Pax6 Expression Research Articles

The subcommissural organ maintains features of neuroepithelial cells in the adult mouse.

The subcommissural organ (SCO) is a part of the circumventricular organs located in the dorsocaudal region of the third ventricle at the entrance of the aqueduct of Sylvius. The SCO comprises epithelial cells and produces high molecular weight glycoproteins, which are secreted into the third ventricle and become part of Reissner's fibre in the cerebrospinal fluid. Abnormal development of the SCO has been linked with congenital hydrocephalus, a condition characterized by excessive accumulation of cerebrospinal fluid in the brain. In the present study, we characterized the SCO cells in the adult mouse brain to gain insights into the possible role of this brain region. Immunohistochemical analyses revealed that expression of Pax6, a transcription factor essential for SCO differentiation during embryogenesis, is maintained in the SCO at postnatal stages from P0 to P84. SCO cells in the adult brain expressed known neural stem/progenitor cell (NSPC) markers, Sox2 and vimentin. The adult SCO cells also expressed proliferating marker PCNA, although expression of another proliferation marker Ki67, indicating a G2/M phase, was not detected. The SCO cells did not incorporate BrdU, a marker for DNA synthesis in the S phase. Therefore, the SCO cells have a potential for proliferation but are quiescent for cell division in the adult. The SCO cells also expressed GFAP, a marker for astrocytes or NSPCs, but not NeuN (for neurons). A few cells positive for Iba1 (microglia), Olig2 (for oligodendrocytes) and PDGFRα (oligodendrocyte progenitors) existed within or on the periphery of the SCO. These findings revealed that the SCO cells have a unique feature as secretory yet immature neuroepithelial cells in the adult mouse brain.

Characterization of myogenic regulatory factors, myod and myf5 from Megalobrama amblycephala and the effect of lipopolysaccharide on satellite cells in skeletal muscle

Myod and Myf5 are muscle-specific basic helix-loop-helix (bHLH) transcription factors that play essential roles in regulating skeletal muscle development and growth. In order to investigate potential function of myod and myf5 of Megalobrama amblycephala, an economically important freshwater fish species, in the present study, we characterized the sequences and expression profiles of M. amblycephala myod and myf5. The open reading frame (ORF) sequences of myod and myf5 encoded 275 and 240 amino acids, respectively, possessing analogous structure with the highly conserved domains, bHLH and C-terminal helix III domains. Spatio-temporal expression patterns revealed that myod and myf5 were predominant in skeletal muscle with the highest expression in white muscle, and the highest at 10 days post-hatching (dph) and the segmentation period, respectively. Furthermore, we evaluated the effects of lipopolysaccharide (LPS) on the expression of muscle-related genes in white and red muscle, and proliferation and differentiation of satellite cells. The myod, myf5 and pax-7 expression generally increased and then decreased with increase of LPS concentration and treatment time in red muscle, while these genes showed inconsistent expression patterns in white muscle. In addition, LPS administration caused the frequency increase of satellite cells in red and white muscle especially at 3 and 7 days after LPS-injection.

Thirty Years' History since the Discovery of Pax6: From Central Nervous System Development to Neurodevelopmental Disorders.

Pax6 is a sequence-specific DNA binding transcription factor that positively and negatively regulates transcription and is expressed in multiple cell types in the developing and adult central nervous system (CNS). As indicated by the morphological and functional abnormalities in spontaneous Pax6 mutant rodents, Pax6 plays pivotal roles in various biological processes in the CNS. At the initial stage of CNS development, Pax6 is responsible for brain patterning along the anteroposterior and dorsoventral axes of the telencephalon. Regarding the anteroposterior axis, Pax6 is expressed inversely to Emx2 and Coup-TF1, and Pax6 mutant mice exhibit a rostral shift, resulting in an alteration of the size of certain cortical areas. Pax6 and its downstream genes play important roles in balancing the proliferation and differentiation of neural stem cells. The Pax6 gene was originally identified in mice and humans 30 years ago via genetic analyses of the eye phenotypes. The human PAX6 gene was discovered in patients who suffer from WAGR syndrome (i.e., Wilms tumor, aniridia, genital ridge defects, mental retardation). Mutations of the human PAX6 gene have also been reported to be associated with autism spectrum disorder (ASD) and intellectual disability. Rodents that lack the Pax6 gene exhibit diverse neural phenotypes, which might lead to a better understanding of human pathology and neurodevelopmental disorders. This review describes the expression and function of Pax6 during brain development, and their implications for neuropathology.

