Expression Of Oct4 Research Articles

From mitochondria to tumor suppression: ACAT1's crucial role in gastric cancer.

Acetyl CoA acetyltransferase 1 (ACAT1), a mitochondrial enzyme, is mainly involved in the formation and decomposition of ketones, isoleucine, and fatty acids. Previous clinical studies showed that mutations in the ACAT1 gene lead to ketoacidosis, Notably the role of ACAT1 in human cancer' pathogenesis varies depending on cancer type, and its specific role in gastric cancer remains largely unknown. In the current study, we found that the expression of ACAT1 in primary late-stage gastric cancer tumor tissues was significantly lower than in early-stage tumors. This observation was further confirmed in high-grade gastric cancer cell line MKN45. The expression of CD44 and OCT4 was decreased, while CD24 expression was increased by overexpressing ACAT1 in MKN45 gastric cancer cells. Moreover, the ability of gastric cancer cells to form colonies on soft agar was also reduced by ACAT1 overexpression. Likewise, overexpression of ACAT1 inhibited epithelial mesenchymal transition (EMT) in gastric cancer cells evidenced by increased expression of the epithelial marker E-Cadherin, decreased expression of mesenchymal marker vimentin, and decreased expression levels of SNAI 1/3. In addition, ACAT1 overexpression inhibited cell migration and invasion, improved the response to 5-Fluorouracil (5-FU) and etoposide. In contrast, inhibition of ACAT1 activity promoted the proliferation of gastric cancer cells. The xenotransplantation results in nude mice showed that overexpression of ACAT1 in gastric cancer cells inhibited tumor growth in vivo. In addition, the low expression of ACAT1 in gastric cancer was further validated by searching public databases and conducting bioinformatic analyses. Mechanistically, bioinformatic analysis found that the inhibitory effect of ACAT1 in gastric cancer may be related to the Adipocytokine Signaling Pathway, Ppar Signaling Pathway, Propanoate Metabolism and P53 Signaling Pathway. Correlation analysis indicated ACAT1 mRNA expression was correlated with immune infiltrates. Collectively, our data show that ACAT1 induces pronounced inhibitory effects on gastric cancer initiation and development, which may impact future strategies to treat this aggressive cancer.

Effects of Topoisomerase II alpha Inhibition on Oral Cancer Cell Metabolism and Cancer Stem Cell Function.

Topoisomerase IIα (TOP2A), is an enzyme involved in DNA replication, transcription, recombination, and chromatin remodeling and is found in a variety of cancers. However, the role of TOP2A regulation in oral cancer progression is not fully explained. We investigated the effect of TOP2A inhibition on cell survival, metabolism, and cancer stem cell self-renewal function in oral cancer cells. Oral carcinoma cell line SCC25 was cultured in complete DMEM/F12 media and treated with 5μM of Etoposide (Topoisomerase II inhibitor) for 48h. The critical parameters of cellular metabolism, including extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation based on the oxygen consumption rate of cancer cells were assessed using Seahorse assay. Western blotting was performed to assess the proteins that are associated with proliferation (Survivin, IL-6) and cancer stem cell function (Oct4, Sox2) in cell lysates prepared from control and etoposide treated groups. Statistical analysis was performed using One-way ANOVA with Dunnett's multiple comparisons test. The protein expression of TOP2A was significantly (P<0.05) inhibited by etoposide. Additionally, TOP2A inhibition decreased the mitochondrial respiratory parameters including basal respiration, maximal respiration and ATP production. However, TOP2A inhibition has no impact on glycolytic function. Moreover, the proliferative marker survivin and IL-6 showed a significant (P<0.05) decrease after TOP2A inhibition. Conversely, the protein expression of cancer stem cell markers Oct-4 and Sox 2 were not altered. These results indicate that inhibition of TOP2A is more efficacious by decreasing the mitochondrial metabolic reprogramming and thereby downregulating the key anti-apoptotic and pro-survival mediators. Thus, TOP2A represents an ideal therapeutic target and offers a potential treatment strategy for OSCC.

THBS2 promotes gastric cancer progression and stemness via the Notch signaling pathway.

