Pax6 Expression Research Articles

MiR-193b-3p Promotes Proliferation of Goat Skeletal Muscle Satellite Cells through Activating IGF2BP1

As a well-known cancer-related miRNA, miR-193b-3p is enriched in skeletal muscle and dysregulated in muscle disease. However, the mechanism underpinning this has not been addressed so far. Here, we probed the impact of miR-193b-3p on myogenesis by mainly using goat tissues and skeletal muscle satellite cells (MuSCs), compared with mouse C2C12 myoblasts. miR-193b-3p is highly expressed in goat skeletal muscles, and ectopic miR-193b-3p promotes MuSCs proliferation and differentiation. Moreover, insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) is the most activated insulin signaling gene when there is overexpression of miR-193b-3p; the miRNA recognition element (MRE) within the IGF1BP1 3' untranslated region (UTR) is indispensable for its activation. Consistently, expression patterns and functions of IGF2BP1 were similar to those of miR-193b-3p in tissues and MuSCs. In comparison, ectopic miR-193b-3p failed to induce PAX7 expression and myoblast proliferation when there was IGF2BP1 knockdown. Furthermore, miR-193b-3p destabilized IGF2BP1 mRNA, but unexpectedly promoted levels of IGF2BP1 heteronuclear RNA (hnRNA), dramatically. Moreover, miR-193b-3p could induce its neighboring genes. However, miR-193b-3p inversely regulated IGF2BP1 and myoblast proliferation in the mouse C2C12 myoblast. These data unveil that goat miR-193b-3p promotes myoblast proliferation via activating IGF2BP1 by binding to its 3' UTR. Our novel findings highlight the positive regulation between miRNA and its target genes in muscle development, which further extends the repertoire of miRNA functions.

Are birds pseudoteeth and denticulations related to touch papilla? An investigation in parrot, goose, and chicken

We first studied the morphology and the development of goose denticulations, which develop mainly by a ripple process, and the touch papillae of the bill tip organ, which appears through an evagination process at the end of the beak. During their development, we observed the specific expression of PAX9, PITX2, and BMP4, while SHH was expressed mainly in the basal layer of the epithelium in a non-specific manner. Adult goose denticulations are associated with numerous columns. The goose denticulations and columns were filled with numerous Herbst and Grandry corpuscles, as well the touch papillae of the bill tip organ. Histological analysis of adult parrot pseudoteeth revealed that the osseous pseudoteeth were extended by similar columns filled with Herbst and Grandry corpuscles. We also examined adult and embryonic chicken beaks. During ontogeny, we observed a process of rostral evagination with folding associated with discrete ripples in the anterior part of the beak rudiment, in which PAX9, PITX2, and BMP4 are expressed. In the corresponding adult areas, there were numerous sensory corpuscles with rostral columns, which were similar to the features observed in goose. These observations support the hypothesis that pseudoteeth and denticulations constitute sensory organs, and that the touch papillae exhibit some similarities with pseudoteeth.

Evaluation of CRISPR/Cas9 exon-skipping vector for choroideremia using human induced pluripotent stem cell-derived RPE.

Exon-skipping is a powerful genetic tool, especially when delivering genes using an AAV-mediated full-length gene supplementation strategy is difficult owing to large length of genes. Here, we used engineered human induced pluripotent stem cells and artificial intelligence to evaluate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9-based exon-skipping vectors targeting genes of the retinal pigment epithelium (RPE). The model system was choroideremia; this is an X-linked inherited retinal disease caused by mutation of the CHM gene. We explored whether artificial intelligence detected differentiation of human OTX2, PAX6 and MITF (hOPM) cells, in which OTX2, PAX6 and MITF expression was induced by doxycycline treatment, into RPE. Plasmid encoding CHM exon-skipping modules targeting the splice donor sites of exons 6 were constructed. A clonal hOPM cell line with a frameshift mutation in exon 6 was generated and differentiated into RPE. CHM exon 6-skipping was induced, and the effects of skipping on phagocytic activity, cell death and prenylation of Rab small GTPase (RAB) were evaluated using flow cytometry, an in vitro prenylation assay and western blotting. Artificial intelligence-based evaluation of RPE differentiation was successful. Retinal pigment epithelium cells with a frameshift mutation in exon 6 showed increased cell death, reduced phagocytic activity and increased cytosolic unprenylated RABs only when oxidative stress was in play. The latter two phenotypes were partially rescued by exon 6-skipping of CHM. CHM exon 6-skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress.