Reevaluation of dietary condensed tannins on growth, antioxidant and immune response and muscle quality of Chinese seabass (Lateolabrax maculatus)

Chinese seabass (Lateolabtax maculatus) is a popular carnivorous fish that is widely cultured in China in recent years. Previous studies suggested that low dose of CT enhanced antioxidant and immune response, but high dose of CT inhibited growth and induced tissue damage of fish. Meanwhile, information regarding effects of CT on muscle quality of L. maculatus is limited. A 63-day feeding trial was performed to assess the effects of dietary CT on growth, antioxidant and immune response and muscle quality of L. maculatus. Four diets were formulated to contain 0 g/kg of CT (G1), 1 g/kg of CT (G2), 2 g/kg of CT (G3), and 2 g/kg of CT plus 4 g/kg of polyethylene glycol (PEG) (G4, as positive control). PEG specifically binds with CT to neutralize CT activity. Fish were randomly distributed into 16 tanks with 4 tanks per diet and 40 fish per tank, and were fed to apparent satiation twice daily during the feeding trial. Results indicated that dietary CT at the concentration of 1 g/kg did not alter (P > 0.05) growth and feed utilization of L. maculatus. Fish fed G3 had lower (P < 0.05) final body weight, weight gain rate, thermal growth coefficient and feed intake than those of fish fed G1, G2 and G4. Fish fed G3 had lower (P < 0.05) total feed intake than those fed other diets in the 1, 2, 3, 4 and 5 week of the feeding trial. As compared with G1, serum catalase and superoxide dismutase activities were higher (P < 0.05) in G2, lower (P < 0.05) in G3, and similar (P > 0.05) in G4. Serum lysozyme activity and immune globulin M concentration were higher (P < 0.05) in G2 and G3 than those in G1 and G4. Muscle fiber density was increased (P < 0.05) as dietary CT increasing from 0 to 2 g/kg, but was similar (P > 0.05) between G1 and G4. Muscle water loss rate of post thawing was higher (P < 0.05) in G2 and G3 than that in G1 and G4, but was similar (P > 0.05) between G1 and G4 as well as G2 and G3. All fish had similar (P > 0.05) muscle amino acid compositions. Fish fed G3 had lower (P < 0.05) mRNA relative expressions of GH, IGF-1, Myf5, MyoD and Pax7 than those of fish fed other diets. The mRNA relative expressions of GH, IGF-1, Myf5, MyoD and Pax7 were similar (P > 0.05) among G1, G2 and G4. All fish had similar (P > 0.05) RNA relative expressions of IGF-2, MyoG and Mstn2 among groups. In summary, inclusion of 1 g/kg CT in L. maculatus diets did not alter growth but enhanced serum antioxidant and immune response, whereas 2 g/kg of CT declined feed intake and growth, decreased antioxidant enzyme activities but enhanced immune response of fish. Supplementation of CT increased muscle fiber density, decreased muscle water holding capacity but did not affect muscle amino acid compositions of L. maculatus. Dietary CT at the concentration of 2 g/kg down-regulated the majority of muscle growth-related genes expression, which may account for the decreased growth performance of L. maculatus.

The Effect of Voluntary Physical Activity in an Enriched Environment and Combined Exercise Training on the Satellite Cell Pool in Developing Rats.

Aim: Postnatal skeletal muscle growth is strongly associated with a satellite cell pool. Early adolescence might be a crucial period when different exercise training interventions have specific consequence on satellite cells. Pax7 and MyoD have been suggested as the leading indicators of satellite cell activation. Methods: In this study, pre-adolescent male rats (n = 18) were either subjected to an enriched environment that facilitated physical activities or combined training or control for three weeks. The flexor hallucis longus muscle was removed for biochemical and histochemical analysis. Results: Findings demonstrated that exercise trained rats displayed high levels of serum IGF-1 (p <0.05). There was an increase in Pax7 (p <0.05) and MyoD (p <0.001) mRNA expression. A significant increase in the mean fiber area (p <0.01), satellite cell (p <0.001), and myonuclear numbers (p <0.01) were also observed in both intervention groups. Importantly, enriched rats showed lower corticosterone levels (p <0.05) compared to training ones. Regarding performance, trained and enriched rats had significant improvement in forelimb grip strength (p <0.01) and load-carrying capacity (p <0.05). Conclusion: Type of physical exercise is an essential part in changing satellite cells pool. Different and frequent physical activities in an enriched environment can be effective for muscle development.