Thrombospondin-2 (THBS2), a secreted extracellular matrix protein, plays a crucial role in various biological processes including angiogenesis, tissue remodeling, and inflammation. Our study focuses on its function in human gastric cancer (GC). Through bioinformatics and tumor tissue analysis, we compared THBS2 expression in GC tissues and adjacent tissues, and predicted regulatory upstream and downstream molecules. The direct regulatory effect of miR-29b-3p on THBS2 was evaluated through dual-luciferase reporter assays, showing that miR-29b-3p targets the 3'-UTR of THBS2 mRNA, reducing its expression in GC cells. The influence of THBS2 on tumorigenesis and stemness was examined on protein expression levels via Western blot. In vivo, THBS2's role was investigated through xenograft and metastasis assays in nude mice, demonstrating that downregulation of THBS2 impairs GC tumorigenesis and liver metastasis. Our study identified THBS2 as a highly expressed prognostic factor in GC patients. Functionally, THBS2 promotes GC progression through the Notch signaling pathway by regulating Notch3, NEY1, and HES1 proteins, and sustains cancer stem cell-like characteristics by Notch3, including the expression of CD44, Nanog, OCT4, and SOX2. In sum, our study reveals that THBS2 promotes GC progression and stemness, modulated negatively by miR-29b-3p. This suggests potential therapeutic targets within the THBS2/Notch signaling axis for combating gastric cancer.

Metadichol, a Natural Ligand for the Expression of Yamanaka Reprogramming Factors in Human Cardiac, Fibroblast, and Cancer Cell Lines

The reprogramming of somatic cells into embryonic-like stem cells requires the activation of four essential transcription factors, referred to as the Yamanaka factors: Sox2, Oct4, Klf4, and c-Myc. However, the utilization of these genes and viral vectors for their delivery presents a risk of carcinogenesis, which renders induced pluripotent stem cell (iPSC) lines unsuitable for clinical applications. Typically, direct reprogramming involves the use of viral vectors to induce the expression of these factors. In contrast, metadichol, a novel approach, enhances the expression of Yamanaka factors in cells using a dose ranging from 1 pg to 100 ng, thereby preventing the need for viral vectors. This non-viral method renders cellular reprogramming safer and more clinically viable. Treatment with metadichol within the specific dose range resulted in a substantial augmentation of the fibroblast expression of OCT4, SOX2, KLF4, KLF2, and Nanog, which was confirmed through qRT-PCR and western blot analyses. The expression levels of OCT4, KLF4, Nanog, and Sox2, exhibited an increase of 4.01-, 3.51-, 1.26-, and 2.5-fold, respectively, compared to the controls. Notably, A549 and Colo-205 cancer cells demonstrated a marked elevation in expression levels. The reprogramming of primary human cancer cells presents considerable challenges. However, in triple-negative primary breast cancer cells, metadichol treatment led to the substantial upregulation of the expression levels of OCT4, KLF4, Nanog, and Sox2 by 19.6-, 8.07-, 2.45-, and 6.91-fold, respectively, across the dose range of 1 pg to 100 ng. Klotho, an antiaging gene that is modulated by metadichol, down regulates TP53, a crucial factor for the production of somatic cell iPSCs. Additionally, metadichol enhances the availability of vitamin C, which is essential for generating iPSCs from somatic cells. Increased levels of alkaline phosphatase, indicative of cellular differentiation, were observed in the treated group compared to the controls. Metadichol is a nontoxic nanoemulsion that is derived from long-chain C26–C28 alcohols from food sources and activates Yamanaka factors across various cell types without requiring the use of viral or CRISPR-based methodologies, thus substantially advancing somatic cell reprogramming.

Stimulating effect of nanocurcumin and crocin on proliferation and pluripotency of bone marrow-derived mesenchymal stem cells.

Enhancement of proliferation, pluripotency, and self-renewal capacity as the unique features of MSCs can improve their therapeutic potential to regenerate tissues. In this context, crocin and curcumin, carotenoid compounds with outstanding medicinal properties, could be promising for cell protection and growth. This study aimed to evaluate the impact of nanocurcumin and crocin on BM-MSCs proliferation and pluripotency in vitro. BM-MSC were isolated from the iliac crest of SCI patients who were candidates for stem cell therapy. The effect of crocin and nanocurcumin on MSC proliferation was evaluated using MTT and PDT assay. The percentage of apoptotic MSCs was measured by flow cytometry. Furthermore, mRNA and protein expression of OCT4 and SOX2 as the proliferation and self-renewal related genes were quantified by real-time PCR and western blotting, respectively. Our findings demonstrated that only low concentrations of nanocurcumin (0.3 and 0.7 µM) and crocin (2.5 5 µM) significantly affected MSCs proliferation and protected them from apoptosis. Also, crocin and nanocurcumin at low doses caused an elevation in the mRNA and protein expression levels of OCT4 and SOX2 genes. In contrast, high concentrations decreased the survival of MSCs and led to increased apoptosis compared with the untreated group. Our results suggest that using nanocurcumin and crocin separately in culturing MSCs can be considered proliferative agents to prepare the more advantageous tool for cell therapies. However, more in vitro and preclinical research is needed in this area.

