Changes In Expression Patterns Research Articles

Abnormal amino acid synthesis and glutathione metabolism may affect PCOS blastocyst development: an examination of in vitro mouse blastocysts model utilizing RNA-sequencing

BackgroundExtensive research has been conducted on embryonic developmental disorders linked to Polycystic Ovary Syndrome (PCOS), a pathological condition that affects 5−10% of women and is characterized by irregularities in the menstrual cycle and infertility. By employing RNA sequencing (RNA-seq), we performed an in-depth investigation of PCOS-related changes in gene expression patterns at the mouse blastocyst stage.MethodsThe zygotes of female B6D2 mice were obtained and then differentiated into blastocysts in K + Simplex Optimised Medium (KSOM) cultures containing exo-NC (negative control for exosomes) or exo-LIPE-AS1 (a novel exosomal marker of PCOS). Subsequently, blastocysts were collected for RNA-seq. The bioinformatics was performed to analyze and compare the differences of gene expression profile between blastocysts of control and PCOS group.ResultsThere were 1150 differentially expressed genes (DEGs) between the two groups of mouse blastocysts; 243 genes were upregulated and 907 downregulated in the blastocysts of the exo-LIPE-AS1 group compared to those of the exo-NC group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the genes involved in amino acid synthesis and glutathione metabolic pathways were down-regulated in exo-LIPE-AS1 group.ConclusionThis study has revealed that blastocyst developmental retardation may be associated with the downregulation of amino acid synthesis and glutathione metabolism, which may affect energy metabolism, biosynthesis, cellular osmotic pressure, antioxidant synthesis, ROS clearance or mitochondrial function, and ultimately cause blastocyst cell development abnormalities. Our research offers encouraging data on the mechanisms underlying aberrant embryonic development in patients with PCOS as well as potential treatment strategies.

Nerve entry points in the mimic musculature of the horse head.

Facial expressions are important in pain recognition in horses, but current observation-based pain scales remain subjective. A promising technique to quantitatively measure subtle changes in expression patterns, including changes invisible to the human eye, is surface electromyography (sEMG). To achieve high-quality and reliable sEMG signals, unilateral placement of bipolar electrodes is required in relation to the motor endplates (MEP). We aimed to localize the nerve entry points (NEPs; where the nerve branch first pierced the muscle belly) and the direction of the terminal nerve endings to estimate MEP locations of the innervating nerves in five equine facial muscles involved in pain expression. Three cadaveric Dutch Warmblood horse heads were dissected to identify the NEPs in the musculi caninus, levator anguli oculi medialis, nasolabialis, masseter and zygomaticus. These points were marked with pins and measured in relation to a reference line between two anatomical landmarks near the origin and insertion of the respective muscle. Relative distances were calculated from the most caudally situated landmark. NEPs were located at 33%-38% (caninus), 69%-86% (levator anguli oculi medialis) and 0%-18% (zygomaticus) from the caudal landmark. The nasolabialis showed two innervations zones. Its NEPs were located at 47%-72% (dorsal muscle branch) and 52%-91% (ventral branch). All terminal nerve endings were found to run in rostral direction. The masseter showed numerous NEPs diffusely spread within the muscle belly. Therefore, calculation of relative positions was not performed. These results could form the basis for feasibility studies and standardization of bipolar electrode positioning invivo to measure facial muscle activity patterns in horses.

Get Spliced: Uniting Alternative Splicing and Arthritis.