Malignant transformation of endometrial hyperplastic processes: immunohistochemical features

Background. Endometrial hyperplasia is one of the most common pathologies of the female reproductive system. There is a high risk of transformation of an atypical form of endometrial hyperplasia into endometrial cancer, which takes a leading position in oncogynecological morbidity. Immunohistochemical markers can become predictors of endometrial malignancy, their role is currently being actively studied.Aim. Search for molecular predictors of malignant transformation of endometrial hyperplasia.Materials and methods. Histological and immunohistochemical studies were performed in 107 patients with diagnoses of endometrial hyperplasia without atypia (EH), endometrial atypical hyperplasia (EIN), endometrioid adenocarcinoma (EC). The tumor receptor status, MSI, expression of ARID1A, PTEN, β-catenin, PAX2, proliferation index (Ki-67) were determined by the immunohistochemical method according to the standard protocol.Results. Loss of PAX2 expression was most common in precancerous and malignant endometrial cells. Loss of PAX2 expression was found in 89 % of cases in the EIN samples, in 86 % of cases in the EC samples and only in 2 cases of EH. Loss of PTEN expression was less common: with an equal proportion in 67 % of the samples of EIN and EC, while practically not occurring in women with benign GE. MSI was detected in 36 % of endometrial cancer samples. There was a deficiency in the DNA repair system in 1 case of EIN. Loss of ARID1A expression was detected in endometrial cancer with a frequency of 33 %. This gene functioned normally in all cases of EIN and EH. The expression of β-catenin was more pronounced in EC than in cases of EH. The Ki-67 proliferation index was statistically higher in the EC group than in EH and EIN.Conclusion. Evaluation of morphological data and expression of the panel of markers PAX2, PTEN, ARID1A, β-catenin, Ki-67 index, PMS2 and MLH1 will improve diagnostic search in case of suspected malignancy of endometrial hyperplasia.

Mechanism of selective induction of apoptosis of HCT116 tumor cells in circulating blood by riboflavin photochemistry

The removal or inactivation of circulating tumor cells (CTCs) can prevent distant metastasis by hematogenous route, but there is still a lack of mature and effective technical means. In the previous research, our team has initially established a method of riboflavin photosensitized treatment (RPT) for continuous treatment of peripheral blood in vitro for the inactivation of CTCs. The core of this technology is that it can selectively induce apoptosis of CTCs (HCT116 cells) without damaging immunocyte (mainly Peripheral Blood Mononuclear Cells，PBMCs) under specific parameters. To clarify the specific mechanism, firstly, the enrichment of riboflavin in HCT116 cells and PBMCs was observed under fluorescence microscope. Secondly, the apoptotic signaling pathways in HCT116 cells and PBMCs in response to RPT treatment were analyzed by transcriptomics. Finally, the mitochondrial damage in HCT116 cells and PBMCs before and after RPT treatment was observed under electron microscope. The results showed that under the same treatment conditions, HCT116 cells were significantly enriched in riboflavin compared with PBMCs. Besides, RPT treatment reduced the expression of long non﹣coding RNA (lncRNA) NEAT1, an effector gene of HCT116 cells, which further down-regulated the expression of target gene PAX2 and promoted the expression of Bax, leading to mitochondrial outer membrane permeabilization (MOMP), and consequently increased the release of pro-apoptotic factors such as cytochrome c(Cyt C), high-temperature requirement protein A2(HTRA2), apoptosis-inducing factor (AIF), endonuclease G(ENDOG), finally leading to apoptosis of HCT116 cells. In contrast, lncRNA NEAT1 remained unchanged in PBMCs before and after RPT treatment, and was unable to stimulate the PBMCs apoptotic signaling pathway. The results of the study indicated that under the specific treatment conditions, RPT technology could selectively induce apoptosis of HCT116 cells by activating the mitochondrial apoptosis pathway, which would further provide a theoretical and technical support for the effective inactivation of CTCs by RPT technology, thereby reducing the risk of recurrence of malignant tumors and improving the cure rate of malignant tumors.

The Cytogenetic Profile of Spindle Cell–Predominant Mucinous Tubular and Spindle Cell Carcinoma

A man in his 30s presented with a 7-cm left renal mass for which he underwent a radical nephrectomy. Histopathologic examination revealed a neoplasm predominantly composed of spindled cells (Figure 1A, magnification ×40). On higher magnification, these cells had a relatively bland appearance with minimal cytologic atypia and mitotic activity (Figure 1B, magnification ×200). Focal areas of this tumor (<5%) had a tubular component with extravasated mucin (Figure 1C, magnification ×100). Immunohistochemistry showed expression of the transcription factor PAX8 (Figure 1D, magnification ×200) as well as keratin 7 in both the epithelial and spindle cell component.

