ig) transcripts in transcriptome data. The immune response in DS remains unknown while they represent a major problem in commercial aquaculture. Here, we characterized the diversity and clonal composition of B cells in DS. Sixteen gene markers of immune cells and antigen presentation were analyzed with RT-qPCR. All genes expression showed a positive correlation with DS area and intensity. The flatter the DS, the higher the expression of cd28, csfr, ctla, igt, and sigm, the lower expression of cd83 and btla, and the larger the cumulative frequency within DS. The expression of most of the analyzed immune genes, including three Ig types and markers of B cells was lower in DS than in the lymphatic organs, head kidney and spleen, but significantly higher compared to skeletal muscle. High levels of ctla4 and cd28 in DS might indicate the recruitment of T cells. Sequencing of IgM repertoire (Ig-seq) assessed migration of B cells by co-occurrence of identical CDR3 sequences in different tissues. The combination of gene expression and Ig-seq revealed the presence of several stages of B cell differentiation in DS. B cells at the earliest stage, with high ratio of membrane to secretory IgM (migm and sigm), showed minor Ig repertoire overlap with other tissues. Further differentiation stage (increased sigm to migm ratio and high expression of pax5 and cd79) was associated with active movement of B cells from DS towards lymphatic organs and visceral fat. Traffic and expression of immune genes decreased at later stages. These B cells could be involved in a response directed against viruses, pathogenic or opportunistic bacteria in DS. Seven of eight fish were positive for salmon alphavirus, and levels were higher in DS than in unstained muscle. PCR with universal primers to the 16S rRNA gene did not detect bacteria in DS. Although the evolution of DS most likely implies local exposure to antigens, neither this nor previous studies have found a necessary association between DS and pathogens or self-antigens.

Co-Expression of Multiple PAX Genes in Renal Cell Carcinoma (RCC) and Correlation of High PAX Expression with Favorable Clinical Outcome in RCC Patients.

Renal cell carcinoma (RCC) is the most common form of kidney cancer, consisting of multiple distinct subtypes. RCC has the highest mortality rate amongst the urogenital cancers, with kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and kidney chromophobe carcinoma (KICH) being the most common subtypes. The Paired-box (PAX) gene family encodes transcription factors, which orchestrate multiple processes in cell lineage determination during embryonic development and organogenesis. Several PAX genes have been shown to be expressed in RCC following its onset and progression. Here, we performed real-time quantitative polymerase chain reaction (RT-qPCR) analysis on a series of human RCC cell lines, revealing significant co-expression of PAX2, PAX6, and PAX8. Knockdown of PAX2 or PAX8 mRNA expression using RNA interference (RNAi) in the A498 RCC cell line resulted in inhibition of cell proliferation, which aligns with our previous research, although no reduction in cell proliferation was observed using a PAX2 small interfering RNA (siRNA). We downloaded publicly available RNA-sequencing data and clinical histories of RCC patients from The Cancer Genome Atlas (TCGA) database. Based on the expression levels of PAX2, PAX6, and PAX8, RCC patients were categorized into two PAX expression subtypes, PAXClusterA and PAXClusterB, exhibiting significant differences in clinical characteristics. We found that the PAXClusterA expression subgroup was associated with favorable clinical outcomes and better overall survival. These findings provide novel insights into the association between PAX gene expression levels and clinical outcomes in RCC patients, potentially contributing to improved treatment strategies for RCC.

SIX1+PAX3+ identify a progenitor for myogenic lineage commitment from hPSCs.

The earliest skeletal muscle progenitor cells (SMPCs) derived from human pluripotent stem cells (hPSCs) are often identified by factors expressed by a diverse number of progenitors. An early transcriptional checkpoint that defines myogenic commitment could improve hPSC differentiation to skeletal muscle. Analysis of several myogenic factors in human embryos and early hPSC differentiations found SIX1+PAX3+ co-expression was most indictive of myogenesis. Using dCas9-KRAB hPSCs, we demonstrate that early inhibition of SIX1 alone significantly decreased PAX3 expression, reduced PAX7+ SMPCs, and myotubes later in differentiation. Emergence of SIX1+PAX3+ precursors can be improved by manipulating seeding density, monitoring metabolic secretion and altering the concentration of CHIR99021. These modifications resulted in the co-emergence of hPSC-derived sclerotome, cardiac and neural crest that we hypothesized enhanced hPSC myogenic differentiation. Inhibition of non-myogenic lineages modulated PAX3 independent of SIX1. To better understand SIX1 expression, we compared directed differentiations to fetal progenitors and adult satellite cells by RNA-seq. Although SIX1 continued to be expressed across human development, SIX1 co-factor expression was dependent on developmental timing. We provide a resource to enable efficient derivation of skeletal muscle from hPSCs.

Pax6 affects Ras-Raf-ERK1/2 in mouse aging brain.

Pax6, a transcription factor and multifunctional protein, changes during aging. It also interacts with regulator proteins involved in cell metabolism and survival signalling pathways including Ras-GAP. Many forms of Ras, Raf and ERK1/2 are known but information on their region-specific expression patterns are unavailable from brain during aging. Therefore, it has been intended to evaluate expressions of Pax6 and forms of Ras, Raf, ERK1/2 in hippocampus, caudate nucleus, amygdale, cerebral cortex, cerebellumand olfactory lobe. Association of Pax6 with Ras, Raf and ERK1/2 was evaluated in co-culture (PC-12, C6-glia, U-87 MG) of neuroglia cell lines. Impacts of Pax6 were evaluated by siRNA mediated knockdown and expression patterns Ras-Raf-Erk1/2. Analysis of activities of Pax6 and impacts of 5'AMP, wild-type and mutant ERK were done by RT-PCR and luciferase reporter assay. Results indicate age-dependent changes of Pax6, Ras, Raf, ERK1/2 in different regions of brain of young and old mice. Erk1/2 shows synergistic activities to Pax6.

