Epigenetically associated IGF2BP3 upregulation promotes cell proliferation by regulating E2F1 expression in hepatocellular carcinoma.

RNA-binding proteins (RBPs) are a class of proteins that primarily function by interacting with different types of RNAs and play a critical role in regulating the transcription and translation of cancer-related genes. However, their role in the progression of hepatocellular carcinoma (HCC) remains unclear. In this study, we analyzed RNA sequencing data and the corresponding clinical information of patients with HCC to screen for prognostic RBPs. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) was identified as an independent prognostic factor for liver cancer. It is upregulated in HCC and is associated with a poor prognosis. Elevated IGF2BP3 expression was validated via immunohistochemical analysis using a tissue microarray of patients with HCC. IGF2BP3 knockdown inhibited the proliferation of Hep3B and HepG2 cells, whereas IGF2BP3 overexpression promoted the expansion of HuH-7 and MHCC97H cells. Mechanistically, IGF2BP3 modulates cell proliferation by regulating E2F1 expression. DNA hypomethylation of the IGF2BP3 gene may increase the expression of IGF2BP3, thereby enhancing cell proliferation in HCC. Therefore, IGF2BP3 may act as a novel prognostic biomarker and a potential therapeutic target for HCC.

Identifying and evaluating a disulfidptosis-related gene signature to predict prognosis in colorectal adenocarcinoma patients.

Disulfidptosis, a regulated form of cell death, has been recently reported in cancers characterized by high SLC7A11 expression, including invasive breast carcinoma, lung adenocarcinoma, and hepatocellular carcinoma. However, its role in colon adenocarcinoma (COAD) has been infrequently discussed. In this study, we developed and validated a prognostic model based on 20 disulfidptosis-related genes (DRGs) using LASSO and Cox regression analyses. The robustness and practicality of this model were assessed via a nomogram. Subsequent correlation and enrichment analysis revealed a relationship between the risk score, several critical cancer-related biological processes, immune cell infiltration, and the expression of oncogenes and cell senescence-related genes. POU4F1, a significant component of our model, might function as an oncogene due to its upregulation in COAD tumors and its positive correlation with oncogene expression. In vitro assays demonstrated that POU4F1 knockdown noticeably decreased cell proliferation and migration but increased cell senescence in COAD cells. We further investigated the regulatory role of the DRG in disulfidptosis by culturing cells in a glucose-deprived medium. In summary, our research revealed and confirmed a DRG-based risk prediction model for COAD patients and verified the role of POU4F1 in promoting cell proliferation, migration, and disulfidptosis.

Screening and characterization of a novel linear B-cell epitope on orf virus F1L protein.

Background: Orf, also known as contagious ecthyma (CE), is an acute, contagious zoonotic disease caused by the orf virus (ORFV). The F1L protein is a major immunodominant protein on the surface of ORFV and can induce the production of neutralizing antibodies. Methods: The prokaryotic expression system was used to produce the recombinant F1L protein of ORFV, which was subsequently purified and used to immunize mice. Positive hybridoma clones were screened using an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity and specificity of the monoclonal antibody (mAb) were verified through Western blot and indirect immunofluorescence (IFA). The linear antigenic epitope specific to the mAb was identified through Western blot, using truncated F1L proteins expressed in eukaryotic cells. A multiple sequence alignment of the ORFV reference strains was performed to evaluate the degree of conservation of the identified epitope. Results: After three rounds of subcloning, a mAb named Ba-F1L was produced. Ba-F1L was found to react with both the exogenously expressed F1L protein and the native F1L protein from ORFV-infected cells, as confirmed by Western blot and IFA. The mAb recognized the core epitope 103CKSTCPKEM111, which is highly conserved among various ORFV strains, as shown by homologous sequence alignment. Conclusion: The mAb produced in the present study can be used as a diagnostic reagent for detecting ORFV and as a basic tool for exploring the mechanisms of orf pathogenesis. In addition, the identified linear epitope may be valuable for the development of epitope-based vaccines.

E2F1 Facilitates the Proliferation and Stemness of Gastric Cancer Cells by Activating CDC25B Transcription and Modulating the MAPK Pathway.