Inhibition of GSK3β Promotes Proliferation and Suppresses Apoptosis of Porcine Muscle Satellite Cells

As the global population increases, interest in cultured meat (a new research field) is gradually increasing. The main raw material for the production of cultured meat is muscle stem cells called satellite cells isolated from livestock. However, how to mass proliferate and maintain satellite cells in vitro without genetic manipulation remains unclear. In the present study, we isolated and purified porcine muscle satellite cells (PMSCs) from the femur of a 1-day-old piglet and cultured PMSCs by treating them with an inhibitor (XAV939, Tankyrase (TNKS) inhibitor) or an activator (CHIR99021, glycogen synthase kinase 3 beta (GSK3β) inhibitor) of Wnt signaling. The CHIR group treated with 3 μM CHIR99021 showed a significantly increased proliferation rate of PMSCs compared to the SC group (control), whereas the XAV group treated with 1 μM XAV939 showed a significantly decreased proliferation rate of PMSCs. CHIR99021 also inhibited the differentiation of PMSCs by reducing the expression of MyoD while maintaining the expression of Pax7 and suppressed apoptosis by regulating the expression of apoptosis-related proteins and genes. RNA sequencing was performed to obtain gene expression profiles following inhibition or activation of the Wnt signaling pathway and various signaling mechanisms related to the maintenance of satellite cells were identified. Our results suggest that inhibition of GSK3β could dramatically improve the maintenance and mass proliferation ability of PMSCs in vitro by regulating the expression of myogenic markers and the cell cycle.

Erythropoietin Administration Delays B Cell Maturation and Promotes B Cell to Erythroid Transdifferentiation in Mice

Background: Erythropoietin (EPO) is a glycoprotein hormone which serves as a key regulator of erythropoiesis. Previous studies have shown that it also has a myriad of extraerythropoietic effects including on immune and skeletal systems. Specifically, it was shown that EPO reduces bone marrow (BM) B cell population, in part, by inducing an egress of IgM+ cells out of BM to the peripheral blood (PB). However, the effect of EPO on early B cells (IgM- Pro- and Pre-B cells) has not been studied. Moreover, a process of transdifferentiation of early B cells into erythroid cells under EPO stimulation has not been addressed. Methods: We used MB1-Cre;R26-eYFP mice for B-cell-specific lineage tracing as well as MB1-Cre; EPO-Rfl/fl and Osx-Cre; EPO-Rfl/fl mouse models for B-cell and osteoblast-specific EPO-R knockdown, respectively. Mice (10-12 week old females) were treated with either diluent or human recombinant EPO (hereafter, EPO) for one week (180IU x3/week). Immunophenotyping of BM cells was performed by multi-color flow cytometry (mFC) and immunofluorescence microscopy (IF) was used to demonstrate B cell-derived erythropoiesis in BM sections. Expression of PAX5 and IL-7 in the BM was determined by real-time quantitative PCR. Results: EPO treatment led to a significant 2.4-fold reduction in BM B cells (B220+/CD19+) manifested by a decrease in both IgM+ and IgM- cell fractions. While the IgM+ B cell fraction in PB after one week of EPO did not change, we did observe an almost 2-fold increase in IgM- B cells in EPO-treated as compared to diluent-treated mice (1.4±0.8% vs 0.7±0.5%, respectively, p<0.05). There was no change in IgM+ or IgM- B cells in the spleen, suggesting that IgM- B cells in PB originate from the BM. Concurring with these data, expression of CXCR4 adhesion molecule by BM Pro-B (IgM-) cells was significantly reduced (1.8-fold) upon EPO treatment. Since a decrease in BM B cells could not be solely explained by their egress from BM, we hypothesized that EPO induced a maturation delay at the level of Pre/Pro-B cell stages. Using both MB1-Cre; EPO-Rfl/fl and Osx-Cre; EPO-Rfl/fl mouse models we showed that this maturation delay is independent of EPO-EPO-R signaling in both early B cells and osteoblasts, i.e. following EPO treatment the amount and subpopulation distribution of BM B cells were similar in the control and corresponding EPO-R knockdown mice. However, we found significant perturbations in the key regulators of B cell lymphopoiesis in EPO-treated mice (EPO-R+/+): a 4-fold and 1.7-fold decrease in the expression of PAX5 and IL-7, respectively, while expression of IL-7 receptor by BM cells (both B220+ and B220-)was modestly but significantly reduced (as measured by mFC). Finally, applying IF on BM sections, we could detect eYFP+ BM cells (derived from MB1-Cre;R26-eYFP mice) that co-expressed the specific erythroid marker TER119 (Figure 1). These findings demonstrate that a fraction of BM B cells can transdifferentiate into erythroid cells upon EPO stimulation. Conclusions: Besides its effect on erythropoiesis, EPO compromises BM B cell compartment by a multifaceted mechanism, including BM egress, delay in maturation as well as transdifferentiation to the erythroid lineage. An understanding of the biological and clinical significance of this phenomenon requires further research. For example, the transdifferentiation might explain an improved erythropoiesis in patients with anemia who already have high EPO levels. This research was funded by Israel Science Foundation (ISF) grant number 1188/21 to DN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Age- and cell cycle-related expression patterns of transcription factors and cell cycle regulators in Müller glia

