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International Journal of Molecular... Apr 2024Pre-mRNA splicing plays a key role in the regulation of gene expression. Recent discoveries suggest that defects in pre-mRNA splicing, resulting from the dysfunction of...
Pre-mRNA splicing plays a key role in the regulation of gene expression. Recent discoveries suggest that defects in pre-mRNA splicing, resulting from the dysfunction of certain splicing factors, can impact the expression of genes crucial for genome surveillance mechanisms, including those involved in cellular response to DNA damage. In this study, we analyzed how cells with a non-functional spliceosome-associated Gpl1-Gih35-Wdr83 complex respond to DNA damage. Additionally, we investigated the role of this complex in regulating the splicing of factors involved in DNA damage repair. Our findings reveal that the deletion of any component within the Gpl1-Gih35-Wdr83 complex leads to a significant accumulation of unspliced pre-mRNAs of DNA repair factors. Consequently, mutant cells lacking this complex exhibit increased sensitivity to DNA-damaging agents. These results highlight the importance of the Gpl1-Gih35-Wdr83 complex in regulating the expression of DNA repair factors, thereby protecting the stability of the genome following DNA damage.
Topics: DNA Damage; DNA Repair; Gene Expression Regulation, Fungal; RNA Precursors; RNA Splicing; RNA Splicing Factors; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Spliceosomes; Multiprotein Complexes
PubMed: 38673778
DOI: 10.3390/ijms25084192 -
International Journal of Molecular... Apr 2024The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not...
The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar ( × ) lines modified in their DXS activity. Single leaves were dynamically labeled with CO in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.
Topics: Populus; Erythritol; Sugar Phosphates; Transferases; Hemiterpenes; Butadienes; Plant Leaves; Plant Proteins; Gene Expression Regulation, Plant; Pentanes; Plants, Genetically Modified
PubMed: 38673766
DOI: 10.3390/ijms25084181 -
Antioxidants (Basel, Switzerland) Mar 2024Leukemia, characterized by the uncontrolled proliferation and differentiation blockage of myeloid or lymphoid precursor cells, presents significant therapeutic... (Review)
Review
Leukemia, characterized by the uncontrolled proliferation and differentiation blockage of myeloid or lymphoid precursor cells, presents significant therapeutic challenges despite current treatment modalities like chemotherapy and stem cell transplantation. Pursuing novel therapeutic strategies that selectively target leukemic cells is critical for improving patient outcomes. Natural products offer a promising avenue for developing effective chemotherapy and preventive measures against leukemia, providing a rich source of biologically active compounds. Telomerase, a key enzyme involved in chromosome stabilization and mainly active in cancer cells, presents an attractive target for intervention. In this review article, we focus on the anti-leukemic potential of natural substances, emphasizing vitamins (such as A, D, and E) and polyphenols (including curcumin and indole-3-carbinol), which, in combination with telomerase inhibition, demonstrate reduced cytotoxicity compared to conventional chemotherapies. We discuss the role of human telomerase reverse transcriptase (hTERT), particularly its mRNA expression, as a potential therapeutic target, highlighting the promise of natural compounds in leukemia treatment and prevention.
PubMed: 38671875
DOI: 10.3390/antiox13040427 -
Molecular Systems Biology Jun 2024Splice-switching oligonucleotides (SSOs) are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). This study demonstrates the value...
Splice-switching oligonucleotides (SSOs) are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). This study demonstrates the value that artificial intelligence/machine learning (AI/ML) provides for the identification of functional, verifiable, and therapeutic SSOs. We trained XGboost tree models using splicing factor (SF) pre-mRNA binding profiles and spliceosome assembly information to identify modulatory SSO binding sites on pre-mRNA. Using Shapley and out-of-bag analyses we also predicted the identity of specific SFs whose binding to pre-mRNA is blocked by SSOs. This step adds considerable transparency to AI/ML-driven drug discovery and informs biological insights useful in further validation steps. We applied this approach to previously established functional SSOs to retrospectively identify the SFs likely to regulate those events. We then took a prospective validation approach using a novel target in triple negative breast cancer (TNBC), NEDD4L exon 13 (NEDD4Le13). Targeting NEDD4Le13 with an AI/ML-designed SSO decreased the proliferative and migratory behavior of TNBC cells via downregulation of the TGFβ pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data.