Immune responses demand the rapid and precise regulation of gene protein expression. Splicing is a crucial step in this process; ~95% of protein-coding gene transcripts are spliced during mRNA maturation. Alternative splicing allows for distinct functional regulation, as it can affect transcript degradation and can lead to alternative functional protein isoforms. There is increasing evidence that splicing can directly regulate immune responses. For several genes, immune cells display dramatic changes in isoform-level transcript expression patterns upon activation. Recent advances in long-read RNA sequencing assays have enabled an unbiased and complete description of transcript isoform expression patterns. With an increasing amount of cell types and conditions that have been analyzed with such assays, thousands of novel transcript isoforms have been identified. Alternative splicing has been associated with autoimmune diseases, including arthritis. Here, GWASs revealed that SNPs associated with arthritis are enriched in splice sites. In this review, we will discuss how alternative splicing is involved in immune responses and how the dysregulation of alternative splicing can contribute to arthritis pathogenesis. In addition, we will discuss the therapeutic potential of modulating alternative splicing, which includes examples of spliceform-based biomarkers for disease severity or disease subtype, splicing manipulation using antisense oligonucleotides, and the targeting of specific immune-related spliceforms using antibodies.

Paralog switching facilitates diadromy: ontogenetic, microevolutionary and macroevolutionary evidence.

Identifying how the demands of migration are met at the level of gene expression is critical for understanding migratory physiology and can potentially reveal how migratory forms evolve from nonmigratory forms and vice versa. Among fishes, migration between freshwater and seawater (diadromy) requires considerable osmoregulatory adjustments, powered by the ion pump Na+, K+-ATPase (NKA) in the gills. Paralogs of the catalytic α-subunit of the pump (NKA α1a and α1b) are reciprocally upregulated in fresh- and seawater, a response known as paralog-switching, in gills of some diadromous species. We tested ontogenetic changes in NKA α-subunit paralog expression patterns, comparing pre-migrant and migrant alewife (Alosa pseudoharengus) sampled in their natal freshwater environment and after 24h in seawater. In comparison to pre-migrants, juvenile out-migrants exhibited stronger paralog switching via greater downregulation of NKA α1a in seawater. We also tested microevolutionary changes in the response, exposing juvenile diadromous and landlocked alewife to freshwater (0 ppt) and seawater (30 ppt) for 2, 5, and 15days. Diadromous and landlocked alewife exhibited salinity-dependent paralog switching, but levels of NKA α1b transcription were higher and the decrease in NKA α1a was greater after seawater exposure in diadromous alewife. Finally, we placed alewife α-subunit NKA paralogs in a macroevolutionary context. Molecular phylogenies show alewife paralogs originated independently of paralogs in salmonids and other teleosts. This study demonstrated that NKA paralog switching is tied to halohabitat profile and that duplications of the NKA gene provided the substrate for multiple, independent molecular solutions that support a diadromous life history.

Skeletal muscle p53-depletion uncovers a mechanism of fuel usage suppression that enables efficient energy conservation.

The ability of skeletal muscle to respond adequately to changes in nutrient availability, known as metabolic flexibility, is essential for the maintenance of metabolic health and loss of flexibility contributes to the development of diabetes and obesity. The tumour suppressor protein, p53, has been linked to the control of energy metabolism. We assessed its role in the acute control of nutrient allocation in skeletal muscle in the context of limited nutrient availability. A mouse model with inducible deletion of the p53-encoding gene, Trp53, in skeletal muscle was generated using the Cre-loxP-system. A detailed analysis of nutrient metabolism in mice with control and knockout genotypes was performed under ad libitum fed and fasting conditions and in exercised mice. Acute deletion of p53 in myofibres of mice activated catabolic nutrient usage pathways even under ad libitum fed conditions, resulting in significantly increased overall energy expenditure (+10.6%; P=0.0385) and a severe nutrient deficit in muscle characterized by depleted intramuscular glucose and glycogen levels (-62,0%; P<0.0001 and -52.7%; P<0.0001, respectively). This was accompanied by changes in marker gene expression patterns of circadian rhythmicity and hyperactivity (+57.4%; P=0.0068). These metabolic changes occurred acutely, within 2-3days after deletion of Trp53 was initiated, suggesting a rapid adaptive response to loss of p53, which resulted in a transient increase in lactate release to the circulation (+46.6%; P=0.0115) from non-exercised muscle as a result of elevated carbohydrate mobilization. Conversely, an impairment of proteostasis and amino acid metabolism was observed in knockout mice during fasting. During endurance exercise testing, mice with acute, muscle-specific Trp53 inactivation displayed an early exhaustion phenotype with a premature shift in fuel usage and reductions in multiple performance parameters, including a significantly reduced running time and distance (-13.8%; P=0.049 and -22.2%; P=0.0384, respectively). These findings suggest that efficient nutrient conservation is a key element of normal metabolic homeostasis that is sustained by p53. The homeostatic state in metabolic tissues is actively maintained to coordinate efficient energy conservation and metabolic flexibility towards nutrient stress. The acute deletion of Trp53 unlocks mechanisms that suppress the activity of nutrient catabolic pathways, causing substantial loss of intramuscular energy stores, which contributes to a fasting-like state in muscle tissue. Altogether, these findings uncover a novel function of p53 in the short-term regulation of nutrient metabolism in skeletal muscle and show that p53 serves to maintain metabolic homeostasis and efficient energy conservation.