Mammalian Müller glia express transcription factors and cell cycle regulators essential for the function of retinal progenitors, indicating the latent neurogenic capacity; however, the role of these regulators remains unclear. To gain insights into the role of these regulators in Müller glia, we analyzed expression of transcription factors (Pax6, Vsx2 and Nfia) and cell cycle regulators (cyclin D1 and D3) in rodent Müller glia, focusing on their age- and cell cycle-related expression patterns. Expression of Pax6, Vsx2, Nfia and cyclin D3, but not cyclin D1, increased in Müller glia during development. Photoreceptor injury induced cell cycle-associated increase of Vsx2 and cyclin D1, but not Pax6, Nfia, and cyclin D3. In dissociated cultures, cell cycle-associated increase of Pax6 and Vsx2 was observed in Müller glia from P10 mice but not from P21 mice. Nfia levels were highly correlated with EdU incorporation suggesting their activation during S phase progression. Cyclin D1 and D3 were transiently upregulated in G1 phase but downregulated after S phase entry. Our findings revealed previously unknown links between cell cycle progression and regulator protein expression, which likely affect the cell fate decision of proliferating Müller glia.

Single Tube Molecular Profiling of ALL Samples Reveals Integrated Molecular Signature and Highlights Gene Regulatory Networks

Background: Over 20 acute lymphoblastic leukemia (ALL) subtypes are defined by molecular signature, which is historically mainly dependent on genomic rearrangements (translocations and gene fusions). Advancements in NGS strategies allow high-resolution profiling, taking in to account also gene expression patterns, single nucleotide variants (SNVs) and insertions and deletions (INDELs). Previous studies showed that molecular signature correlates to prognosis and treatment compatibility, making accrue molecular profiling a major interest point for both clinical and research communities, helping identify novel therapeutic targets and designing clinical management approaches. Many current NGS approaches rely on panels-based assays, which screen DNA or RNA to provide one piece of the puzzle, resulting in higher cost and processing time and requiring larger initial inputs. We report a single tube NGS panel that uses total nucleic acid as input for simultaneous screening of DNA and RNA, allowing for simultaneous profiling of gene expression, fusions and variants, establishing a work frame for comprehensive molecular profiling within a single assay. Methods: This study used 102 ALL clinical samples, 85 bone marrow (BM) and 17 peripheral blood (PB). Libraries were prepared using a custom Qiagen Multimodal NGS panel targeting 302 DNA targets and 229 RNA fusions. Enriched amplicon libraries were sequenced with unique dual indices on Illumina NovaSeq 6000. Data were analyzed on our in-house bioinformatic pipeline. Expression values were normalized to GUSB. Previous in-house studies were used to establish baseline cut-offs for upregulation of 31 genes known to be associated with ALL. Variants enrichment for genes was done by normalizing the number of variants to the size of the gene and genes with elevated variant loads were identified, to highlight molecular pathways within our cohort. Results: Since expression can be modulated by both genomic variants and rearrangements, we performed a principal component analysis (PCA) on our RNA expression data to identify the clustering effect within the samples that could be driven by either or both of these genetic factors. The PCA identified several distinct clusters, with PC1 accounting for 39.3% of the variance, and PC2 accounting for 15.3%. The biological source (BM vs. PB) did not have an impact on the clusters, allowing us to analyze both sample types together. We then investigated whether the predetermined 31 over-expressed genes could be driving the clusters. Three clear groups were observed based on over-expression quantification. One group with low overexpression levels (0-1 genes), the second with medium levels (8-11), and one with high-level of over expression (>16 genes). These clusters also corresponded to T-ALL (high variance)-vs B-ALL (low to medium variance) classification, and showed clear segregation of samples with over-expression of EPOR (high variance cluster) from the rest of the samples. RNA expression data also showed a high correlation between expression of PAX5, RUNX1 and TCF3; all are known to interact as part of a gene regulatory network, highlighting its importance to ALL development. Finally, we investigated SNVs' role within the different clusters. While we could not identify any single variant/gene to be especially enriched with any of the clusters, we observed that KMT2D has an enrichment in variants within our cohort, underscoring that epigenetics is an important component of the disease landscape. Conclusions: We demonstrate the use of a single tube multimodal NGS assay for comprehensive genomic profiling. Our analysis shows that this powerful and cost-effective tool can help identify molecular signatures, classify ALL sub-types, identify regulatory networks and molecular mechanisms, which will help not only properly diagnose but also identify and develop therapeutic targets clinical management strategies.