Gastric cancer (GC) is a health problem that concerns people around the world. CDC25B is an essential cell cycle regulatory factor that is overexpressed in a variety of tumor cells. CDC25B plays a vital part in the progression and proliferation of malignant tumors. However, it is not yet clear that how CDC25B affects the stemness of GC cells. The study used bioinformatics to detect the expression of E2F1 and CDC25B in GC tissues and their correlation, as well as pathways enriched by CDC25B. We detected the expression of E2F1 and CDC25B in GC cell lines using quantitative reverse transcription polymerase chain reaction and tested the combination relationship between E2F1 and CDC25B using chromatin immunoprecipitation (ChIP) and dual-luciferase assays. We measured cell viability using CCK-8 assay, evaluated sphere-forming efficiency using sphere formation assay, and determined cell proliferation ability using colony formation assay. We also analyzed the expression of stemness markers and MAPK pathway-related proteins using western blot. In GC tissues and cells, CDC25B was upregulated. Silencing CDC25B could affect the MAPK pathway, thereby repressing the proliferation and stemness of GC cells. As predicted by bioinformatics, CDC25B had an upstream transcription factor, E2F1, which also had a high expression level in GC. Dual-luciferase and ChIP assays confirmed the combination relationship between the two. Rescue experiments uncovered that overexpression of CDC25B could reverse the impact induced by E2F1 knockdown on proliferation and stemness of cells. In conclusion, E2F1 could activate CDC25B transcription to regulate the MAPK pathway and enhance the proliferation and stemness of GC cells. We revealed a potential regulatory pathway of stemness of GC cells that was mediated by CDC25B, providing new ideas for improving and innovating GC treatment.

The Role of ArtificiaI Intelligence in Brain Tumor Diagnosis: An Evaluation of a Machine Learning Model.

This research study explores of the effectiveness of a machine learning image classification model in the accurate identification of various types of brain tumors. The types of tumors under consideration in this study are gliomas, meningiomas, and pituitary tumors. These are some of the most common types of brain tumors and pose significant challenges in terms of accurate diagnosis and treatment. The machine learning model that is the focus of this study is built on the Google Teachable Machine platform (Alphabet Inc., Mountain View, CA). The Google Teachable Machine is a machine learning image classification platform that is built from Tensorflow, a popular open-source platform for machine learning. The Google Teachable Machine model was specifically evaluated for its ability to differentiate between normal brains and the aforementioned types of tumors in MRI images. MRI images are a common tool in the diagnosis of brain tumors, but the challenge lies in the accurate classification of the tumors. This is where the machine learning model comes into play. The model is trained to recognize patterns in the MRI images that correspond to the different types of tumors. The performance of the machine learning model was assessed using several metrics. These include precision, recall, and F1 score. These metrics were generated from a confusion matrix analysis and performance graphs. A confusion matrix is a table that is often used to describe the performance of a classification model. Precision is a measure of the model's ability to correctly identify positive instances among all instances it identified as positive. Recall, on the other hand, measures the model's ability to correctly identify positive instances among all actual positive instances. The F1 score is a measure that combines precision and recall providing a single metric for model performance. The results of the study were promising. The Google Teachable Machine model demonstrated high performance, with accuracy, precision, recall, and F1 scores ranging between 0.84 and 1.00. This suggests that the model is highly effective in accurately classifying the different types of brain tumors. This study provides insights into the potential of machine learning models in the accurate classification of brain tumors. The findings of this study lay the groundwork for further research in this area and have implications for the diagnosis and treatment of brain tumors. The study also highlights the potential of machine learning in enhancing the field of medical imaging and diagnosis. With the increasing complexity and volume of medical data, machine learning models like the one evaluated in this study could play a crucial role in improving the accuracy and efficiency of diagnoses. Furthermore, the study underscores the importance of continued research and development in this field to further refine these models and overcome any potential limitations or challenges. Overall, the study contributes to the field of medical imaging and machine learning and sets the stage for future research and advancements in this area.

FLMatchQA: a recursive neural network-based question answering with customized federated learning model.