TGFβ1 induces CXCL1 to promote stemness features in lung cancer.

Chemokines critically orchestrate the tumorigenesis, metastasis, and stemness features of cancer cells that lead to poor outcomes. High plasma levels of transforming growth factor-β1 (TGFβ1) correlate with poor prognostic features in advanced lung cancer patients, thus suggesting the importance of TGFβ1 in the lung tumor microenvironment. However, the role of chemokines in TGFβ1-induced tumor stemness features remains unclear. Here, we clarify the previously undocumented role of CXCL1 in TGFβ1-induced lung cancer stemness features. CXCL1 and its receptor CXCR2 were significantly upregulated in TGFβ1-induced lung cancer stem cells (CSCs). CXCL1 silencing (shCXCL1) suppressed stemness gene expression, tumorsphere formation, colony formation, drug resistance, and in vivo tumorigenicity in TGFβ1-induced lung tumorspheres. Immunohistochemistry staining showed that patients with stage II/III lung cancer had higher expression levels of CXCL1. The levels of CXCL1 were positively associated with lymph node metastasis and correlated with the expression of the CSC transcription factor Oct-4. Furthermore, online database analysis revealed that CXCL1 expression was negatively correlated with lung cancer survival in patients. Patients with high TGFβ1/CXCL1/CD44 co-expression had a worse survival rate. We suggest that CXCL1 serves as a crucial factor in TGFβ1-induced stemness features of lung cancer.

Adipose Derived Stem Cell‐Conditioned Media As Alternative of Stem Cell Therapy for Diabetic Wound Healing

Diabetes mellitus (DM) is a set of metabolic disorder primarily characterized by chronic hyperglycaemia state, which is linked with several complications including retinopathy, neuropathy, and delayed wound healing. Adipose derived stem cells conditioned media (ADSCs-CM) contains several cytokines, and growth factors that can be used for treatment of diabetes-associated wounds. This study was conducted to explore the therapeutic potential of ADSCs- CM in excisional wound model in diabetic mice. Diabetes was induced in mice through Streptozotocin (STZ). Then, excisional wound splinting model in diabetic mice was developed. Subcutaneous injections of ADSC-CM (pure) and ADSC-CM conjugated with zinc sulphate were applied on the mice wound bed. Wound diameter, weight, and blood glucose levels of all the mice groups were measured at the start and end of the experiment. Additionally, qPCR was performed to analyse the expression level of some crucial genes (MMP-2, NANOG and OCT4) associated with wound healing. Overall, the results showed reduced wound size, blood glucose level, and weight restoration in ADSCs-CM treated mice as compared with control groups. Furthermore, ADSCs-CM also modulated the gene expression of MMP-2, NANOG, and OCT4, contributing to the enhanced wound healing observed in diabetic mice. This suggests ADSC-CM’s potential as an alternative therapeutic option for achieving improved wound healing outcomes in diabetic patients.

#193 : Comparison of the Different Expression of OCT-4, GATA-3 and Clinical Pregnancy Outcome Between the Day 5 and Day 6 Blastocysts