Osmotic priming-induced cryotolerance uncovers rejuvenation of grapevine cell cultures: morphogenetic changes and gene expression pattern highlighting enhanced embryogenic potential.

Cryopreservation is a reliable technique for the long-term storage and preservation of embryogenic cells, maintaining their viability without loss of their embryogenic capacity. However, the large-scale conservation of grapevine embryogenic lines in cryobanks remains limited. A significant challenge is understanding somatic cell rejuvenation. Here, we investigate the encapsulation/dehydration and encapsulation/vitrification for cryopreserving embryogenic material. Cell rejuvenation and enhanced embryogenic competence were observed after cryopreservation, as evidenced through structural cellular changes observed by histology and electron scanning microscopy. Results showed that cryopreserved samples of 110-Richter, Riesling, and Tempranillo using encapsulation/dehydration had better survival rates, averaging 81%, 62%, and 48%, respectively, while encapsulation/vitrification yielded lower survival rates, averaging 58%, 42%, and 32%, respectively. Cryopreservation also improved post-thaw recovery and regeneration efficiency assessed through regrowth of proembryogenic masses and somatic embryo conversion reaching 54-72% against 11-17% in control samples. Cryopreservation triggered changes in gene expression patterns and exhibited considerable increase at genotype-specific basis of 1.5- to 4.5-fold in SERK1, BBM, and WOX associated to embryogenic competence as well as in ChitIV and LEA involved in stress response. Membrane stability index, hydrogen peroxide, and proline contents were used as indicators of oxidative stress uncovering a key role of an osmotic trans-priming effect leading to cryotolerance. Our finding highlighted that cryopreservation enhances embryogenic capacity in senescent callus and probably acts as a screening process allowing safe maintenance of proembryogenic cells and promoting their recovery. This study provides a high throughput innovation to set up cryolines for cell rejuvenation of grapevine and other important plant species.

A Modeling Approach to Studying the Influence of Grafting on the Anatomical Features and SAUR Gene Expression in Watermelons

Grafting alters the genetic and anatomical features of plants. Although grafting has been widely applied in plant propagation, the underlying processes that govern the effects of the procedure are not fully understood. Samples were collected to study the long-term influence of grafting on the leaf-shoot morphology, leaf-shoot anatomy, and genetic signature of the grafted plants. Citrulus lanatus (Thunb.) Matsum. &amp; Nakai (cv. Lady) was used as the scion, and Lagenaria siceraria (Molina) Standl (cv. Argentario) as a rootstock. In grafted plants, leaf blades and petioles were 20.92% and 12.82% longer, respectively, while the midrib collenchyma was 35.68% thicker, and the diameter of the vessel member was 11.17% larger than in ungrafted plants. In the stem, grafting affected the arrangement and number of vascular bundles (from 1 to 2 rings). The thickness of the epidermis decreased by 69.79%, and the size of the external fascicular phloem decreased by 23.56%. The diameter of the vessel member of the grafted plants increased by 28.94%. Eight out of ten evaluated primers met the requirements (stability in both watermelons and bottle gourd, tissue-specific). In the genetic tests, we examined whether this change in the gene expression pattern is due to the grafting and, if so, to what extent. Seven out of eight tested Small Auxin Up-Regulated RNA (SAUR) genes were expressed in the ungrafted and grafted C. lanatus lines in four cases; the expression increased by more than 10% after grafting. The morpho-anatomical changes and genetic variation reported in this study for grafted lines of C. lanatus contribute to the understanding of the underlying mechanisms of plant growth observations resulting from grafting.