More sophisticated data access is possible with artificial intelligence (AI) techniques such as question answering (QA), but regulations and privacy concerns have limited their use. Federated learning (FL) deals with these problems, and QA is a viable substitute for AI. The utilization of hierarchical FL systems is examined in this research, along with an ideal method for developing client-specific adapters. The User Modified Hierarchical Federated Learning Model (UMHFLM) selects local models for users' tasks. The article suggests employing recurrent neural network (RNN) as a neural network (NN) technique for learning automatically and categorizing questions based on natural language into the appropriate templates. Together, local and global models are developed, with the worldwide model influencing local models, which are, in turn, combined for personalization. The method is applied in natural language processing pipelines for phrase matching employing template exact match, segmentation, and answer type detection. The (SQuAD-2.0), a DL-based QA method for acquiring knowledge of complicated SPARQL test questions and their accompanying SPARQL queries across the DBpedia dataset, was used to train and assess the model. The SQuAD2.0 datasets evaluate the model, which identifies 38 distinct templates. Considering the top two most likely templates, the RNN model achieves template classification accuracy of 92.8% and 61.8% on the SQuAD2.0 and QALD-7 datasets. A study on data scarcity among participants found that FL Match outperformed BERT significantly. A MAP margin of 2.60% exists between BERT and FL Match at a 100% data ratio and an MRR margin of 7.23% at a 20% data ratio.

Efficient CRISPR-Cas12f1-Mediated Multiplex Bacterial Genome Editing via Low-Temperature Recovery.

CRISPR-Cas system is being used as a powerful genome editing tool with developments focused on enhancing its efficiency and accuracy. Recently, the miniature CRISPR-Cas12f1 system, which is small enough to be easily loaded onto various vectors for cellular delivery, has gained attention. In this study, we explored the influence of temperature conditions on multiplex genome editing using CRISPR-Cas12f1 in an Escherichia coli model. It was revealed that when two distinct targets in the genome are edited simultaneously, the editing efficiency can be enhanced by allowing cells to recover at a reduced temperature during the editing process. Additionally, employing 3'-end truncated sgRNAs facilitated the simultaneous single-nucleotide level editing of three targets. Our results underscore the potential of optimizing recovery temperature and sgRNA design protocols in developing more effective and precise strategies for multiplex genome editing across various organisms.

Structural, Biophysical, and Computational Studies of a Murine Light Chain Dimer.

Antibodies are widely used in medicinal and scientific research due to their ability to bind to a specific antigen. Most often, antibodies are composed of heavy and light chain domains. Under physiological conditions, light chains are produced in excess, as compared to the heavy chain. It is now known that light chains are not silent partners of the heavy chain and can modulate the immune response independently. In this work, the first crystal structure of a light chain dimer originating from mice is described. It represents the light chain dimer of 6A8, a monoclonal antibody specific to the allergen Der f 1. Building on the unexpected occurrence of this kind of dimer, we have demonstrated that this light chain is stable in solution alone. Moreover, enzyme-linked immunosorbent assays (ELISA) have revealed that, when the light chain is not partnered to its corresponding heavy chain, it interacts non-specifically with a wide range of proteins. Computational studies were used to provide insight on the role of the 6A8 heavy chain domain in the specific binding to Der f 1. Overall, this work demonstrates and supports the ongoing notion that light chains can function by themselves and are not silent partners of heavy chains.

Up-regulation of NCAPG mediated by E2F1 facilitates the progression of osteosarcoma through the Wnt/ß-catenin signaling pathway.