Ginsenoside Rg1 promotes neurite growth of retinal ganglion cells through cAMP/PKA/CREB pathways

BackgroundMechanisms of synaptic plasticity in retinal ganglion cells (RGCs) are complex and the current knowledge cannot explain. Growth and regeneration of dendrites together with synaptic formation are the most important parameters for evaluating the cellular protective effects of various molecules. The effect of ginsenoside Rg1 (Rg1) on the growth of retinal ganglion cell processes has been poorly understood. Therefore, we investigated the effect of ginsenoside Rg1 on the neurite growth of RGCs. MethodsExpression of proteins and mRNA were detected by Western blot and qPCR. cAMP levels were determined by ELISA. In vivo effects of Rg1 on RGCs were evaluated by hematoxylin and eosin, and immunohistochemistry staining. ResultsThis study found that Rg1 promoted the growth and synaptic plasticity of RGCs neurite by activating the cAMP/PKA/CREB pathways. Meanwhile, Rg1 upregulated the expression of GAP43, Rac1 and PAX6, which are closely related to the growth of neurons. Meantime, H89, an antagonist of PKA, could block this effect of Rg1. In addition, we preliminarily explored the effect of Rg1 on enhancing the glycolysis of RGCs, which could be one of the mechanisms for its neuroprotective effects. ConclusionRg1 promoted neurite growth of RGCs through cAMP/PKA/CREB pathways. This study may lay a foundation for its clinical use of optic nerve diseases in the future.

Pax3 Hypomorphs Reveal Hidden Pax7 Functional Genetic Compensation in Utero

Pax3 and Pax7 transcription factors are paralogs within the Pax gene family that that are expressed in early embryos in partially overlapping expression domains and have distinct functions. Significantly, mammalian development is largely unaffected by Pax7 systemic deletion but systemic Pax3 deletion results in defects in neural tube closure, neural crest emigration, cardiac outflow tract septation, muscle hypoplasia and in utero lethality by E14. However, we previously demonstrated that Pax3 hypomorphs expressing only 20% functional Pax3 protein levels exhibit normal neural tube and heart development, but myogenesis is selectively impaired. To determine why only some Pax3-expressing cell lineages are affected and to further titrate Pax3 threshold levels required for neural tube and heart development, we generated hypomorphs containing both a hypomorphic and a null Pax3 allele. This resulted in mutants only expressing 10% functional Pax3 protein with exacerbated neural tube, neural crest and muscle defects, but still a normal heart. To examine why the cardiac neural crest appears resistant to very low Pax3 levels, we examined its paralog Pax7. Significantly, Pax7 expression is both ectopically expressed in Pax3-expressing dorsal neural tube cells and is also upregulated in the Pax3-expressing lineages. To test whether this compensatory Pax7 expression is functional, we deleted Pax7 both systemically and lineage-specifically in hypomorphs expressing only 10% Pax3. Removal of one Pax7 allele resulted in partial outflow tract defects, and complete loss of Pax7 resulted in full penetrance outflow tract defects and in utero lethality. Moreover, combinatorial loss of Pax3 and Pax7 resulted in severe craniofacial defects and a total block of neural crest cell emigration from the neural tube. Pax7Cre lineage mapping revealed ectopic labeling of Pax3-derived neural crest tissues and within the outflow tract of the heart, experimentally confirming the observation of ectopic activation of Pax7 in 10% Pax3 hypomorphs. Finally, genetic cell ablation of Pax7Cre-marked cells is sufficient to cause outflow tract defects in hypomorphs expressing only 10% Pax3, confirming that ectopic and induced Pax7 can play an overlapping functional genetic compensational role in both cardiac neural crest lineage and during craniofacial development, which is normally masked by the dominant role of Pax3.

Immunohistochemical expression of transcription factors PAX5, OCT2, BCL6 and transcription regulator P53 in Non-Hodgkin lymphomas: A diagnostic cross-sectional study