PAX5-miR-142 feedback loop promotes breast cancer proliferation by regulating DNMT1 and ZEB1

BackgroundBreast cancer is one of the most common malignancies occurred in female around the globe. Recent studies have revealed the crucial characters of miRNA and genes, as well as the essential roles of epigenetic regulation in breast cancer initiation and progression. In our previous study, miR-142-3p was identified as a tumor suppressor and led to G2/M arrest through targeting CDC25C. However, the specific mechanism is still uncertain.MethodsWe identified PAX5 as the upstream regulator of miR-142-5p/3p through ALGGEN website and verified by series of assays in vitro and in vivo. The expression of PAX5 in breast cancer was detected by qRT-PCR and western blot. Besides, bioinformatics analysis and BSP sequencing were performed to analyze the methylation of PAX5 promoter region. Finally, the binding sites of miR-142 on DNMT1 and ZEB1 were predicted by JASPAR, and proved by luciferase reporter assay, ChIP analysis and co-IP.ResultsPAX5 functioned as a tumor suppressor by positive regulation of miR-142-5p/3p both in vitro and in vivo. The expression of PAX5 was regulated by the methylation of its promoter region induced by DNMT1 and ZEB1. In addition, miR-142-5p/3p could regulate the expression of DNMT1 and ZEB1 through binding with their 3’UTR region, respectively.ConclusionIn summary, PAX5-miR-142-DNMT1/ZEB1 constructed a negative feedback loop to regulate the progression of breast cancer, which provided emerging strategies for breast cancer therapy.

Expression of Ras signaling pathway proteins and developmental factors in peripheral nerve sheath tumors of patients with neurofibromatosis type 1.

To characterize expression of factors relevant for Ras signaling and developmental factors in a large series of peripheral nerve sheath tumors (PNST) obtained from patients with neurofibromatosis type 1 (NF1). Tissue micro-array technique was applied to study 520 PNST of 385 NF1 patients by immunohistochemistry for mTor, Rho, phosphorylated MEK, Pax7, Sox9, and periaxin expression. PNST comprised cutaneous neurofibroma (CNF) (n=114), diffuse neurofibroma (DNF) (n=109), diffuse plexiform neurofibroma (DPNF) (n=108), plexiform neurofibroma (PNF) (n=110), and malignant PNST (MPNST) (n=22). All proteins examined showed highest expression levels/highest frequency of expression in MPNST. Benign PNF with potential for malignant dedifferentiation expressed mTor, phosphorylated MEK, Sox9, and periaxin significantly higher/more frequently than other benign neurofibroma subtypes. In NF1-associated PNST, expression of proteins involved in Ras-signaling and development is upregulated not only in MPNST, but also in benign PNF with the potential for malignant dedifferentiation. The differences in protein expression may provide clues for understanding the therapeutic effects of substances applied for reduction of PNST in NF1.

Neurogenesis in Brain Neurogenic Niches in Experimental Alzheimer’s Disease at the Presymptomatic Stage of Neurodegeneration

Deciphering the mechanisms of development of neurodegeneration at the presymptomatic stage is an urgent task. It’s solving allows optimizing the methods of early diagnostics and prevention of Alzheimer’s disease (AD). Goal of the study: to study the features of neurogenesis in brain neurogenic niches in experimental Alzheimer’s disease at the presymptomatic stage of neurodegeneration. Modeling of AD in vivo was carried out in experimental animals (male mice, C57BL/6, 8 months old) as follows: the control group, n = 30, animals were injected with 2 µl of a 0.9% NaCl solution in the CA1 field of the hippocampus; the experimental group, n = 30, animals were injected with a 1M solution of oligomerized beta-amyloid 25–35 (Aβ25–35) (2 μl bilaterally). Cognitive impairments have been assessed with the passive avoidance task (PAT). For immunohistochemical studies, we identified the subgranular zone of the hippocampus (SGZ) and the subventricular zone (SVZ) in frozen sections of the brain tissue. We have analyzed the expression of markers – Nestin, Pax6, NeuroD1, VEGFR2, as well as apoptosis (TUNEL protocol) in neurogenic niches. In the period preceding the manifestation of cognitive dysfunction (from 9 to 17 days after intrahippocampal administration of Aβ25–35), we registered multidirectional changes in the expression of markers of neurogenesis, neoangiogenesis and the severity of apoptosis in the SGZ of the hippocampus and in the SVZ. At 9th day since the beginning of development of Alzheimer’s type neurodegeneration, we found elevated expression of Pax6 and VEGFR2 in the SGZ and higher number of Nestin+ cells in the SVZ. Subsequent application of the PAT protocol with the presentation of an aversive stimulus (day 10) or the corresponding context (days 11 and 17) resulted in dynamic changes in the expression of cell markers at different stages of neurogenesis. In sum, аt the presymptomatic stage of the Alzheimer’s type neurodegeneration, SGZ and SVZ show signs of aberrant neurogenesis associated with a disruption in the pool of stem and progenitor cells and suppression of the production of neuroblasts/immature neurons in the period preceding the evident cognitive dysfunction.