Topics: Humans; Machine Learning; Triple Negative Breast Neoplasms; Artificial Intelligence; Alternative Splicing; Cell Line, Tumor; Nedd4 Ubiquitin Protein Ligases; RNA Precursors; Cell Proliferation; RNA Splicing Factors; Oligonucleotides, Antisense; Cell Movement; Spliceosomes; Oligonucleotides; Female
PubMed: 38664594
DOI: 10.1038/s44320-024-00034-9 -
Sheng Li Xue Bao : [Acta Physiologica... Apr 2024The present study aims to observe the change in expression of heat shock protein 90 (HSP90) along with amyloid-β (Aβ) and phosphorylated Tau (p-Tau) protein levels in...
The present study aims to observe the change in expression of heat shock protein 90 (HSP90) along with amyloid-β (Aβ) and phosphorylated Tau (p-Tau) protein levels in the hippocampus tissue of Alzheimer's disease (AD) transgenic animal model with age. APP/PS1 transgenic mice at age of 6-, 9- and 12-month and C57BL/6J mice of the same age were used. The cognitive abilities of these animals were evaluated using a Morris water maze. Western blot or immunohistochemistry was used to detect the expressions of HSP90 and Aβ, as well as the phosphorylation levels of Tau protein in the hippocampus. The hsp90 mRNA levels and the morphology and number of cells in the hippocampus were detected with real-time quantitative polymerase chain reaction (qRT-PCR) and Nissl staining, respectively. The results showed that compared with C57BL/6J mice of the same age, HSP90 and hsp90 mRNA expression were decreased (P < 0.05 or P < 0.01), while Aβ and p-Tau protein levels were increased (P < 0.05 or P < 0.01) in the hippocampal tissue of APP/PS1 transgenic mice. Meanwhile, the decrease in HSP90 and hsp90 mRNA expression (P < 0.05 or P < 0.01), the increase in Aβ and p-Tau levels (P < 0.01 or P < 0.05) in hippocampal tissue and the reduction in behavioral ability showed a progressive development with the advancing of age in the APP/PS1 transgenic mice. In conclusion, in the hippocampal tissue of APP/PS1 mice, the decrease in HSP90 expression and the increase in Aβ and p-Tau levels together with the decline of their cognitive ability are age-dependent.
Topics: Animals; HSP90 Heat-Shock Proteins; Hippocampus; Mice, Transgenic; Mice; Alzheimer Disease; Mice, Inbred C57BL; tau Proteins; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Male; Disease Models, Animal; Phosphorylation; Age Factors; Aging; RNA, Messenger; Peptide Fragments; Presenilin-1
PubMed: 38658375
DOI: No ID Found -
MAbs 2024The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody...
The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.
Topics: Alternative Splicing; Animals; Protein Subunits; Humans; Chickens; Antibodies, Bispecific; CHO Cells; Exons; Cricetulus; Green Fluorescent Proteins; Antibodies, Monoclonal; RNA Precursors
PubMed: 38650451
DOI: 10.1080/19420862.2024.2342243 -
Theranostics 2024In recent years, nicotinamide adenine dinucleotide (NAD) precursors (Npre) have been widely employed to ameliorate female reproductive problems in both humans and...
In recent years, nicotinamide adenine dinucleotide (NAD) precursors (Npre) have been widely employed to ameliorate female reproductive problems in both humans and animal models. However, whether and how Npre plays a role in the male reproductive disorder has not been fully clarified. In the present study, a busulfan-induced non-obstructive azoospermic mouse model was used, and Npre was administered for five weeks following the drug injection, with the objective of reinstating spermatogenesis and fertility. Initially, we assessed the NAD level, germ cell types, semen parameters and sperm fertilization capability. Subsequently, testis tissues were examined through RNA sequencing analysis, ELISA, H&E, immunofluorescence, quantitative real-time PCR, and Western blotting techniques. The results indicated that Npre restored normal level of NAD in blood and significantly alleviated the deleterious effects of busulfan (BU) on spermatogenesis, thereby partially reestablishing fertilization capacity. Transcriptome analysis, along with recovery of testicular Fe, GSH, NADPH, and MDA levels, impaired by BU, and the fact that Fer-1, an inhibitor of ferroptosis, restored spermatogenesis and semen parameters close to CTRL values, supported such possibility. Interestingly, the reduction in SIRT2 protein level by the specific inhibitor AGK2 attenuated the beneficial effects of Npre on spermatogenesis and ferroptosis by affecting PGC-1α and ACLY protein levels, thus suggesting how these compounds might confer spermatogenesis protection. Collectively, these findings indicate that NAD protects spermatogenesis against ferroptosis, probably through SIRT2 dependent mechanisms. This underscores the considerable potential of Npre supplementation as a feasible strategy for preserving or restoring spermatogenesis in specific conditions of male infertility and as adjuvant therapy to preserve male fertility in cancer patients receiving sterilizing treatments.