BackgroundNon-Hodgkin lymphoma represents a heterogeneous group of tumors that constitute the seventh most common malignancy. Immunohistochemistry plays a major role in the detection of specific cell receptors. Transcription factors are a heterogeneous group of genes that play a critical role in the commitment, differentiation, and proliferation of specific cell types. MethodsParaffin-embedded tissue sections of non-Hodgkin lymphoma cases were selected, classified, and evaluated before staining with immunohistochemical markers (PAX5, OCT2, BCL6, and P53). Expression of the aforementioned markers was compared with histological subtypes and grades of lymphoma cases. Means of expression were also compared among histological subtypes. ResultsA total of 55 cases of NHL including 26 cases of low-grade lymphomas and 29 cases of high-grade lymphomas were included in the study. DLBCL and FL were the most common subtypes of high-grade and low-grade lymphomas respectively. Both PAX5 and OCT2 were positive in 44 cases of NHL (80%) including all cases of B-cell lymphomas. BCL6 and P53 demonstrated positive expression in 29.1% and 67.3% respectively. Interestingly, we found a significant association between the histological subtypes and the aforementioned markers (P-value<0.05). DiscussionExpression of PAX5, OCT2, BCL, and P53 played a major role in the diagnosis and grading of non-Hodgkin lymphomas in our study. Both PAX5 and OCT2 provided more accuracy and specificity in the diagnosis of B-cell neoplasms compared to the classical B-cell markers. BCL6 expression reflected its role in germinal center formation in normal and malignant lymphoid tissues, and expression of P53 mirrored the accumulation of gene mutations in more aggressive lymphoma subtypes. ConclusionIn this manuscript, we aimed to present a unique study that highlights the immunohistochemical expression of all the aforementioned factors among various histological subtypes of non-Hodgkin lymphomas with disparities in histological aggressiveness, highlighting a promising diagnostic and prognostic panel for non-Hodgkin lymphomas.

The utility of PAX8 in comparison with PAX5 immunohistochemical staining in the diagnosis of Hodgkin lymphoma

BackgroundHodgkin's lymphoma (HL) is among the most prevalent lymphomas worldwide. PAX5 has a great value in separating this entity from other T cell lymphoma; however, it is weakly expressed in neoplastic cells. Polyclonal PAX8 was positive in a variety of lymphoid neoplasms in several previous studies and the staining paralleled that of PAX5 but monoclonal PAX8 was negative in the same neoplasms. The aim of this study was to compare immunohistochemical patterns of monoclonal PAX8 with PAX5 in Classical and NLPHL samples. Material and methodsThis retrospective study was conducted on 89 formalin-fixed paraffin embedded blocks from HL patients (69 Classical and 20 NLPHL) admitted to Imam Khomeini and Dr. Shariati hospitals, Tehran, Iran during 2016–2020. Diagnoses were confirmed by reviewing previous immunohistochemistry (IHC) studies, including PAX5. All samples were stained for PAX8 (clone MRQ-50). Expression intensity scoring was made for both antibodies in neoplastic and background cells based on nuclear staining percentage. ResultsPAX8 was positive in neoplastic and background B lymphocytes of all classical and NLPHL samples. PAX8 Expression intensity was significantly higher in neoplastic and background cells compared to PAX5 in classical HL samples (P = 0.001). PAX5 expression intensity in neoplastic cells was significantly higher in NLPHL samples compared to classical HL (P = 0.040); however, no significant difference in PAX8 expression between neoplastic cells of NLPHL and HL was seen. PAX8 expression intensity was not significantly correlated with gender, histologic subtype, tumor location, and relapse. ConclusionsPAX8 monoclonal antibody (clone MRQ-50) showed strong nuclear reactivity in neoplastic and background cells of classical HL and NLPHL samples. Therefore, this marker can be utilized as a valuable alternative for PAX5 in differentiating HL from other T cell lymphoma in challenging cases.

PAX9 reactivation by inhibiting DNA methyltransferase triggers antitumor effect in oral squamous cell carcinoma

Aberrant DNA hypermethylation is associated with oral carcinogenesis. Procaine, a local anesthetic, is a DNA methyltransferase (DNMT) inhibitor that activates anticancer mechanisms. However, its effect on silenced tumor suppressor gene (TSG) activation and its biological role in oral squamous cell carcinoma (OSCC) remain unknown. Here, we report procaine inhibited DNA methylation by suppressing DNMT activity and increased the expression of PAX9, a differentiation gene in OSCC cells. Interestingly, the reactivation of PAX9 by procaine found to inhibit cell growth and trigger apoptosis in OSCC in vitro and in vivo. Likely, the enhanced PAX9 expression after exposure to procaine controls stemness and differentiation through the autophagy-dependent pathway in OSCC cells. PAX9 inhibition abrogated procaine-induced apoptosis, autophagy, and inhibition of stemness. In OSCC cells, procaine improved anticancer drug sensitivity through PAX9, and its deficiency significantly blunted the anticancer drug sensitivity mediated by procaine. Additionally, NRF2 activation by procaine facilitated the antitumor response of PAX9, and pharmacological inhibition of NRF2 by ML385 reduced death and prevented the decrease in the orosphere-forming potential of OSCC cells. Furthermore, procaine promoted antitumor activity in FaDu xenografts in athymic nude mice, and immunohistochemistry data showed that PAX9 expression was significantly enhanced in the procaine group compared to the vehicle control. In conclusion, PAX9 reactivation in response to DNMT inhibition could trigger a potent antitumor mechanism to provide a new therapeutic strategy for OSCC.