Restoration of functional PAX3 transcriptional factor enhanced neuronal differentiation in PAX3b isoform-depleted neuroblastoma cells.

Reexpressed PAX3 transcription factor is believed to be responsible for the differentiation defects observed in neuroblastoma. Although the importance of PAX3 in neuronal differentiation is documented how it is involved in the defective differentiation remains unexplored particularly with its isoforms. Here, first we have analyzed PAX3 expression, its functional status, and its correlation with the neuronal marker expression in SH-SY5Y and its parental SK-N-SH cells. We have found that SH-SY5Y cells which expressed more PAX3 showed increased expression of neuronal marker genes (TUBB, MAP2, NEFL, NEUROG2, SYP) and reported PAX3 target genes (MET, TGFA, and NCAM1) than the SK-N-SH cells that had low PAX3 level. Retinoic acid treatment is unable to induce neuronal differentiation in cells (SK-N-SH) with low PAX3 level/activity. Moreover, ectopic expression of PAX3 in SK-N-SH cells neither induces neuronal marker genes nor its target genes. PAX3 isoform expression analysis revealed the expression of PAX3b isoform that contains only paired domain in SK-N-SH cells, whereas in SH-SY5Y cells, we could also observe PAX3c isoform that contains all functional domains. Further, PAX3b depletion in SK-N-SH cells is not induced PAX3 target genes, and the cells remain poorly differentiated. Interestingly, ectopic PAX3 expression in PAX3b-depleted SK-N-SH cells enhanced neuronal outgrowth along with neuronal marker gene induction. Collectively, these results showed that the PAX3b isoform may be responsible for the differentiation defect observed in SK-N-SH cells and restoration of functional PAX3 in the absence of PAX3b can induce neurogenesis in these cells.

Familial Predisposition to B-Cell Precursor Acute Lymphoblastic Leukemia Mediated By PAX5 Germline Variants

Introduction: Somatic PAX5 alterations are one of the most common features present in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Recently, PAX5alt and PAX5 p.P80R have been identified as two novel PAX5-driven BCP-ALL subtypes (Gu et al., Nat Genet., 2019). PAX5 is a well-known master regulator for B-cell development, hence, deregulated PAX5 expression poses high risks for leukemia development. In this regard, families with germline PAX5 variants are a unique source to study the effect of PAX5 on leukemia development in humans. Methods: We corroborated clinical data with immunophenotyping and genomic analyses of families carrying PAX5 germline variants. Results: Here, we describe two new families with inherited PAX5 germline variants affecting amino acid 183 who developed BCP-ALL, including one family with a so far undescribed amino acid substitution p.Gly183Arg. To identify additional synergizing germline variants, whole exome sequencing of both families, together with one family previously reported by Auer et al., (Leukemia, 2014) was carried out. This analysis yielded no common germline variants apart from PAX5, which could explain the incomplete penetrance in the observed leukemia development, but confirmed the families’ diverse backgrounds in different ancestry. Interestingly, an in-depths B-cell differentiation study comparing PBMCs from PAX5 variant carriers (n=8) in the respective families and unrelated non-carriers (n=44) revealed a significant decrease of intermittent B-cells (Student's T-test, 5.796% vs 9.820%, p= 0.017) and memory B-cells (Mann-Whitney U, 10.760% vs 17.350%, p= 0.003) in carriers versus non-carriers. Moreover, bone marrow analysis from one of the patients carrying PAX5 p.G183S showed a reduction in the number of mature B cells, suggesting deregulation of the B-cell differentiation at the pre-B stage. Altogether, a total of 21 patients from seven families carrying three different heterozygous germline PAX5 variants have been reported. Statistically significant differences are observed between the age of onset of patients harboring the PAX5 p.Arg38His variant compared with patients carrying p.Gly183Ser/Arg (Student's T-test, 17.75 vs. 2.70 years old, respectively; p=0.03). Furthermore, all patients who developed BCP-ALL showed somatic chromosome 9p alterations with loss of heterozygosity of the Wildtype PAX5 locus. Concordantly, all families display incomplete penetrance, with PAX5 p.Gly183Arg and p.Arg38His being by far more penetrant than p.Gly183Ser. Regarding the outcome, a high rate of overall survival (72%) and a high rate of relapse (75%) were observed. 54% of the patients received hematopoietic stem cell transplantation, and of these, 71% survived. Conclusion: Taken together, our data provide evidence for deregulated B-cell development mediated by reduced PAX5 levels in the germline of families carrying PAX5 germline variants. Our results emphasize the benefit of PAX5 germline variant screening in patients diagnosed with BCP-ALL and somatic alterations on chromosome 9p, regardless of a family history of leukemia.