Background: In recent years, there are few reports on non-SMC condensin I complex subunit G (NCAPG) in osteosarcoma. Our study aims to explore the biological role of NCAPG in osteosarcoma and its underlying molecular mechanism and to further clarify the reasons for the abnormal expression of NCAPG in osteosarcoma. Methods: Here, we mined The Cancer Genome Atlas (TCGA) Program public database through bioinformatics methods, analyzed the differential expression of NCAPG in sarcoma tissue and normal tissue, and explored the relationship between NCAPG expression level and sarcoma tissue differentiation, including tumor recurrence, metastasis, and patient survival. Next, the transcription factors responsible for the abnormal expression of NCAPG in osteosarcoma tumors were predicted by multiple online website tools and verified via cellular experiments. Subsequently, loss of function and cell phenotype experiments were performed to confirm the effect of NCAPG on the malignant biological behavior of osteosarcoma cells. Mechanistically, by reviewing the literature, we found that NCAPG can affect the malignant progression of many solid tumors by regulating the Wnt/ß-catenin signaling pathway. Therefore, we preliminarily investigated the potential effect of NCAPG on this pathway via western blot experiments in osteosarcoma. Results: Increased expression of NCAPG was found in sarcoma compared to normal tissues, which was positively correlated with poor differentiation, metastasis, and poor prognosis. Combining the transcription factor prediction results, correlation analysis, and expression level in the TCGA public database with validation outcomes of in vitro cell assays, we found that E2F transcription factor 1 (E2F1) regulated the increased expression of NCAPG in osteosarcoma. The results of cell phenotype experiments showed that silencing NCAPG could inhibit the proliferation, migration, and invasion of osteosarcoma cells. The preliminary mechanistic investigation suggested that NCAPG may affect osteosarcoma progression through the Wnt/ß-catenin pathway. Conclusions: Our data reveal that E2F1 facilitates NCAPG expression in osteosarcoma by regulating the transcription of the NCAPG gene. Up-regulation of NCAPG promotes osteosarcoma progression via the Wnt/ß-catenin signaling axis.

CircMYBL2 facilitates hepatocellular carcinoma progression by regulating E2F1 expression.

Circular RNAs (circRNAs) have been recognized as pivotal regulators in tumorigenesis, yet the biological functions as well as molecular mechanisms of the majority of circRNAs in hepatocellular carcinoma (HCC) remain elusive. We sought to unveil the expression profile and biological role of circMYBL2 in HCC. Initial microarray analyses were conducted to probe the expression profile of circMYBL2 in HCC cells, and qRT‒PCR analysis was then performed in HCC cell lines and tissues, revealing significant upregulation of circMYBL2. Subsequent experiments were conducted to evaluate the biological function of circMYBL2 in HCC progression. Furthermore, bioinformatics analysis, qRT‒PCR analysis, luciferase reporter assays, and western blot analysis were employed to investigate the interplay among circMYBL2, miR-1205, and E2F1. CircMYBL2 was found to exhibit marked upregulation in tumor tissues as well as HCC cell lines. Elevated expression of circMYBL2 increased the proliferation and migration of HCC cells, whereas circMYBL2 knockdown elicited contrasting effects. Mechanistically, our results indicated that circMYBL2 promoted E2F1 expression and facilitated HCC progression by sponging miR-1205. Our findings revealed that circMYBL2 contributed to HCC progression through the circMYBL2/miR-1205/E2F1 axis, suggesting the potential of circMYBL2 as a novel target for HCC treatment or a prognostic biomarker for HCC.

The primitive endoderm supports lineage plasticity to enable regulative development.

Mammalian blastocyst formation involves the specification of the trophectoderm followed by the differentiation of the inner cell mass into embryonic epiblast and extra-embryonic primitive endoderm (PrE). During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the PrE alone was sufficient to regenerate a complete blastocyst and continue post-implantation development. We identify an in vitro population similar to the early PrE in vivo that exhibits the same embryonic and extra-embryonic potency and can form complete stem cell-based embryo models, termed blastoids. Commitment in the PrE is suppressed by JAK/STAT signaling, collaborating with OCT4 and the sustained expression of a subset of pluripotency-related transcription factors that safeguard an enhancer landscape permissive for multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlight the importance of the PrE in perturbed development.

Size matters in metabolic scaling: Critical role of the thermodynamic efficiency of ATP synthesis and its dependence on mitochondrial H+ leak across mammalian species.