Identification of protein markers for skeletal muscle‐derived extracellular vesicles (SkM‐EVs) by quantitative proteomics reveals how SkM‐EVs function in vivo

BackgroundSkeletal muscle serves as a secretory organ; it releases various soluble factors called myokine. They act as mediators for cell‐cell communications in autocrine, paracrine, and endocrine fashions. Extracellular vesicles (EVs) have been proposed to be a partial paracrine and/or endocrine effectors by facilitating the exchange of biological cargoes among cells and tissue. Some in vitro study showed that skeletal muscle released EVs and skeletal muscle‐derived EVs (SkM‐EVs) could induce biological effects in recipient cells. However, whether the skeletal muscle actively releases EVs in vivo, how much proportion of plasma EVs are derived from this tissue, and where SkM‐EVs are delivered and exert their roles remain largely unknown. Here, we aimed to identify SkM‐EV marker proteins by quantitative proteomics on human and mouse myocyte‐derived EVs to evaluate SkM‐EVs abundance in vivo. In addition, we explored whether SkM‐EVs isolated from mice could exert biological functions.MethodsProteomic analysis of EVs released from mouse C2C12 myotubes and human iPS‐derived myocytes was performed to identify potential SkM‐EVs markers. The levels of SkM‐EVs in plasma and muscle interstitium were evaluated by the abundance of identified SkM‐EVs protein markers. Based on previous methods, we developed a modified protocol to isolated SkM‐EVs from muscle interstitium. We confirmed that muscle interstitium‐EVs were incorporated into C2C12 myoblast and investigated the expression levels of myogenesis‐related genes.Results&amp; DiscussionWe first determined proteomic profiles of EVs released from C2C12 myoblasts, C2C12 myotubes, and hiPSC‐derived myocytes and identified several proteins that serve as potential markers for SkM‐EVs. Among them, we identified ATP2A1, β‐enolase, and desmin as reliable SkM‐EV marker proteins. Using these protein markers, we confirmed that SkM‐EVs were present in muscle interstitium but little in the blood, which were supported by our electron microscopy. Unexpectedly, the abundance of SkM‐EVs present in plasma and muscle interstitium was not changed in response to acute exercise. Furthermore, we showed that SkM‐EVs accumulated in muscle interstitium space were incorporated into C2C12 myoblast and promoted muscle differentiation by regulating gene expression of Pax7 and myosin heavy chain at least partially through myomiRs (miRs‐1, ‐206, ‐431, and ‐486). We expect that molecular characterization and in vivo distribution of SkM‐EVs could provide a conceptually new basis how skeletal muscle EVs exert their functions.

Sex-Based Differences in the Myogenic Response and Inflammatory Gene Expression Following Eccentric Contractions in Humans.

After muscle injury, the interaction between muscle satellite cells (SC) and the immune response is instrumental for the repair and regeneration of skeletal muscle tissue. Studies have reported sex-based differences in the skeletal muscle inflammatory and regenerative response following injury. However, many of these studies investigated such differences by manipulating the concentration of estradiol, in rodents and humans, without directly comparing males to females. We sought to explore differences in the myogenic and inflammatory response following unaccustomed eccentric exercise in males and females. We hypothesized that females would have a blunted myogenic and inflammatory response as compared to males. Methods: 26 (13 male, 13 female) healthy young adults (22 ± 0.4 years [mean ± SEM]) performed 300 maximal eccentric contractions (180°/s) of the knee extensors. Muscle biopsies were taken before (pre) and 48 h (post) following eccentric damage. SC content and activation were determined by immunohistochemical and real time-polymerase chain reaction (rt-PCR) analysis. Inflammatory markers were analyzed using rt-PCR. Results: Following eccentric damage, males had a greater expansion of type I-associated SC (p < 0.05), and there was a trend for a greater expansion in total SC (type I + II fibers) (p = 0.06) compared to females. There was a trend for a greater increase in Pax7 and CCL2 gene expression in males compared to females (p = 0.09). Conclusion: We conclude that there are sex-based differences in the myogenic and inflammatory response, where females have a blunted SC and inflammatory response.