Hemophagocytic Lymphohistiocytosis Associated with Diffuse Large B-Cell Lymphoma: A Case Report

Abstract Introduction/Objective Secondary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of immune hyperactivation and dysregulation that has been observed in some malignancies. In HLH, natural killer (NK) cells and cytotoxic T-cells fail to control activated macrophages, leading to cytokine-mediated tissue injury and multiorgan failure. Persistent tumor antigenic stimulation and cytokine hypersecretion can trigger HLH. Diffuse large B- cell lymphoma (DLBCL) is uncommonly associated with HLH. Methods/Case Report A 50-year-old woman presented with daily fevers and malaise of one-month duration. Imaging showed marked hepatosplenomegaly with no lymphadenopathy. Laboratory studies were significant for pancytopenia (WBC: 3.8 K/µL, Hb: 6.8 g/dL, Plt: 74 K/µL), hypertriglyceridemia (330 mg/dL), hyperferritinemia (1460 ng/mL), and elevated soluble interleukin-2 receptor (CD25) (42351.5 pg/mL). These findings, along with NK cell activity level (not obtained in this case), fever, splenomegaly and hemophagocytosis (bone marrow, spleen or lymph nodes) represent the eight diagnostic criteria by Histiocyte Society HLH-2004. A splenic biopsy showed a nodular proliferation of medium-to-large neoplastic lymphocytes with CD20, PAX5, BCL6, MUM1, BCL2, CD5 (dim) and CD10 (dim) expression and negative for cyclin D1 and p53. CD21 did not highlight any dendritic cell meshwork. The bone marrow biopsy provided similar results. Overall, these findings were consistent with a DLBCL, germinal center subtype. Rearrangements of MYC, BCL2 or BCL6 genes were not detected. Although hemophagocytosis was not noted in the bone marrow or spleen, the patient met six of the eight HLH-2004 criteria (minimum five are required). The H-score was 174 reflecting up to 70% probability of HLH. Treatment with daily intravenous dexamethasone 10 mg/m2 was initiated as a temporizing measure prior to R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy which resulted in complete remission. Results (if a Case Study enter NA) NA Conclusion Physicians should be cognizant of secondary HLH as a rare complication of DLBCL. Prompt initiation of the appropriate therapy could result in rapid and complete resolution.

Optimal Pre-Plating Method of Chicken Satellite Cells for Cultured Meat Production.

To establish a pre-plating method of chicken satellite cells with high purity, pre-plating was performed under culture conditions of 37°C and 41°C, and the pre-plating time was set from a total of 3 hours to 6 hours in consideration of the cell attachment time. The purity of the cells was confirmed by staining paired box protein 7 (Pax7) after proliferation, and Pax7 expression was the highest in culture flasks shaken for 2 hours after incubation at 41°C for 2 hours to prevent the attachment of satellite cells (p<0.05). Also, when pre-plating and proliferation were performed at 37°C and 41°C, the Pax7 expression rate was higher at 41°C. The differentiation capabilities of the three groups (T3, T6, and T7) with high Pax7 expression were compared and the fusion index (%) and myotube formation area (%) determined by myosin heavy chain (MHC) staining was calculated. The T6 and T7 groups, which were cultured at 41°C, showed significantly higher values than the T3 group (p<0.05). There was no significant difference in the expression of Pax7 and MHC between the T6 and T7 groups (p>0.05). These results suggest that pre-plating at 41°C for a total of 4 hours was the most efficient in terms of cost and time for purifying chicken satellite cells for cultured meat.