In this last article of the trilogy, the unified biothermokinetic theory of ATP synthesis developed in the previous two papers is applied to a major problem in comparative physiology, biochemistry, and ecology-that of metabolic scaling as a function of body mass across species. A clear distinction is made between intraspecific and interspecific relationships in energy metabolism, clearing up confusion that had existed from the very beginning since Kleiber first proposed his mouse-to-elephant rule almost a century ago. It is shown that the overall mass exponent of basal/standard metabolic rate in the allometric relationship [Formula: see text] is composed of two parts, one emerging from the relative intraspecific constancy of the slope (b), and the other (b') arising from the interspecific variation of the mass coefficient, a(M) with body size. Quantitative analysis is shown to reveal the hidden underlying relationship followed by the interspecific mass coefficient, a(M)=P0M0.10, and a universal value of P0=3.23 watts, W is derived from empirical data on mammals from mouse to cattle. The above relationship is shown to be understood only within an evolutionary biological context, and provides a physiological explanation for Cope's rule. The analysis also helps in fundamentally understanding how variability and a diversity of scaling exponents arises in allometric relations in biology and ecology. Next, a molecular-level understanding of the scaling of metabolism across mammalian species is shown to be obtained by consideration of the thermodynamic efficiency of ATP synthesis η, taking mitochondrial proton leak as a major determinant of basal metabolic rate in biosystems. An iterative solution is obtained by solving the mathematical equations of the biothermokinetic ATP theory, and the key thermodynamic parameters, e.g. the degree of coupling q, the operative P/O ratio, and the metabolic efficiency of ATP synthesis η are quantitatively evaluated for mammals from rat to cattle. Increases in η (by ∼15%) over a 2000-fold body size range from rat to cattle, primarily arising from an ∼3-fold decrease in the mitochondrial H+ leak rate are quantified by the unified ATP theory. Biochemical and mechanistic consequences for the interpretation of basal metabolism, and the various molecular implications arising are discussed in detail. The results are extended to maximum metabolic rate, and interpreted mathematically as a limiting case of the general ATP theory. The limitations of the analysis are pointed out. In sum, a comprehensive quantitative analysis based on the unified biothermokinetic theory of ATP synthesis is shown to solve a central problem in biology, physiology, and ecology on the scaling of energy metabolism with body size.

Residence of the Nucleotide Sugar Transporter Family Members SLC35F1 and SLC35F6 in the Endosomal/Lysosomal Pathway.

The SLC35 (Solute Carrier 35) family members acting as nucleotide sugar transporters are typically localized in the endoplasmic reticulum or Golgi apparatus. It is, therefore, intriguing that some reports document the presence of orphan transporters SLC35F1 and SLC35F6 within the endosomal and lysosomal system. Here, we compared the subcellular distribution of these proteins and found that they are concentrated in separate compartments; i.e., recycling endosomes for SLC35F1 and lysosomes for SLC35F6. Swapping the C-terminal tail of these proteins resulted in a switch of localization, with SLC35F1 being trafficked to lysosomes while SLC35F6 remained in endosomes. This suggested the presence of specific sorting signals in these C-terminal regions. Using site-directed mutagenesis, fluorescence microscopy, and cell surface biotinylation assays, we found that the EQERLL360 signal located in the cytoplasmic tail of human SLC35F6 is involved in its lysosomal sorting (as previously shown for this conserved sequence in mouse SLC35F6), and that SLC35F1 localization in the recycling pathway depends on two YXXΦ-type signals: a Y367KQF sequence facilitates its internalization from the plasma membrane, while a Y392TSL motif prevents its transport to lysosomes, likely by promoting SLC35F1 recycling to the cell surface. Taken together, these results support that some SLC35 members may function at different levels of the endosomal and lysosomal system.

Comparison of Age-Related Decline and Behavioral Validity in C57BL/6 and CB6F1 Mice.

Variability in physical resilience to aging prompts a comprehensive examination of underlying mechanisms across organs and individuals. We conducted a detailed exploration of behavioral and physiological differences between C57BL/6 and CB6F1 mice across various age groups. In behavioral assays, B6 mice displayed superior performance in rotarod tasks but higher anxiety while CB6F1 mice exhibited a decline in short-term memory with age. Grip strength, long-term memory, and voluntary wheel running declined similarly with age in both strains. Examining physiological phenotypes, B6 mice exhibited lower body fat percentages across ages compared to CB6F1 mice, though cataract severity worsened with age in both strains. Analysis of cardiac functions revealed differences between strains, with worsening left ventricular hypertrophy and structural heart abnormalities with age in CB6F1 mice along with higher blood pressure than B6. Lesion scores showed an age-related increase in heart, kidney, and liver lesions in both strains, while lung lesions worsened with age only in CB6F1 mice. This study underscores the validity of behavioral assays and geropathology assessment in reflecting age-related decline and emphasizes the importance of considering strain specificity when using mouse models to study human aging.