Topics: Animals; Busulfan; Male; Spermatogenesis; Mice; NAD; Ferroptosis; Sirtuin 2; Disease Models, Animal; Testis; Azoospermia
PubMed: 38646657
DOI: 10.7150/thno.92416 -
Research Square Apr 2024Diffuse midline glioma, -altered (DMG-Alt) are highly aggressive malignancies of the central nervous system (CNS) that primarily affect the pediatric population. Large...
Transcriptomic and Proteomic Spatial Profiling of Pediatric and Adult Diffuse Midline Glioma H3 K27-Altered, Reveals Region Specific Differences and Limited Overlap between mRNA and Protein.
Diffuse midline glioma, -altered (DMG-Alt) are highly aggressive malignancies of the central nervous system (CNS) that primarily affect the pediatric population. Large scale spatial transcriptomic studies have implicated that tumor microenvironmental landscape plays an important role in determining the phenotypic differences in tumor presentation and clinical course, however, data connecting overall transcriptomic changes to the protein level is lacking. The NanoString GeoMx Digital Spatial Profiler platform was used to determine the spatial transcriptomic and proteomic landscape in a cohort of both pediatric and adult -altered DMG biopsy samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells and a nucleic acid stain (SYTO-13) were used to establish regions of interest (ROI) for genomic and proteomic analysis. We found genetic alterations within the tumor which can be delineated across patient age and spatial location. We show that the H3 K27M mutation itself has a profound impact on tumor cells transcriptomics and interestingly we found limited fidelity between overall transcriptome and proteome. Our data also validate the previously described OPC like precursor signature at the proteomic level and reveal a special shift in the signature based on the local TME composition.
PubMed: 38645012
DOI: 10.21203/rs.3.rs-4139314/v1 -
Biochemical and Biophysical Research... Jun 2024The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is...
The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3-4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 μM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.
Topics: Humans; Cathelicidins; Antimicrobial Cationic Peptides; Osteoblasts; Vitamin D; THP-1 Cells; Proteasome Endopeptidase Complex; Cell Survival
PubMed: 38642493
DOI: 10.1016/j.bbrc.2024.149962 -
Scientific Reports Apr 2024Lung adenocarcinoma (LUAD), a leading cause of cancer-related mortality worldwide, demands a deeper understanding of its molecular mechanisms and the identification of...
Lung adenocarcinoma (LUAD), a leading cause of cancer-related mortality worldwide, demands a deeper understanding of its molecular mechanisms and the identification of reliable biomarkers for better diagnosis and targeted therapy. Leveraging data from the Cancer Genome Atlas (TCGA), the Clinical Proteomic Tumor Analysis Consortium (CPTAC), and the Human Protein Atlas (HPA), we investigated the mRNA and protein expression profiles of TIMM17A and assessed its prognostic significance through Kaplan-Meier survival curves and Cox regression analysis. Through Gene Set Enrichment Analysis, we explored the regulatory mechanisms of TIMM17A in LUAD progression and demonstrated its role in modulating the proliferative capacity of A549 cells, a type of LUAD cell, via in vitro experiments. Our results indicate that TIMM17A is significantly upregulated in LUAD tissues, correlating with clinical staging, lymph node metastasis, overall survival, and progression-free survival, thereby establishing it as a critical independent prognostic factor. The construction of a nomogram model further enhances our ability to predict patient outcomes. Knockdown of TIMM17A inhibited the growth of LUAD cells. The potential of TIMM17A as a biomarker and therapeutic target for LUAD presents a promising pathway for improving patient diagnosis and treatment strategies.
Topics: Humans; Adenocarcinoma of Lung; Lung Neoplasms; Nomograms; Prognosis; Proteomics; Mitochondrial Precursor Protein Import Complex Proteins; Gene Expression Regulation, Neoplastic; Biomarkers, Tumor; A549 Cells
PubMed: 38632467
DOI: 10.1038/s41598-024-59526-1