OR09-4 Novel Murine Models of BrafV600E driven Papillary Thyroid Cancer

Abstract Thyroid cancer is the most prevalent endocrine malignancy and is estimated to have affected 52,890 individuals in the United States in the year 2020. Papillary thyroid cancer (PTC) accounts for up to 80% of all thyroid cancer diagnoses. Within PTC, BRAFV600E is the most common mutation in adults and the second most common mutation in pediatrics. While there are several validated human thyroid cancer cell lines harboring BRAF mutations, no pediatric derived cell line has been established to date, limiting generalizations in pediatric disease. Patient-derived pre-clinical models are valuable tools but are limited due to their need to be studied in vitro or in an immunocompromised host. However, in vivo mouse models recapitulate the complex interactions between tumor cells, the immune system, and components of the microenvironment, allowing more comprehensive investigation of thyroid oncogenesis and response to therapy. In this study we describe a new subcutaneous mouse model that can be used to understand differences between pediatric onset and adult onset PTC. We have developed multiple independent, congenic murine cell lines from different stages of thyroid cancer progression. Two independent BRAFV600E-driven cell lines harboring the same genetic mutations were utilized in this study, one representing a well-differentiated tumor (WD) and one representing a poorly-differentiated tumor (PD). Despite coming from different stages of disease, both cell lines showed similar expression of the thyroid specific genes: Pax8, Ttf1, Tg, and Slc5a5, but expression of Pax8, Tg, and Slc5a5 was lower compared to WT thyrocytes. Activation of the Pi3Kinase pathway and the Akt Pathway were assessed via western blot analysis. The WD cell line had increased pERK activation compared to the PD cell line, and the PD cell line had increased pAKT compared to WD cell line. Both cell lines were injected subcutaneously into the hind flank of Wt SJV129 mice of pediatric age (4-5 weeks) and adult age (20-22 weeks). These animals were monitored for 10 weeks post injection. While the PD cell line developed tumors at approximately the same rate and penetrance in both age groups, tumors reached end point more rapidly in the adult cohort. Adult hosts injected with the WD cell line developed tumors more rapidly than in pediatric hosts, however, tumors receded in both cohorts. While adult and pediatric thyroid cancer share common driving mutations, there are distinct differences in disease pathogenesis between these patient populations. There are known differences in the immune system, metabolism, and other variables between pediatrics and adults. However, it is unknown what causes the difference in PTC pathogenesis between pediatric versus adult patients. We believe these new models provide a great opportunity to evaluate the role of age in PTC development and progression. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

Insect peptide CopA3 promotes proliferation and PAX7 and MYOD expression in porcine muscle satellite cells

Insects are a valuable natural source that can produce a variety of bioactive compounds due to their increasing species diversity. CopA3 is an antimicrobial peptide derived from Copris tripartitus (i.e., the dung beetle). It is known to increase the proliferation of colonic epithelial and neuronal stem cells by regulating cell cycle. This research hypothesized that CopA3 can promote the proliferation of porcine muscle satellite cells (MSCs). The effects of CopA3 on porcine MSCs, which are important for muscle growth and regeneration, remain unclear. Here, we investigated the effects of CopA3 on porcine MSCs. According to viability results, we designed four groups: control (without CopA3) and three treatment groups (treated with 5,10, and 25 μg/mL of CopA3). At a CopA3 concentration of 5 μg/mL and 10 μg/mL, the proliferation of MSCs increased more than that observed in the control group. Furthermore, compared to that in the control, CopA3 treatment increased the S phase but decreased the G0/G1 phase ratio. Additionally, early and late apoptotic cells were found to be decreased in the 5 μg/mL group. The expressions of the myogenesis-related transcription factor PAX7 and MYOD proteins were significantly upregulated in the 5 μg/mL and 10 μg/mL groups, whereas the MYOG protein remained undetected in all group. This study suggested that CopA3 promotes muscle cell proliferation by regulating the cell cycle of MSCs and can regulate the activity of MSCs by increasing the expressions of PAX7 and MYOD.

Immunophenotypic and molecular characterization of pancreatic neuroendocrine tumors producing serotonin