Background and Aims: In common experience of in vitro-fertilization (IVF) cycle, blastocysts formed at Day 5 (D5) are regarded as higher quality and are more desirable for embryo transfer compared with those formed at Day 6 (D6). How those delayed D6 embryo differs from D5 embryo is less discussed. This study aimed to elucidate the cell content distinguished by immunofluorescent (IF) staining between the same grade of D5 and D6 blastocysts that might link to the clinical pregnancy outcome in frozen–thawed embryo from different day of blastocyst formation. Method: The retrospective study included 1327 frozen-thawed single blastocyst transfer (SBT) cycles in the Taipei Fertility Center during March 2020 to December 2022. All D5 (D5-SBT) and D6 (D6-SBT) frozen-thawed SET cycles (n=1327) were separated to non-PGS (n=671) and PGS-euploid (n=656) groups, then assigned to high grade (AA, AB, BA, BB) and low grade (BC, CB, CC). Aneuploidy and mosaicism embryo are excluded. For cell content analysis, pluripotent marker OCT-4 initially expressed in all blastomeres but restricted to the inner cell mass (ICM) of the blastocyst and downregulated in the trophectoderm (TE). GATA-3 is a TE specific marker which increases during pre-implantation embryo development. The cell number, OCT-4 and GATA-3 expression of same grade frozen-thawed D5 (n=14) and D6 (n=14) blastocysts were analyzed with IF staining. D5 extensively cultured to D6 (n=10) were used as the control group of regularly development. Cell numbers of TE and ICM region were examined by OCT-4 and GATA-3 IF detection. The total cell number was visualized with DAPI staining. Results: According to staining results, there were no significant differences in total cell number, TE and ICM cell number between the same grade D5 and D6 blastocysts. However, TE cell number significantly increased in D5 extensively cultured blastocysts, indicating higher proliferative capacity in TE cells from D5 blastocysts. In the clinical data of the non-PGS group, chemical pregnancy rate (HCG+) and clinical pregnancy rate (SAC+) of high grade D5-SBT were significantly increased compared with high grade D6-SBT (HCG+: 69.1% versus 47.0%, P&lt;0.001; SAC+: 63.0% versus 43.4%, P=0.001). In the PGS-euploid group, the chemical pregnancy rate (HCG+) showed significantly increased in high grade D5-SBT compared with high grade D6-SBT (69.5% versus 60.0%, P=0.047). The clinical pregnancy rate (SAC+) was significantly higher in high grade D5-SBT compared with high grade D6-SBT (65.5% versus 52.5%, P=0.009). The ongoing pregnancy rate was significantly higher in high grade D5-SBT compared with high grade D6-SBT (57.6% versus 43.3%, P=0.005). Conclusion: According to the IF data in this study, we demonstrated that the number of GATA3-positive TE cells were significantly decreased in D6 blastocyst compared with D5 extensively cultured blastocysts. Due to decreased proliferative capacity in high grade D6 blastocyst, transfer of high grade D5 blastocyst would exhibit higher ongoing pregnancy rate than D6 blastocyst.

Angiotensin-(1-7) Improves Islet β-cell Dedifferentiation by Activating PI3K/Akt/FoxO1 Pathway.

Islet β-cell dedifferentiation may be the main cause of reduced insulin secretion. Angiotensin-(1-7) [Ang-(1-7)] can attenuate high glucose-induced apoptosis and dedifferentiation of pancreatic β-cell, but the specific signal transduction pathway and mechanism are not yet clear. This study aimed to investigate the effects of Ang-(1-7) on high glucose-induced islet β-cell dedifferentiation by activating the phosphatidylinositol-3-kinase/Protein kinase B/ Forkhead box transcription factor O1 (PI3K/Akt/FoxO1) signaling pathway. The mouse islet β-cell line MIN6 cells were passaged and cultured and randomly divided into five groups: control (Con) group, high glucose (HG) group, HG with Ang-(1-7) group, HG with Ang-(1-7) and specific MasR antagonist A-779 group, and HG with Ang-(1-7) and PI3K inhibitor LY294002 group. After 48 hours, glucose-stimulated insulin secretion (GSIS) was detected by Enzyme-Linked Immunosorbent Assay (ELISA). The mRNA and protein expression levels of β-cell-specific factors (Pancreatic duodenal homeobox-1 (Pdx1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A(MafA)) and endocrine progenitor cell-specific factors (Octamer binding transcription factor 4(Oct4), Nanog) were measured by Real Time-PCR and Western blot. The factors of protein expression levels of PI3K/Akt/FoxO1 signaling pathway (Akt, p-Akt, Fox- O1, p-FoxO1) were determined by Western blot. We observed for the first time that high glucotoxicity can induce dedifferentiation of pancreatic islet β-cell, causing a decrease in insulin secretion levels and expression of Pdx1, MafA, p-- FoxO1, and p-Akt and an increase in expression of Oct4 and Nanog. After Ang-(1-7) intervention, insulin secretion levels and expression of Pdx1, MafA, p-FoxO1 and p-Akt were increased, and the levels of Oct4 and Nanog were reduced. However, A-779 and LY294002 could reverse this effect. During these processes, the total Akt and total FoxO1 expression did not change significantly. Ang-(1-7) may prevent high glucose-induced pathological dedifferentiation of pancreatic β-cell by activating the PI3K/Akt/FoxO1 signaling pathway.