Multifunctional Roles of Zinc in Cadmium Transport in Soil-Rice Systems: Novel Insights from Stable Isotope Fractionation and Gene Expression.

The effect of Zn on Cd accumulation in rice varies under flooding and drainage conditions, and the underlying mechanism during uptake and transport from the soil to grains remains unclear. Isotope fractionation and gene expression were investigated using pot experiments under distinct water regimes and with Zn addition to gain a deeper understanding of the molecular effects of Zn on Cd uptake and transport in rice. The higher OsHMA2 expression but constitutively lower expression of zinc-regulated, iron-regulated transporter-like protein (ZIP) family genes in roots under the drainage regime than the flooding regime caused the enrichment of nonheavy Zn isotopes in the shoots relative to roots but minimally affected Cd isotopic fractionation. Drainage regime seem to exert a striking effect on the root-to-shoot translocation of Zn rather than Cd, and increased Zn transport via OsHMA2. The changes in expression patterns in response to Zn addition were similar to those observed upon switching from the flooding to drainage regime, except for OsNRAMP1 and OsNRAMP5. However, soil solution-to-rice plants and root-to-shoot fractionation toward light Zn isotopes with Zn addition (Δ66Znriceplant-soilsolution = -0.49 to -0.40‰, Δ66Znshoot-root = -0.36 to -0.27‰) indicated that Zn transport occurred via nonspecific uptake pathways and OsHMA2, respectively. Accordingly, the less pronounced and minimally varied Cd isotope fractionation suggested that OsNRAMP5 and OsHMA2 are crucial for Cd uptake and root-to-shoot transport, respectively, facilitating Cd accumulation in grains. This study demonstrated that a high Zn supply promotes Cd uptake and root-to-shoot transport in rice by sharing distinct pathways, and by utilizing a non-Zn-sensitive pathway with a high affinity for Cd.

Abnormal genes and pathways that drive muscle contracture from brachial plexus injuries: Towards machine learning approach

In order to clarify the pathways closely linked to denervated muscle contracture, this work uses IoMT-enabled healthcare stratergies to examine changes in gene expression patterns inside atrophic muscles following brachial plexus damage. The gene expression Omnibus (GEO) database searching was used to locate the dataset GSE137606, which is connected to brachial plexus injuries. Strict criteria (|logFC|≥2 & adj.p < 0.05) were used to extract differentially expressed genes (DEGs). To identify dysregulated activities and pathways in denervated muscles, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and Gene Set Enrichment Analysis (GSEA) were used. Hub genes were found using Cytoscape software's algorithms, which took into account parameters like as proximity, degree, and MNC. Their expression, enriched pathways, and correlations were then examined. The results showed that 316 DEGs were predominantly concentrated in muscle-related processes such as tissue formation and contraction pathways. Of these, 297 DEGs were highly expressed in denervated muscles, whereas 19 DEGs were weakly expressed. GSEA showed improvements in the contraction of striated and skeletal muscles. In addition, it was shown that in denervated muscles, Myod1, Myog, Myh7, Myl2, Tnnt2, and Tnni1 were elevated hub genes with enriched pathways such adrenergic signaling and tight junction. These results point to possible therapeutic targets for denervated muscular contracture, including Myod1, Myog, Myh7, Myl2, Tnnt2, and Tnni1. This highlights treatment options for this ailment which enhances the mental state of patient.

