Changes In Protein Expression Levels Research Articles

Recent Insights into Hippocampal Dysfunction and Neuroplasticity in Sleep Disorders: An Update from Preclinical Studies.

Sleep disorders are prevalent neurological conditions linked to neurocognitive impairments. Understanding the neuroplasticity changes in the hippocampus, which plays a central role in regulating neurocognitive function, is crucial in the context of sleep disorders. However, research on neurodegenerative disorders and the influence of sleep disorders on hippocampal neuroplasticity remains largely unclear. Therefore, this review aims to highlight the latest advancements regarding hippocampal neuroplasticity and functional changes during sleep disorders, drawing insights from clinical and preclinical research involving sleep-deprived animal models. These articles were gathered through comprehensive literature searches across databases, including Google Scholar, PubMed, Web of Science, and Scopus. Maternal sleep deprivation has been observed to cause neurocognitive impairment in offspring, along with changes in protein expression levels associated with neuroplasticity. Similarly, sleep deprivation in adult mice has been shown to affect several cognitive functions and fear extinction without influencing the acquisition of fear conditioning. While mechanistic research on neurocognitive dysfunction induced by maternal and adult sleep deprivation is limited, it suggests the involvement of several signaling pathways, including neurotrophic factors, synaptic proteins, and inflammatory molecules, which are triggered by sleep deprivation. Further studies are needed to clarify the mechanistic pathways underlying hippocampal dysfunction and synaptic alterations associated with sleep disturbances.

Global regulation via modulation of ribosome pausing by the ABC-F protein EttA

Having multiple rounds of translation of the same mRNA creates dynamic complexities along with opportunities for regulation related to ribosome pausing and stalling at specific sequences. Yet, mechanisms controlling these critical processes and the principles guiding their evolution remain poorly understood. Through genetic, genomic, physiological, and biochemical approaches, we demonstrate that regulating ribosome pausing at specific amino acid sequences can produce ~2-fold changes in protein expression levels which strongly influence cell growth and therefore evolutionary fitness. We demonstrate, both in vivo and in vitro, that the ABC-F protein EttA directly controls the translation of mRNAs coding for a subset of enzymes in the tricarboxylic acid (TCA) cycle and its glyoxylate shunt, which modulates growth in some chemical environments. EttA also modulates expression of specific proteins involved in metabolically related physiological and stress-response pathways. These regulatory activities are mediated by EttA rescuing ribosomes paused at specific patterns of negatively charged residues within the first 30 amino acids of nascent proteins. We thus establish a unique global regulatory paradigm based on sequence-specific modulation of translational pausing.

Bioinformatics analysis and experimental verification of NLRX1 as a prognostic factor for esophageal squamous cell carcinoma.

Nucleotide binding and oligomeric domain-like receptor X1 (NLRX1), a member of the NLR family, is associated with the physiological and pathological processes of inflammation, autophagy, immunity, metabolism and mitochondrial regulation, and has been demonstrated to have pro- or antitumor effects in various tumor types. However, the biological function of NLRX1 in esophageal squamous cell carcinoma (ESCC) has remained elusive. In the present study, by using bioinformatics methods, the differential expression of NLRX1 at the mRNA level was examined. Overall survival, clinical correlation, receiver operating characteristic curve, Cox regression, co-expression, enrichment, immune infiltration and drug sensitivity analyses were carried out. A nomogram and a calibration curve were constructed. Changes in protein expression levels were investigated by immunohistochemistry and western blotting. The impact of NLRX1 on i) cell proliferation was evaluated by Cell Counting Kit-8 assays; ii) migration was examined by wound-healing assays; iii) migration and invasion were evaluated by Transwell assays; and iv) apoptosis was assessed by Annexin V/PI staining and flow cytometry. The results revealed that, compared to normal adjacent tissue, NLRX1 was lowly expressed in ESCC, and patients with low NLRX1 expression had a shorter survival time. NLRX1 was an independent prognostic factor for ESCC and was associated with tumor grading. Patients in the low-NLRX1 group showed a decrease in the infiltration of activated natural killer cells, monocytes and M0 macrophages, and these immune-cell infiltration levels were positively correlated with NLRX1 expression. Knocking down NLRX1 promoted the proliferation of KYSE450 cells, while overexpression of NLRX1 inhibited the proliferation of ECA109 cells. NLRX1 negatively regulated the PI3K/AKT signaling pathway in ESCC. These findings indicate that, through several mechanisms, NLRX1 suppresses tumor growth in ESCC, which offers new insight for investigating the causes and progression of ESCC, as well as for identifying more efficient therapeutic approaches.

Elucidating the role of the GC/GR/GLUT1 axis in steroid-induced osteonecrosis of the femoral head: A proteomic approach