A paradigm for post-embryonic Oct4 re-expression: E7-induced hydroxymethylation regulates Oct4 expression in cervical cancer.

The Octamer-binding transcription factor-4 (Oct4) is upregulated in different malignancies, yet a paradigm for mechanisms of Oct4 post-embryonic re-expression is inadequately understood. In cervical cancer, Oct4 expression is higher in human papillomavirus(HPV)-related than HPV-unrelated cervical cancers and this upregulation correlates with the expression of the E7 oncogene. We have reported that E7 affects the Oct4-transcriptional output and Oct4-related phenotypes in cervical cancer, however, the underlying mechanism remains elusive. Here, we characterize the Oct4-protein interactions in cervical cancer cells via computational analyses and Mass Spectrometry and reveal that Methyl-binding proteins (MBD2 and MBD3), are determinants of Oct4-driven transcription. E7 triggers MBD2 downregulation and TET1 upregulation, thereby disrupting the methylation status of the Oct4 gene. This coincides with an increase in the total DNA hydroxymethylation leading to the re-expression of Oct4 in cervical cancer and likely affecting broader transcriptional patterns. Our findings reveal a previously unreported mechanism by which the E7 oncogene can regulate Oct4 re-expression and global transcriptional patterns by increasing DNA hydroxymethylation and lowering the barrier to cellular plasticity during carcinogenesis.

Exploring the Influence of miR-597-5p on the Tumor Stem Cell Characteristics of Gastric Cancer Cells Through the STAT3/NF-κB Signaling Pathway

In this study, we investigated the impact of miR-597-5p on the tumor stem cell characteristics of gastric cancer cells and the underlying mechanisms. Gastric cancer cell lines were transfected with miR-597-5p mimics to elevate miR-597-5p expression, confirmed by qRT-PCR. Overexpression of miR-597-5p led to reduced cell viability, proliferation, migration, invasion, and sphere-forming ability, while increasing apoptosis. Additionally, the expression of stem cell markers OCT4 and SOX2, as well as the phosphorylation of STAT3 and NF-κB, decreased with miR-597-5p overexpression. Co-treatment with a STAT3 activator reversed these effects, promoting cell proliferation, invasion, migration, sphere formation, and increasing p-STAT3, p-NF-κB, SOX2, and OCT4 expression. Importantly, miR-597-5p inhibited the STAT3/NF-κB signaling pathway, preventing gastric cancer cells from exhibiting stem-like characteristics. These findings suggest that miR-597-5p may have therapeutic potential in suppressing the stem cell properties of gastric cancer cells through STAT3/NF-κB pathway regulation.

Methoxychlor induces oxidative stress and impairs early embryonic development in pigs.

Introduction: Methoxychlor (MXC) is an organochlorine pesticide (OCP) that was formerly used worldwide as an insecticide against pests and mosquitoes. However, MXC is not biodegradable and has lipophilic characteristics; thus, it accumulates in organisms and affects reproductive function. MXC, as an estrogenic compound, promotes oxidative stress, induces oxidative stress damage to ovarian follicles, and causes miscarriages and stillbirths in females. In this research endeavor, our primary objective was to explore the ramifications of MXC regarding the developmental processes occurring during the initial stages of embryogenesis in pigs. Methods: In this study, we counted the blastocyst rate of early embryos cultured in vitro. We also examined the reactive oxygen species level, glutathione level, mitochondrial membrane potential, mitochondrial copy number and ATP level in four-cell stage embryos. Finally, apoptosis and DNA damage in blastocyst cells, as well as pluripotency-related and apoptosis-related genes in blastocyst cells were detected. The above experiments were used to evaluate the changes of MXC damage on early parthenogenetic embryo development. Results and Discussion: The results showed that early embryos exposed to MXC had a significantly lower cleavage rate, blastocyst rate, hatching rate, and total cell count compared with the control group. It was also of note that MXC not only increased the levels of reactive oxygen species (ROS), but also decreased the mitochondrial membrane potential (ΔΨm) and mitochondrial copy number during the development of early embryos. In addition, after MXC treatment, blastocyst apoptosis and DNA damage were increased, decreased cell proliferation, and the expression of pluripotency-related genes SOX2, NANOG, and OCT4 was down-regulated, while the expression of apoptosis-related genes BAX/BCL-2 and Caspase9 was up-regulated. Our results clearly show that MXC can have deleterious effects on the developmental processes of early porcine embryos, establishing the toxicity of MXC to the reproductive system. In addition, the study of this toxic effect may lead to greater concern about pesticide residues in humans and the use of safer pesticides, thus potentially preventing physiological diseases caused by chemical exposure.