Sustained Depolarization Induces Gene Expression Pattern Changes Related to Synaptic Plasticity in a Human Cholinergic Cellular Model

Sustained Depolarization Induces Gene Expression Pattern Changes Related to Synaptic Plasticity in a Human Cholinergic Cellular Model

CREG1 promotes bovine placental trophoblast cells exosome release by targeting IGF2R and participates in regulating organoid differentiation via exosomes transport

BackgroundPlacental exosomes are a kind of intercellular communication media secreted by placental cells during pregnancy, exosomogenesis and release are regulated by many secretory glycoproteins. CREG1 is a kind of secreted glycoprotein widely expressed in various organs and tissues of the body, which inhibits cell proliferation and enhances cell differentiation. The aim of this study was to explore the role of CREG1 in regulating exosomogenesis during the proliferation and differentiation of placental trophoblast cells in early pregnant dairy cows by targeting IGF2R and participating in regulating organoid differentiation via exosomes transport. MethodsMolecular biological methods were firstly used to investigate the expression patterns of CREG1, IGF2R and exosomal marker proteins in early placental development of pregnant dairy cows. Subsequently, the effects of CREG1 on the formation and release of bovine placental trophoblast (BTCs) derived exosomes by targeting IGF2R were investigated. Further, the effects of CREG1 on the change of gene expression patterns along with the transport of exosomes to recipient cells and participate in regulating the differentiation of organoids were explored. ResultsThe expression of CREG1, IGF2R and exosomal marker proteins increased with the increase of pregnancy months during the early evolution of placental trophoblast cells in dairy cows. Overexpression of Creg1 enhanced the genesis and release of exosomes derived from BTCs, while knocking down the expression of Igf2r gene not only inhibited the genesis of exosomes, but also inhibited the genesis and release of exosomes induced by overexpression of CREG1 protein. Interestingly, IGF2R can regulate the expression of CREG1 through reverse secretion. What's more, the occurrence and release of trophoblast-derived exosomes are regulated by CREG1 binding to IGF2R, which subsequently binds to Rab11. CREG1 can not only promote the formation and release of exosomes in donor cells, but also regulate the change of gene expression patterns along with the transport of exosomes to recipient cells and participate in regulating the early development of placenta. ConclusionsOur study confirmed that CREG1 is involved in the exosomogenesis and release of exosomes during the proliferation and differentiation of placental trophoblast cells in early pregnant dairy cows by targeting IGF2R, and is involved in the regulation of organoid differentiation through exosome transport.

Understanding the Endocrine and Molecular Signaling Cascade Regulation Pathways in Children with Hypospadias.

Hypospadias (HS) is a congenital defect that occurs due to defective androgenization. It is characterized by the aberrant location of the urinary meatus on the ventral aspect of the penis with various degrees of severity. The molecular mechanisms and genetic associations underlying the condition remain largely unknown. Existing literature revolves around surgical and medical management of the condition. Human chorionic gonadotropin pretreatment in HS is proposed to decrease the severity of the anomaly and improve the clinical outcome of surgery. The underlying mechanisms that drive these outcomes have not been explored. Few studies have explored the endocrine signaling and pathways which lead to the development of the condition. Hence, a prospective study was conducted to understand the same. Eighteen children with mid or proximal penile HS were included as cases, and nine children undergoing circumcision for phimosis (nonpathological) were included as controls. Serum samples from all these children and preputial skin samples taken during surgery were used in the analysis. The hormonal milieu was normal in all children in our cohort. A comparison of previously reported genes with our cohort sequencing revealed changes in several major pathways involved in cell proliferation and differentiation, cell signaling, angiogenesis, and immune response pathways. Compared with healthy controls, HS subjects had 152 differentially expressed genes. Of these, 93 genes were up-regulated, and 59 genes were found to be significantly down-regulated. The gene expression evaluation also showed changes in expression patterns in inflammatory genes and link RNAs, unlike previously reported genes.