Glyphosate-based herbicide induces long-lasting impairment in neuronal and glial differentiation.

Glyphosate‐based herbicides (GBH) are among the most sold pesticides in the world. There are several formulations based on the active ingredient glyphosate (GLY) used along with other chemicals to improve the absorption and penetration in plants. The final composition of commercial GBH may modify GLY toxicological profile, potentially enhancing its neurotoxic properties. The developing nervous system is particularly susceptible to insults occurring during the early phases of development, and exposure to chemicals in this period may lead to persistent impairments on neurogenesis and differentiation. The aim of this study was to evaluate the long‐lasting effects of a sub‐cytotoxic concentration, 2.5 parts per million of GBH and GLY, on the differentiation of human neuroepithelial stem cells (NES) derived from induced pluripotent stem cells (iPSC). We treated NES cells with each compound and evaluated the effects on key cellular processes, such as proliferation and differentiation in daughter cells never directly exposed to the toxicants. We found that GBH induced a more immature neuronal profile associated to increased PAX6, NESTIN and DCX expression, and a shift in the differentiation process toward glial cell fate at the expense of mature neurons, as shown by an increase in the glial markers GFAP, GLT1, GLAST and a decrease in MAP2. Such alterations were associated to dysregulation of key genes critically involved in neurogenesis, including PAX6, HES1, HES5, and DDK1. Altogether, the data indicate that subtoxic concentrations of GBH, but not of GLY, induce long‐lasting impairments on the differentiation potential of NES cells.

Cyclosporin A Prevents Ovarian Graft Rejection, and Permits Normal Germ Cell Maturation Within the First 5 Weeks Post-transplantation, in the Domestic Turkey (Meleagris gallopavo).

Biobanked ovaries collected from recently hatched poults can only be revived through transplantation, using a recipient bird. The main hurdle in transplantation is preventing graft rejection, which appears as lymphocytic infiltration upon histologic evaluation of the graft. In this study, the condition of the transplants [immunological compatibility (auto- vs. allotransplants), donor age, time in holding media, and temperature of holding media] and treatment of recipient poults with varying immunosuppressants [mycophenolate mofetil (MFM), cyclophosphamide (CY), and cyclosporin A (CsA)] were studied to determine which factors could reduce lymphocytic infiltration, during the first 35 days post-transplantation. Lymphocytic infiltration was determined via cytoplasmic CD3 (T cell) and nuclear PAX5 (B cell) expression. There was no significant difference in the percent of cytoplasmic CD3 or nuclear PAX5 immunostained area between the unoperated group and the autotransplants, by 6 days post-transplantation. However, the allotransplants had more (P < 0.05) positive cytoplasmic and nuclear immunostained areas compared to autotransplants, irrespective of donor age, time in holding media or temperature of the media. By 14 days post-transplantation, the CsA 25 and 50 mg/kg/day treatment groups had less (P < 0.05) CD3 and PAX5 positive areas in their allotransplants, compared to the unsuppressed group. At 35 days post-transplantation, the CsA 25 mg/kg/day allotransplant group also had less (P < 0.05) CD3 and PAX5 positive areas compared to the unsuppressed group. The CsA 25 mg/kg/day transplants also had a similar ovarian follicular size compared to the unoperated group, although they contained fewer (P < 0.05) follicles based on follicular density. Donor age, duration in holding media, temperature of media, and treatment of recipients with MFM or CY had no effect on reducing lymphocytic infiltration. However, immunological compatibility was associated with decreased lymphocytic infiltration, as autotransplants had little lymphocytic infiltration. Treatment of recipients with CsA at 25 mg/kg/day was also associated with reduced lymphocytic infiltration and allowed transplants to develop normally during the first 35 days post transplantation.