In vivo reprogramming leads to premature death linked to hepatic and intestinal failure.

The induction of cellular reprogramming via expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM) can drive dedifferentiation of somatic cells and ameliorate age-associated phenotypes in multiple tissues and organs. However, the benefits of long-term in vivo reprogramming are limited by detrimental side-effects. Here, using complementary genetic approaches, we demonstrated that continuous induction of the reprogramming factors in vivo leads to hepatic and intestinal dysfunction resulting in decreased body weight and contributing to premature death (within 1 week). By generating a transgenic reprogrammable mouse strain, avoiding OSKM expression in both liver and intestine, we reduced the early lethality and adverse effects associated with in vivo reprogramming and induced a decrease in organismal biological age. This reprogramming mouse strain, which allows longer-term continuous induction of OSKM with attenuated toxicity, can help better understand rejuvenation, regeneration and toxicity during in vivo reprogramming.

Analysis of OCT4 and PGP9.5 gene expression in prenatal and postnatal buffalo (Bubalus bubalis) testes

This study aimed to investigate and characterize the spermatogonial stem cells (SSCs) in buffaloes at different stages of development, including prenatal, neonatal, prepubertal, and adult testes. We sought a comprehensive understanding of these cells through a combination of histological, immunohistochemical, and ultrastructural analyses. Specifically, we examined changes in the expression of two potential SSC markers, OCT4 and PGP9.5, using immunohistochemistry. Additionally, we conducted a real-time quantitative polymerase chain reaction (RT-qPCR) to assess the relative gene expression of OCT4 and PGP9.5. The relative expression of the OCT4 gene was down-regulated in the adult testes compared to its expression during prepubertal and neonatal life. The relative expression of the PGP9.5 gene was up-regulated in the neonatal testes and down-regulated in the prepubertal and adult testes. The spermatogonia were round, oval-to-ellipsoidal cells lying over the basement membrane (BM) with a round-to-oval nucleus. Based on the immunoexpression of the putative SSC markers, OCT4 and PGP9.5, we concluded that the proportion of stem cells was highest during the neonatal stage, followed by the prepubertal and prenatal stages. This finding sheds light on the dynamics of spermatogonial stem cells in buffalo testes at different developmental stages, providing valuable insights into these cells’ regulation and potential applications.

Transcription factor LHX9 (LIM Homeobox 9) enhances pyruvate kinase PKM2 activity to induce glycolytic metabolic reprogramming in cancer stem cells, promoting gastric cancer progression