Efficient biocompatible drug carrier: Lignin-based nanogel amplifies Curcumin's cytotoxic effects and induces apoptosis in brain and lung cancer cell lines

This study focuses on the biological effects of size-tunable nanogel based on the natural polymer named lignin. The flexibility of the nanogel to act under variable conditions makes it a promising smart carrier in nanomedicine. Through the “grafting from” approach, it was synthesized by atom transfer radical polymerization (ATRP) method, loaded with curcumin (an anticancer herbal compound) and has been applied to treat two cell lines (A549 and U87MG). Treatment of cells with free curcumin and curcumin-loaded nanogel demonstrated significant differences in IC50 values. The values of free curcumin were 48.23 μM for A549 cells and 36.54 μM for U87MG cells, while the values of curcumin-loaded nanogel were 25.95 μM for A549 cells and 25.65 μM for U87MG cells, respectively. This indicates that the nanogels enhanced the cytotoxic effects of curcumin on both cancerous cell lines.Furthermore, the expression of apoptosis associated genes was evaluated. Under the effects of curcumin-loaded nanogel, the expression of caspase 3 and caspase 9 genes in the A549 cell line increased by 2.3-fold and 1.41-fold, respectively. Additionally, the expression of the pro-apoptotic gene Bax increased by 1.29-fold, while the anti-apoptotic gene Bcl2 decreased by 0.315-fold. Similarly, in U87MG cells, the expression of caspase 3 and caspase 9 genes increased by 6.49-fold and 2.08-fold, respectively, while Bcl2 decreased by 0.023-fold. The changes in gene expression patterns indicate the induction of apoptosis in both cell lines when exposed to curcumin-loaded nanogels. The results of flow cytometry confirmed an increase in apoptosis in the presence of curcumin-loaded nanogels.

Spatial Chromosome Organization and Adaptation of Escherichia coli under Heat Stress.

The spatial organization of bacterial chromosomes is crucial for cellular functions. It remains unclear how bacterial chromosomes adapt to high-temperature stress. This study delves into the 3D genome architecture and transcriptomic responses of Escherichia coli under heat-stress conditions to unravel the intricate interplay between the chromosome structure and environmental cues. By examining the role of macrodomains, chromosome interaction domains (CIDs), and nucleoid-associated proteins (NAPs), this work unveils the dynamic changes in chromosome conformation and gene expression patterns induced by high-temperature stress. It was observed that, under heat stress, the short-range interaction frequency of the chromosomes decreased, while the long-range interaction frequency of the Ter macrodomain increased. Furthermore, two metrics, namely, Global Compactness (GC) and Local Compactness (LC), were devised to measure and compare the compactness of the chromosomes based on their 3D structure models. The findings in this work shed light on the molecular mechanisms underlying thermal adaptation and chromosomal organization in bacterial cells, offering valuable insights into the complex inter-relationships between environmental stimuli and genomic responses.

Aging and putative frailty biomarkers are altered by spaceflight

Human space exploration poses inherent risks to astronauts’ health, leading to molecular changes that can significantly impact their well-being. These alterations encompass genomic instability, mitochondrial dysfunction, increased inflammation, homeostatic dysregulation, and various epigenomic changes. Remarkably, these changes bear similarities to those observed during the aging process on Earth. However, our understanding of the connection between these molecular shifts and disease development in space remains limited. Frailty syndrome, a clinical syndrome associated with biological aging, has not been comprehensively investigated during spaceflight. To bridge this knowledge gap, we leveraged murine data obtained from NASA’s GeneLab, along with astronaut data gathered from the JAXA and Inspiration4 missions. Our objective was to assess the presence of biological markers and pathways related to frailty, aging, and sarcopenia within the spaceflight context. Through our analysis, we identified notable changes in gene expression patterns that may be indicative of the development of a frailty-like condition during space missions. These findings suggest that the parallels between spaceflight and the aging process may extend to encompass frailty as well. Consequently, further investigations exploring the utility of a frailty index in monitoring astronaut health appear to be warranted.

Prognostic implications of preoperative, postoperative, and dynamic changes of alpha-fetoprotein and des-gamma (γ)-carboxy prothrombin expression pattern for hepatocellular carcinoma after hepatic resection: a multicenter observational study.