The effect of IGFBP3 gene knockout by the CRISPR/Cas9 system on the IGF-1 pathway in murine cells.

BACKGROUND: The IGF-1 signaling pathway has been deeply involved in the aging mechanism. The insulin-like growth factor binding protein 3 (IGFBP-3) is a protein that binds to IGF-1 that regulates growth, survival, and aging. OBJECTIVE: The purpose of this study was to investigate the impact of the IGFBP3 gene knockout (KO) on the expressions of aging-related proteins and genes using the CRISPR/Cas9 system. METHODS: The IGFBP3 gene knockout (KO) was performed by the CRISPR/Cas9 system. Sanger DNA sequencing and Indel analyses were used to verify the induction of mutation. RESULTS: First, Sanger DNA sequencing was used to analyze the IGFBP3 gene knockout in murine cells (B16F1). The isolation of three colonies with the mutated DNA sequences in the IGFBP3 gene was validated. In addition, the expression levels of the IGFBP3 gene and protein in the edited B16F1 cells were lower than in those of normal B16F1 cells in western blot analysis as well as RT-PCR and qPCR. Moreover, IGFBP3 gene KO cells enhanced the level of SA-ß-gal staining and short telomere length compared to normal B16F1 cells. In particular, it was found that the expression levels of senescence-related proteins such as PI3K, AKT1, PDK1, and p53 were higher in IGFBP3 gene KO cells than in normal cells in both the absence and presence of IGF-1. CONCLUSIONS: Therefore, the above findings could provide a clue that IGFBP3 could play a key role in the aging mechanism.

Sex differences in immune protection in mice conferred by heterologous vaccines for pneumonic plague.

Background: Yersinia pestis is the etiological agent of plague, which can manifest as bubonic, septicemic, and/or pneumonic disease. Plague is a severe and rapidly progressing illness that can only be successfully treated with antibiotics initiated early after infection. There are no FDA-approved vaccines for plague, and some vaccine candidates may be less effective against pneumonic plague than bubonic plague. Y. pestis is not known to impact males and females differently in mechanisms of pathogenesis or severity of infection. However, one previous study reported sex-biased vaccine effectiveness after intranasal Y. pestis challenge. As part of developing a safe and effective vaccine, it is essential that potential sex differences are characterized. Methods: In this study we evaluated novel vaccines in male and female BALB/c mice using a heterologous prime-boost approach and monitored survival, bacterial load in organs, and immunological correlates. Our vaccine strategy consisted of two subcutaneous immunizations, followed by challenge with aerosolized virulent nonencapsulated Y. pestis. Mice were immunized with a combination of live Y. pestis pgm- pPst-Δcaf1, live Y. pestis pgm- pPst-Δcaf1/ΔyopD, or recombinant F1-V (rF1-V) combined with adjuvants. Results: The most effective vaccine regimen was initial priming with rF1-V, followed by boost with either of the live attenuated strains. However, this and other strategies were more protective in female mice. Males had higher bacterial burden and differing patterns of cytokine expression and serum antibody titers. Male mice did not demonstrate synergy between vaccination and antibiotic treatment as repeatedly observed in female mice. Conclusions: This study provides new knowledge about heterologous vaccine strategies, sex differences in plague-vaccine efficacy, and the immunological factors that differ between male and female mice.

Usefulness of alien sterilizing cytoplasms for the hybrid breeding of triticale (xTriticosecale Wittmack): preliminary results.