BackgroundGlycolytic metabolic reprogramming is a phenomenon in which cells undergo altered metabolic patterns during malignant transformation, mainly involving various aspects of glycolysis, electron transport chain, oxidative phosphorylation, and pentose phosphate pathway. This reprogramming phenomenon can be used as one of the markers of tumorigenesis and development. Pyruvate kinase is the third rate-limiting enzyme in the sugar metabolism process by specifically catalyzing the irreversible conversion of PEP to pyruvate.PurposeThis study aimed to reveal the critical mediator(s) that regulate glycolytic metabolism reprogramming in gastric cancer and their underlying molecular mechanism and then explore the molecular mechanisms by which LHX9 may be involved in regulating gastric cancer (GC) progression.MethodsFirstly, we downloaded the GC and glycolysis-related microarray datasets from TCGA and MSigDB databases and took the intersection to screen out the transcription factor LHX9 that regulates GC glycolytic metabolic reprogramming. Software packages were used for differential analysis, single gene predictive analysis, and Venn diagram. In addition, an enrichment analysis of the glycolytic pathway was performed. Immunohistochemical staining was performed for LHX9 and PKM2 protein expression in 90 GC patients, and the association between their expressions was evaluated by Spearman's correlation coefficient method. Three human GC cell lines (AGS, NCI-N87, HGC-27) were selected for in vitro experimental validation. Flow cytometry was utilized to determine the stem cell marker CD44 expression status in GCSCs. A sphere formation assay was performed to evaluate the sphere-forming capabilities of GCSCs. In addition, RT-qPCR and Western blot experiments were employed to investigate the tumor stem cell markers OCT4 and SOX2 expression levels in GCSCs. Furthermore, a lentiviral expression vector was constructed to assess the impact of downregulating LHX9 or PKM2 on the glycolytic metabolic reprogramming of GCSCs. The proliferation, migration, and invasion of GCSCs were then detected by CCK-8, EdU, and Transwell assays. Subsequently, the mutual binding of LHX9 and PKM2 was verified using chromatin immunoprecipitation and dual luciferase reporter genes. In vivo experiments were verified by establishing a subcutaneous transplantation tumor model in nude mice, observing the size and volume of tumors in vivo in nude mice, and obtaining fresh tissues for subsequent experiments.ResultsBioinformatics analysis revealed that LHX9 might be involved in the occurrence and development of GC through regulating glycolytic metabolism. High LHX9 expression could be used as a reference marker for prognosis prediction of GC patients. Clinical tissue assays revealed that LHX9 and PKM2 were highly expressed in GC tissues. Meanwhile, GC tissues also highly expressed glycolysis-associated protein GLUT1 and tumor cell stemness marker CD44. In vitro cellular assays showed that LHX9 could enhance its activity and induce glycolytic metabolic reprogramming in GCSCs through direct binding to PKM2. In addition, the knockdown of LHX9 inhibited PKM2 activity and glycolytic metabolic reprogramming and suppressed the proliferation, migration, and invasive ability of GCSCs. In vivo animal experiments further confirmed that the knockdown of LHX9 could reduce the tumorigenic ability of GCSCs in nude mice by inhibiting PKM2 activity and glycolytic metabolic reprogramming.ConclusionThe findings suggest that both LHX9 and PKM2 are highly expressed in GCs, and LHX9 may induce the reprogramming of glycolytic metabolism through transcriptional activation of PKM2, enhancing the malignant biological properties of GCSCs and ultimately promoting GC progression.

Three-dimensional glass scaffolds improve the In Vitro maturation of porcine cumulus-oocyte complexes and subsequent embryonic development after parthenogenetic activation

In vitro maturation (IVM) methods for porcine oocytes are still deficient in achieving full developmental capacity, as the currently available oocyte in vitro culture systems still have limitations. In vitro embryo production must also improve the porcine oocyte IVM system to acquire oocytes with good developmental potential. Herein, we tested a three-dimensional (3D) glass scaffold culture system for porcine oocyte maturation. After 42 h, we matured porcine cumulus-oocyte complexes (COCs) on either two-dimensional glass dishes (2D-B), two-dimensional microdrops (2D-W), or 3D glass scaffolds. The 3D glass scaffolds were tested for porcine oocyte maturation and embryonic development. Among these culture methods, the extended morphology of the 3D group maintained a 3D structure better than the 2D-B and 2D-W groups, which had flat COCs that grew close to the bottom of the culture vessel. The COCs of the 3D group had a higher cumulus expansion index and higher first polar body extrusion rate, cleavage rate, and blastocyst rate of parthenogenetic embryos than the 2D-B group. In the 3D group, the cumulus-expansion-related gene HAS2 and anti-apoptotic gene Bcl-2 were significantly upregulated (p < 0.05), while the pro-apoptotic gene Caspase3 was significantly downregulated (p < 0.05). The blastocysts of the 3D group had a higher relative expression of Bcl-2, Oct4, and Nanog than the other two groups (p < 0.05). The 3D group also had a more uniform distribution of mitochondrial membrane potential and mitochondria (p < 0.05), and its cytoplasmic active oxygen species content was much lower than that in the 2D-B group (p < 0.05). These results show that 3D glass scaffolds dramatically increased porcine oocyte maturation and embryonic development after parthenogenetic activation, providing a suitable culture model for porcine oocytes.