AbstractSerotonin producing pancreatic neuroendocrine tumors (SP-PanNET) account for 0.58–1.4% of all pancreatic neuroendocrine tumors (PanNET). They may present with atypical symptoms, such as acute pancreatitis and are often radiologically characterized by main pancreatic duct dilatation. SP-PanNET are well differentiated neuroendocrine tumors (NET) distinct from classical PanNET by atypical serotonin secretion and abundant dense stroma deposition, like serotonin producing ileal NET leading in some cases to difficulties to reliably distinguish SP-PanNET from ileal NET metastases. The biology and molecular profile of SP-PanNET remain poorly characterized and the cell of origin within the pancreas is unclear. To address these questions, we analyzed a large cohort of SP-PanNET by immunohistochemistry (n = 29; ATRX, DAXX, MENIN, Islet1, PAX6, PDX1, ARX, CDX2), whole genome copy number array (Oncoscan™) and a large NGS panel (NovoPM™) (n = 10), FISH (n = 13) and RNA sequencing (n = 24) together with 21 ileal NET and 29 nonfunctioning PanNET (NF-PanNET). These analyses revealed a unique genomic profile with frequent isolated loss of chromosome 1 (14 cases-61%) and few pathogenic mutations (KMT2C in 2 cases, ARID1A in 1 case). Unsupervised RNAseq-based clustering showed that SP-PanNET were closer to NF-PanNET than ileal NET with an exclusive beta cell-like signature. SP-PanNET showed TGF-β pathway activation signatures associated with extracellular matrix remodeling and similar signature were reproduced in vitro when pancreatic stellate cells were exposed to serotonin. SP-PanNET immunohistochemical profile resemble that of ileal NET except for PDX1 and PAX6 expression to a lesser extend suggesting that these two markers may be useful to diagnose SP-PanNET. Taken together, this suggests that SP-PanNET are a very specific PanNET entity with a peculiar biology leading to the characteristic fibrotic aspect.

Immune function modulation during artificial ovarian maturation in Japanese eel (Anguilla japonica): A transcriptome profiling approach

The Japanese eel (Anguilla japonica) experiences dramatic internal and external environmental changes during its transoceanic reproductive migrations. Here, we assess immune function changes in the primary and secondary immune organs (head kidney and spleen) of A. japonica during artificial ovarian maturation at the previtellogenic (PV), midvitellogenic (MV), and ovulating (OV) stages by transcriptome analyses. Stress responses were also assessed by determining the serum concentrations of lysozyme, alkaline phosphatase, acid phosphatase, total antioxidant capacity, and superoxide dismutase. Our results showed that together with increased serum 17β-estrogen and testosterone, lysozyme activity and antioxidant capacity were suppressed during artificial ovarian maturation. Comparisons across these developmental stages identified 60 (head kidney) and 36 (spleen) differentially expressed genes associated with the immune system. Genes related to the key activation markers of innate immune function, such as CXCL10, CXCL11, CCL20, HSP90B, MMP9, and MMP13, were upregulated and significantly enriched in the interleukin-17 signaling pathway. Adaptive immune function-related genes (IGM and MHC1) were upregulated in the head kidney from PV to MV, and their levels increased thereafter in the spleen. Moreover, a correlation between Pax5 expression and IGM expression in the spleen of MV (IGM+/Pax5+) and OV (IGM++/Pax5−) stage suggests that adaptive immune function was enhanced during ovarian maturation. To our knowledge, the present study is the first to describe transcriptome profiling of immune organs during ovarian maturation in teleost. Our findings suggest that the interleukin-17 pathway and IgM may play important roles in spawning.

An overview of PAX1: Expression, function and regulation in development and diseases.

Transcription factors play multifaceted roles in embryonic development and diseases. PAX1, a paired-box transcription factor, has been elucidated to play key roles in multiple tissues during embryonic development by extensive studies. Recently, an emerging role of PAX1 in cancers was clarified. Herein, we summarize the expression and functions of PAX1 in skeletal system and thymus development, as well as cancer biology and outline its cellular and molecular modes of action and the association of PAX1 mutation or dysregulation with human diseases, thus providing insights for the molecular basis of congenital diseases and cancers.

Effects of the Probiotic Enterococcus faecium on Muscle Characteristics of Chickens.

The use of antibiotics in farm animals is one of the main reasons for the development of resistant bacterial strains (e.g., zoonotic pathogens). Therefore, save alternatives are needed. Here, we examined how post-hatch application (day one to seven of life) of the probiotic Enterococcus faecium AL41 (EF) affects the development and tissue properties of the broiler pectoralis major muscle (PM). Expression of regulators, namely IGF-1, PAX7, and MYF5, was also investigated. At day 1 (n = 6), and days 5, 8, and 12 (n = 10), muscle samples were taken from control and EF supplemented chicks. From day 5 on, myonuclei number per fiber was elevated in EF chicks. Improved capillarization (from day 8), larger myofibers, increased body and PM weights (day 12) were found in the EF group. Part of our findings is explainable by higher intramuscular expression of IGF-1 and lower MYF5 expression in EF birds. In both groups IGF-1 expression decreases with age, thereby increasing the cellular myogenic potential. However, a strong increase in PAX7 expression and more PAX7-positive nuclei were found in EF chicks at day 12. We conclude that EF supplementation improves PM growth and health due to positive effects on bioavailability and fusion capacity of SATC progeny and better tissue perfusion.