To be useful for cereal breeding, cytoplasmic male sterility (CMS) should express the complete sterility of maternal lines and the full restoration of the male fertility of F1 hybrids. The most reliable source of sterilizing cytoplasm for triticale is Triticum timopheevi; however, due to the low frequency of efficient non-restorer genotypes for this cytoplasm, new sources of CMS are needed. In this study, aside from T. timopheevi (T) cytoplasm, three alternative CMS sources were tested: Pampa (P) from Secale cereale L., Aegilops sharonensis (A), and Ae. ventricosa (V). The suitability of these cytoplasms for breeding was assessed based on the male fertility/sterility of F1 hybrids obtained through the manual pollination of CMS maternal lines with 36 triticale cultivars and breeding strains. About half of the hybrids with each type of cytoplasm were fully fertile and produced more than 30 grains per bagged spike. The highest percentage was found in hybrids with P cytoplasm (58.33%) and the lowest in hybrids with A cytoplasm (44.44%). Male sterility was observed in hybrids with P cytoplasm (16.67%) and A cytoplasm (16.67%) but not in hybrids with T or V cytoplasm. In terms of practical aspects, male sterility systems with P or A cytoplasm exhibit similarity in their ability to restore male fertility that differ from the T and V cytoplasms. Although all studied cytoplasms exhibited some disadvantages for breeding purposes, none should be definitively classified as unacceptable for future breeding programs regarding the development of triticale hybrid cultivars.

CKB Promotes Mitochondrial ATP Production by Suppressing Permeability Transition Pore.

Creatine kinases are essential for maintaining cellular energy balance by facilitating the reversible transfer of a phosphoryl group from ATP to creatine, however, their role in mitochondrial ATP production remains unknown. This study shows creatine kinases, including CKMT1A, CKMT1B, and CKB, are highly expressed in cells relying on the mitochondrial F1F0 ATP synthase for survival. Interestingly, silencing CKB, but not CKMT1A or CKMT1B, leads to a loss of sensitivity to the inhibition of F1F0 ATP synthase in these cells. Mechanistically, CKB promotes mitochondrial ATP but reduces glycolytic ATP production by suppressing mitochondrial calcium (mCa2+) levels, thereby preventing the activation of mitochondrial permeability transition pore (mPTP) and ensuring efficient mitochondrial ATP generation. Further, CKB achieves this regulation by suppressing mCa2+ levels through the inhibition of AKT activity. Notably, the CKB-AKT signaling axis boosts mitochondrial ATP production in cancer cells growing in a mouse tumor model. Moreover, this study also uncovers a decline in CKB expression in peripheral blood mononuclear cells with aging, accompanied by an increase in AKT signaling in these cells. These findings thus shed light on a novel signaling pathway involving CKB that directly regulates mitochondrial ATP production, potentially playing a role in both pathological and physiological conditions.

Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence.

MCPH1 has been identified as the causal gene for primary microcephaly type 1, a neurodevelopmental disorder characterized by reduced brain size and delayed growth. As a multifunction protein, MCPH1 has been reported to repress the expression of TERT and interact with transcriptional regulator E2F1. However, it remains unclear whether MCPH1 regulates brain development through its transcriptional regulation function. This study showed that the knockout of Mcph1 in mice leads to delayed growth as early as the embryo stage E11.5. Transcriptome analysis (RNA-seq) revealed that the deletion of Mcph1 resulted in changes in the expression levels of a limited number of genes. Although the expression of some of E2F1 targets, such as Satb2 and Cdkn1c, was affected, the differentially expressed genes (DEGs) were not significantly enriched as E2F1 target genes. Further investigations showed that primary and immortalized Mcph1 knockout mouse embryonic fibroblasts (MEFs) exhibited cell cycle arrest and cellular senescence phenotype. Interestingly, the upregulation of p19ARF was detected in Mcph1 knockout MEFs, and silencing p19Arf restored the cell cycle and growth arrest to wild-type levels. Our findings suggested it is unlikely that MCPH1 regulates neurodevelopment through E2F1-mediated transcriptional regulation, and p19ARF-dependent cell cycle arrest and cellular senescence may contribute to the developmental abnormalities observed in primary microcephaly.

Oct4 redox sensitivity potentiates reprogramming and differentiation.

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.