A calibration and uncertainty quantification analysis of classical, fractional and multiscale logistic models of tumour growth

Background and Objective: The validation of mathematical models of tumour growth is frequently hampered by the lack of sufficient experimental data, resulting in qualitative rather than quantitative studies. Recent approaches to this problem have attempted to extract information about tumour growth by integrating multiscale experimental measurements, such as longitudinal cell counts and gene expression data. In the present study, we investigated the performance of several mathematical models of tumour growth, including classical logistic, fractional and novel multiscale models, in terms of quantifying in-vitro tumour growth in the presence and absence of therapy. We further examined the effect of genes associated with changes in chemosensitivity in cell death rates. Methods: The multiscale expansion of logistic growth models was performed by coupling gene expression profiles to the cell death rates. State-of-the-art Bayesian inference, likelihood maximisation and uncertainty quantification techniques allowed a thorough evaluation of model performance. Results: The results suggest that the classical single-cell population model (SCPM) was the best fit for the untreated and low-dose treatment conditions, while the multiscale model with a cell death rate symmetric with the expression profile of OCT4 (Sym-SCPM) yielded the best fit for the high-dose treatment data. Further identifiability analysis showed that the multiscale model was both structurally and practically identifiable under the condition of known OCT4 expression profiles. Conclusions: Overall, the present study demonstrates that model performance can be improved by incorporating multiscale measurements of tumour growth when high-dose treatment is involved.

Vitamin B12 is a limiting factor for induced cellular plasticity and tissue repair

Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair.

Female Bovine Donor Age Influence on Quality Markers’ Expression and PPARS Abundance in Day 7 Blastocysts

Abstract In bovine females, the production of embryos derived from oocytes obtained by ovum pick-up (OPU) is becoming a frequent procedure. Collection of oocytes from prepubertal animals enables shortening the genetic distance, especially in high-value animals. Nevertheless, the oocyte and later embryo developmental potential differ between oocytes derived from prepubertal and adult heifers. The aim of this study was to determine the possible underlying causes for differences in quality between embryos obtained from prepubertal and pubertal heifers. Cumulus oocyte complexes were collected via OPU. The expression of PLAC8, IFN τ, IGFR1, SOX2, and OCT4 in blastocysts was evaluated. The transcriptome of peroxisome proliferator-activated receptors (PPARs), the major genes involved in the metabolism and development of the embryo, and the embryo quality marker gene expression (PLAC8, IFN τ, IGFR1, SOX2, and OCT4) in the day 7 blastocysts derived from oocytes collected from prepubertal or pubertal heifers differed significantly. The expression of genes involved in basic biological processes of early embryonic development differed between embryos derived from oocytes collected from prepubertal and pubertal heifers. The obtained data may be used to adjust culture conditions to the biological requirements of the embryos derived from oocytes collected from prepubertal heifers, and to predict the possibility of successful development of the embryo.

Transgenic Lines of Human Induced Pluripotent Stem Cells ICGi022-A-6 and ICGi022-A-7 with Doxycycline-Inducible Variants of Programmable Nuclease AsCas12a

Genome editing in human pluripotent stem cells using programmable nucleases makes it possible to create models of hereditary pathologies using directed transgenesis, gene knockout, and replacement of individual nucleotides in DNA sequences. Using CRISPR/SpCas9-mediated homologous recombination at the AAVS1 locus, clones of human induced pluripotent stem cells (iPSCs) ICGi022-A (Malakhova et al., 2020) were obtained, which carry transgenes of two variants of the nuclease AsCas12a (also known as AsCpf1), recognizing different PAM consensuses, and the reverse doxycycline transgene-dependent transactivator – M2rtTA. For each AsCas12a variant, the lines ICGi022-A-6 (AsCas12a, PAM 5'-TTTV-3') and ICGi022-A-7 (AsCas12a, PAM 5'-TYCV-3') were obtained. Using Western blot analysis, it was shown that the addition of doxycycline to the culture medium causes activation of the expression of AsCas12a(TTTV) and AsCas12a(TYCV) proteins. The resulting transgenic iPSC clones were subjected to molecular and cytogenetic analysis. Using quantitative PCR and immunocytochemical analysis, it was shown that they have a high level of mRNA expression of gene markers of pluripotent cells, namely OCT4, NANOG and SOX2, as well as specific expression of protein marker OCT4, SOX2, SSEA-4 and TRA-1-60. In addition, using iPSCs spontaneous differentiation into embryoid bodies, it was found that transgenic clones can give derivatives of all three primitive germ layers: ectoderm, mesoderm and endoderm. Cytogenetic analysis showed that transgenic iPSC clones have a normal karyotype, 46,XX.