Modulatory effects of cell–cell interactions between porcine skeletal muscle satellite cells and fibroblasts on the expression of myogenesis-related genes

ABSTRACT Co-culture is mimicked in vivo condition, which is influenced by adjacent cells. This study aimed to determine different myogenic genes in mono- and co-culture of satellite cells and fibroblasts. Fibroblasts and satellite cells were isolated from the piglet’s thigh muscles. The expression of PAX-7, MyoD, MyoG, and PAX-3 was downregulated in 24 h co-culture satellite cells than in monoculture. The expression of Myf-5 and CAV-1 was upregulated in 24 h co-culture satellite cells than in 24 h monoculture. The expression of PAX-7, MyoD, and Myf-5 was downregulated in 24 and 48 h co-culture fibroblasts than 24 h monoculture. The expression of MyoG and PAX-3 was upregulated in 24 and 48 h co-culture than 48 h monoculture fibroblasts. The Cyto-C was downregulated in 24 and 48 h co-culture fibroblast than 24 h monoculture. The expression of MyoG and CAV-1 was upregulated in 48 h incubation period co-culture fibroblasts than 24 h monoculture. The MyoD and CAV-1 expression were upregulated in 24 h co-culture than 24 h satellite cells and fibroblasts. The Bcl-2 and BAX were downregulated in 48 h co-culture than 24 h. This result recommended that the satellite cells and fibroblasts interaction enhance the myogenesis marker expression without cellular morphology change.

Mucinous Tumor Coexisting With Mesonephric-like Proliferation/Tumor in the Ovary: A Novel Association.

The literature indicates that mesonephric carcinoma (MC) and mesonephric-like adenocarcinoma (MLA) typically lack mucinous and squamous features/differentiation. We report 4 cases of ovarian mucinous tumors (1 mucinous cystadenofibroma and 3 mucinous borderline tumors/atypical proliferative mucinous tumors [MBT/APMT]) co-existing with mesonephric-like lesions which were highlighted by Gata3 and Pax8 expression. All cases contained benign mesonephric-like proliferations (MLP) which focally displayed gastrointestinal-type mucinous metaplasia/differentiation and some were intimately admixed with mucinous glands associated with the mucinous tumor. Metaplastic mucinous epithelium retained expression of Gata3 and Pax8 in some areas while 1 mucinous cystadenofibroma and 1 MBT/APMT were focally positive for Pax8. Along with these mesonephric components, case 1 exhibited features of mesonephric hyperplasia and in 2 cases, 3 and 4, MLA was identified. In case 4, a KRAS c.35G>T (p.Gly12Val) somatic mutation was detected in both the MBT/APMT and the MLA, indicating a clonal origin. This same mutation was also detected in the benign MLP, indicating that it was likely an early genetic event. A CTNNB1 c.98C>T (p.Ser33Phe) somatic mutation, FGFR2 amplification, and CDKN2A/p16 deletion were only detected in the MLA but not in the MBT/APMT. Our result provides evidence to demonstrate the clonal relationship between these morphologically distinct components. Although speculative, we postulate that benign MLPs may give rise to lineage-specific mucinous and mesonephric-like lesions and propose that the MLPs are a new possible origin of some ovarian mucinous tumors. Whether these MLPs arise through transdifferentiation of Müllerian tissue or represent true mesonephric remnants, however, remains largely unknown.

Evolutionary Transition in the Regulation of Vertebrate Pronephros Development: A New Role for Retinoic Acid.

The anterior-posterior (AP) axis in chordates is regulated by a conserved set of genes and signaling pathways, including Hox genes and retinoic acid (RA), which play well-characterized roles in the organization of the chordate body plan. The intermediate mesoderm (IM), which gives rise to all vertebrate kidneys, is an example of a tissue that differentiates sequentially along this axis. Yet, the conservation of the spatiotemporal regulation of the IM across vertebrates remains poorly understood. In this study, we used a comparative developmental approach focusing on non-conventional model organisms, a chondrichthyan (catshark), a cyclostome (lamprey), and a cephalochordate (amphioxus), to assess the involvement of RA in the regulation of chordate and vertebrate pronephros formation. We report that the anterior expression boundary of early pronephric markers (Pax2 and Lim1), positioned at the level of somite 6 in amniotes, is conserved in the catshark and the lamprey. Furthermore, RA, driving the expression of Hox4 genes like in amniotes, regulates the anterior pronephros boundary in the catshark. We find no evidence for the involvement of this regulatory hierarchy in the AP positioning of the lamprey pronephros and the amphioxus pronephros homolog, Hatschek’s nephridium. This suggests that despite the conservation of Pax2 and Lim1 expressions in chordate pronephros homologs, the responsiveness of the IM, and hence of pronephric genes, to RA- and Hox-dependent regulation is a gnathostome novelty.