The utility of pre- and post-operative alpha-fetoprotein (AFP) and des-gamma (γ)-carboxy prothrombin (DCP) expression patterns and their dynamic changes as predictors of the outcome of hepatic resection for hepatocellular carcinoma (HCC) has yet to be well elucidated. From a multicenter database, AFP and DCP data during the week prior to surgery and the first post-discharge outpatient visit (within 1-2 months after surgery) were collected from patients with HCC who underwent hepatectomy. AFP-DCP expression patterns were categorized according to the number of positive tumor markers (AFP ≥ 20ng/mL, DCP ≥ 40mAU/mL), including double-negative, single-positive, and double-positive. Changes in the AFP-DCP expression patterns were delineated based on variations in the number of positive tumor markers when comparing pre- and post-operative patterns. Preoperatively, 53 patients (8.3%), 337 patients (52.8%), and 248 patients (38.9%) exhibited double-negative, single-positive, and double-positive AFP-DCP expression patterns, respectively. Postoperatively, 463 patients (72.6%), 130 patients (20.4%), and 45 patients (7.0%) showed double-negative, single-positive, and double-positive AFP-DCP expression patterns, respectively. Survival analysis showed a progressive decrease in recurrence-free (RFS) and overall survival (OS) as the number of postoperative positive tumor markers increased (both P < 0.001). Multivariate analysis showed that postoperative AFP-DCP expression pattern, but not preoperative AFP-DCP expression pattern, was an independent risk factor for RFS and OS. Further analysis showed that for patients with positive preoperative markers, prognosis gradually improves as positive markers decrease postoperatively. In particular, when all postoperative markers turned negative, the prognosis was consistent with that of preoperative double-negative patients, regardless of the initial number of positive markers. AFP-DCP expression patterns, particularly postoperative patterns, serve as vital sources of information for prognostic evaluation following hepatectomy for HCC. Moreover, changes in AFP-DCP expression patterns from pre- to post-operation enable dynamic prognostic risk stratification postoperatively, aiding the development of individualized follow-up strategies.

Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun.

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the βγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.

Tumor-matched and unmatched cancer associated fibroblasts exhibit differential effect on proliferation and FMOD and MMP9 gene expression in head and neck squamous cell carcinoma cells when cocultured in spheroids

BackgroundCancer-associated fibroblasts (CAFs) are the major cellular component of the tumor microenvironment and are known to affect tumor growth and response to various treatments. This study was undertaken to investigate the crosstalk between tumor-matched or unmatched CAFs and head and neck squamous cell carcinoma (HNSCC) cells regarding tumor growth and treatment response.MethodsThree HNSCC cell lines (LK0412, LK0902 and LK0923), were cocultured in 2D or in 3D with their tumor-matched CAFs, site matched CAFs from other tumors or normal oral fibroblasts (NOFs). Cell proliferation was assessed as the amount of Ki67 positive cells/ spheroid area in formalin-fixed- paraffin-embedded 3D spheroids stained with Ki67 antibody. Viability after seven days of cisplatin treatment was measured with CellTiter-Glo 3D Viability Assay. The mRNA expression of CAF-associated markers (ACTA2, COL1A2, FAP, PDGFRα, PDGFRβ, PDPN, POSTN and S100A4) in CAFs before and after coculture with tumor cells as well as mRNA expression of CAF-induced genes (MMP1, MMP9 and FMOD) in tumor cells separated from CAFs after co-culture was measured with RT-qPCR. The expression of selected protein biomarkers was validated with immunohistochemistry based on previous mRNA expression results.ResultsThe proliferation of the LK0412 and LK0902 tumor spheroids varied significantly when cocultured with different CAFs and NOFs as shown by Ki-67 positive cells. RT‒qPCR analysis revealed different molecular profile of the analyzed HNSCC-derived CAFs concerning the expression of CAF-associated markers. The interaction between CAFs and HNSCC cells was more pronounced after coculture with unmatched CAFs as shown by changes in mRNA expression pattern of CAF-specific markers. Additionally, the unmatched CAFs significantly upregulated the mRNA expression of MMP1, MMP9 and FMOD in tumor cells compared to tumor-matched CAFs.ConclusionOur results indicate that tumor-matched CAFs are unique for each tumor and affect the proliferation and the gene/protein expression of tumor cells in a distinct manner. The interaction between tumor unmatched CAFs and HNSCC cells in the tumor spheroids is associated with significant changes in the mRNA expression of CAF-specific markers and significant increases in FMOD and MMP9 in tumor cells compared to when cocultured with tumor-matched CAFs. Taken together, our results show how important the selection of CAFs is to get a reliable in vitro model that mimics the patients’ tumor.

The Specific ROCK2 Inhibitor KD025 Alleviates Glycolysis through Modulating STAT3-, CSTA- and S1PR3-Linked Signaling in Human Trabecular Meshwork Cells.

To investigate the biological significance of Rho-associated coiled-coil-containing protein kinase (ROCK) 2 in the human trabecular meshwork (HTM), changes in both metabolic phenotype and gene expression patterns against a specific ROCK2 inhibitor KD025 were assessed in planar-cultured HTM cells. A seahorse real-time ATP rate assay revealed that administration of KD025 significantly suppressed glycolytic ATP production rate and increased mitochondrial ATP production rate in HTM cells. RNA sequencing analysis revealed that 380 down-regulated and 602 up-regulated differentially expressed genes (DEGs) were identified in HTM cells treated with KD025 compared with those that were untreated. Gene ontology analysis revealed that DEGs were more frequently related to the plasma membrane, extracellular components and integral cellular components among cellular components, and related to signaling receptor binding and activity and protein heterodimerization activity among molecular functions. Ingenuity Pathway Analysis (IPA) revealed that the detected DEGs were associated with basic cellular biological and physiological properties, including cellular movement, development, growth, proliferation, signaling and interaction, all of which are associated with cellular metabolism. Furthermore, the upstream regulator analysis and causal network analysis estimated IL-6, STAT3, CSTA and S1PR3 as possible regulators. Current findings herein indicate that ROCK2 mediates the IL-6/STAT3-, CSTA- and S1PR3-linked signaling related to basic biological activities such as glycolysis in HTM cells.

Proteomic profiling of peanut hairy root culture unveils distinctive adaptive responses induced by elicitor treatment across diverse time intervals

Plants employ a diverse array of responses to counteract and defend against stressors. Utilizing quantitative proteomic analysis allows for the exploration of global proteomic dynamics in cells and tissues at specific time points. In order to comprehend the intricate mechanisms underlying plant responses, we conducted a proteomic investigation of peanut hairy root cultures induced with a combination of chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD) at multiple time points. The study identified 292, 317, and 327 differentially expressed proteins (DEPs) in hairy root tissues at 24-, 48-, and 72 h intervals post-elicitation, respectively. Principal component analysis (PCA) distinctly clustered each timepoint of treatment and the control group. Gene ontology (GO) enrichment analysis highlighted terms such as "responding to biotic stimuli" and "defense response" during the early response, with the upregulation of Heat shock cognate 70 kDa protein, Wound-induced protein 1, and Disease resistance-like protein. The 72 h time point specifically showed an upregulation of superoxide dismutase (SOD) and auxin response factor, indicating a delayed response and illustrating how hairy roots adapt to stress in later stages. Furthermore, GO enrichment at later stages implicated alterations in protein production, involving both synthesis and degradation processes. Proteolytic enzymes, including those in the ubiquitin/proteasome pathway, were identified as facilitating protein degradation. The detection of small molecular weight (MW) peptides in the culture medium of elicited hairy roots suggested their origin from the proteolytic cleavage of precursor proteins. The GO enrichment of culture medium peptides pointed towards involvement in signaling pathways. The suggested signaling cascade acts as a potential link that connects the recognition of stress to the initiation of appropriate response to external stressors. This approach delves into dynamic changes in protein expression and secretion patterns over various intervals, providing insights into the plant's adaptive response to external stress.