Microplastics exposure affects neural development of human pluripotent stem cell-derived cortical spheroids

Plastics have been part of our ecosystem for about a century and their degradation by different environmental factors produce secondary microplastics (MPs). To date, the impact of MPs on human health has not been well investigated. To understand the possible effects of polystyrene-MPs (PS-MPs) on the human brain, a 3D model of human forebrain cortical spheroids has been derived, which mimics early development of human cerebral cortex. The spheroids were exposed to 100, 50, and 5 µg/mL of 1 µm and 10 µm PS-MPs during day 4–10 and day 4–30. The short-term MP exposure showed the promoted proliferation and high gene expression of Nestin, PAX6, ATF4, HOXB4 and SOD2. For long-term exposure, reduced cell viability was observed. Moreover, changes in size and concentration of PS-MPs altered the gene expression of DNA damage and neural tissue patterning. In particular, β-tubulin III, Nestin, and TBR1/TBR2 gene expression decreased in PS-MP treated conditions compare to the untreated control. The results of this study suggest that the size- and concentration-dependent exposure to PS-MPs can adversely affect embryonic brain-like tissue development in forebrain cerebral spheroids. This study has significance in assessing environmental factors in neurotoxicity and degeneration in human.

Unusual staining of immunohistochemical markers PAX8 and CDX2 in breast carcinoma: a potential diagnostic pitfall

Knowing the sensitivity and specificity of tissue-specific immunohistochemical markers is crucial for accurate determination of the primary tumor site. PAX8 has been used as a diagnostic marker for carcinomas of the gynecologic tract, kidney, and thyroid gland, and CDX2 has been used as a marker of gastrointestinal carcinoma. Neither is considered a marker for breast carcinoma (BC). However, we have encountered BCs that express PAX8 or CDX2, some of which caused diagnostic confusion. We investigated the immunohistochemical staining frequency of PAX8 and CDX2 in BC. We identified 237BCs for which PAX8 staining results were reported (102 primary and 135 metastatic BCs); seven primary and four metastatic BCs (4.6%) were positive for PAX8, with various intensities and staining patterns. CDX2 staining results were reported for 271BCs (78 primary and 193 metastatic); four primary BCs and one metastatic BC (1.8%) were positive for CDX2, ranging from focal and weak to diffuse and strong. We also stained primary invasive BCs with PAX8 and CDX2 using tissue microarrays. None of the 332 PAX8-stained cases was positive, while one of 143 CDX2-stained cases was positive. Four PAX8-positive and three CDX2-positive cases were stained with TRPS1, and all were positive for TRPS1. In addition, we reviewed the literature for PAX8 and CDX2 expression in BCs and found 5.5% PAX8-positive BCs (90/1625) in 17 studies and 0.8% CDX-2 positive BCs (7/909) in 20 studies. PAX8 and CDX2 are infrequently expressed in BC by immunohistochemistry, and in rare cases, the staining can be strong and diffuse. Additional diagnostic markers are necessary and helpful in distinguishing breast from other primary origins.

Myoblast deactivation within engineered human skeletal muscle creates a transcriptionally heterogeneous population of quiescent satellite-like cells

Satellite cells (SCs), the adult Pax7-expressing stem cells of skeletal muscle, are essential for muscle repair. However, in vitro investigations of SC function are challenging due to isolation-induced SC activation, loss of native quiescent state, and differentiation to myoblasts. In the present study, we optimized methods to deactivate in vitro expanded human myoblasts within a 3D culture environment of engineered human skeletal muscle tissues (“myobundles”). Immunostaining and gene expression analyses revealed that a fraction of myoblasts within myobundles adopted a quiescent phenotype (3D-SCs) characterized by increased Pax7 expression, cell cycle exit, and activation of Notch signaling. Similar to native SCs, 3D-SC quiescence is regulated by Notch and Wnt signaling while loss of quiescence and reactivation of 3D-SCs can be induced by growth factors including bFGF. Myobundle injury with a bee toxin, melittin, induces robust myofiber fragmentation, functional decline, and 3D-SC proliferation. By applying single cell RNA-sequencing (scRNA-seq), we discover the existence of two 3D-SC subpopulations (quiescent and activated), identify deactivation-associated gene signature using trajectory inference between 2D myoblasts and 3D-SCs, and characterize the transcriptomic changes within reactivated 3D-SCs in response to melittin-induced injury. These results demonstrate the ability of an in vitro engineered 3D human skeletal muscle environment to support the formation of a quiescent and heterogeneous SC population recapitulating several aspects of the native SC phenotype, and provide a platform for future studies of human muscle regeneration and disease-associated SC dysfunction.