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a prevalent and incapacitating condition that affects the hip joint. Unfortunately, early diagnostic and treatment measures are limited. MethodsOur study employed Tandem Mass Tag (TMT) labeling mass spectrometry (MS)-based quantitative proteome to compare the proteins of femoral head tissues in patients with SONFH with those of patients who sustained femoral neck fracture (FNF). We investigated the level and effects of glucose transporter member 1 (GLUT1) in SONFH patients and MC3T3-E1 cells and examined the function and molecular mechanism of GLUT1 in the context of SONFH using in vivo and in vitro approaches. ResultsThe SONFH group exhibited significant changes in protein expression levels compared to the fracture group. Specifically, we observed the up-regulation of 86 proteins and the down-regulation of 138 proteins in the SONFH group. Among the differentially expressed proteins, GLUT1 was down-regulated and associated with glucose metabolic processes in the SONFH group. Further analysis using Parallel Reaction Monitoring (PRM), WB, and PCR confirmed that the protein was significantly down-regulated in both femoral head tissue samples from SONFH patients and dexamethasone-treated MC3T3-E1 cells. Moreover, overexpression of GLUT1 effectively reduced glucocorticoid (GC)-induced apoptosis and the suppression of osteoblast proliferation and osteogenic differentiation in MC3T3-E1 cells, as well as GC-induced femoral head destruction in GC-induced ONFH rat models. Additionally, our research demonstrated that GC down-regulated GLUT1 transcription via glucocorticoid receptors in MC3T3-E1 cells. ConclusionsGLUT1 was down-regulated in patients with SONFH; furthermore, down-regulated GLUT1 promoted apoptosis and inhibited osteoblast ossification in dexamethasone-induced MC3T3-E1 cells and contributed to GC-induced femoral head destruction in a SONFH rat model. Glucocorticoids inhibited the transcriptional activity of GLUT1, leading to a reduction in the amount and activity of GLUT1 in the cells and ultimately promoting apoptosis and inhibiting osteoblast ossification via the GC/GR/GLUT1 axis in SONFH.

Understanding the Mechanisms of Diminished Ovarian Reserve: Insights from Genetic Variants and Regulatory Factors.

Delaying childbearing age has become a trend in modern times, but it has also led to a common challenge in clinical reproductive medicine-diminished ovarian reserve (DOR). Since the mechanism behind DOR is unknown and its clinical features are complex, physicians find it difficult to provide targeted treatment. Many factors affect ovarian reserve function, and existing studies have shown that genetic variants, upstream regulatory genes, and changes in protein expression levels are present in populations with reduced ovarian reserve function. However, existing therapeutic regimens often do not target the genetic profile for more individualized treatment. In this paper, we review the types of genetic variants, mutations, altered expression levels of microRNAs, and other related factors and their effects on the regulation of follicular development, as well as altered DNA methylation. We hope this review will have significant implications for the future treatment of individuals with reduced ovarian reserve.

Mechanism of resveratrol in protecting ovary in poor ovarian response mice by regulating Hippo signaling pathway

This study observed the protective effect of resveratrol(Res) on ovarian function in poor ovarian response(POR) mice by regulating the Hippo signaling pathway and explored the potential mechanism of Res in inhibiting ovarian cell apoptosis. Female mice with regular estrous cycles were randomly divided into a blank group, a model group, and low-and high-dose Res groups(20 and 40 mg·kg~(-1)), with 20 mice in each group. The blank group received an equal volume of 0.9% saline solution by gavage, while the model group and Res groups received suspension of glycosides of Triptergium wilfordii(GTW) at 50 mg·kg~(-1) by gavage for two weeks to induce the model. After modeling, the low-and high-dose Res groups were continuously treated with drugs by gavage for two weeks, while the blank group and the model group received an equal volume of 0.9% saline solution by gavage. Ovulation was induced in all groups on the day following the end of treatment. Finally, 12 female mice were randomly selected from each group, and the remaining eight female mice were co-housed with male mice at a ratio of 1∶1. Changes in the estrous cycle of mice were observed using vaginal cytology smears. The number of ovulated eggs, ovarian wet weight, ovarian index, and pregnancy rate of mice were measured. The le-vels of anti-Mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in serum were determined using enzyme-linked immunosorbent assay(ELISA). Ovarian tissue morphology and ovarian cell apoptosis were observed using hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining, respectively. The protein expression levels of yes-associated protein(YAP) 1 and transcriptional coactivator with PDZ-binding motif(TAZ) were detected by immunohistochemistry(IHC), while the changes in protein expression levels of mammalian sterile 20-like kinase(MST) 1/2, large tumor suppressor(LATS) 1/2, YAP1, TAZ, B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were determined by Western blot. The results showed that compared with the blank group, the model group had an increased rate of estrous cycle disruption in mice, a decreased number of normally developing ovarian follicles, an increased number of blocked ovarian follicles, increased ovarian granulosa cell apoptosis, decreased ovulation, reduced ovarian wet weight and ovarian index, increased serum FSH and LH levels, decreased AMH and E_2 levels, decreased protein expression levels of YAP1 and TAZ in ovarian tissues, increased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and decreased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Additionally, the number of embryos per litter significantly decreased after co-housing. Compared with the model group, the low-and high-dose Res groups exhibited reduced estrous cycle disruption rates in mice, varying degrees of improvement in the number and morphology of ovarian follicles, reduced numbers of blocked ovarian follicles, improved ovarian granulosa cell apoptosis, increased ovulation, elevated ovarian wet weight and ovarian index, decreased serum FSH and LH levels, increased AMH and E_2 levels, elevated protein expression levels of YAP1 and TAZ in ovarian tissues, decreased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and increased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Furthermore, the number of embryos per litter increased to varying degrees after co-housing. In conclusion, Res effectively inhibits ovarian cell apoptosis in mice and improves ovarian responsiveness. Its mechanism may be related to the regulation of key molecules in the Hippo pathway.

Indomethacin and 20-hydroxyecdysone influence protein expression in a Spodoptera frugiperda nervous system cell line.

Insecticide mode of action studies provide insights into how new insecticidal actives function and contribute to assessing safety to humans and nontarget organisms. Insect cell lines that express potential target sites can serve as valuable tools in this effort. In this paper, we report on the influence of two signaling molecules on protein expression in a nervous system cell line established from Spodoptera frugiperda (Bayer/BCIRL-SfNS2-0714-TR). We selected this line because we established it in our laboratory and we are experienced in using it. Cells were exposed to the insect developmental hormone (1 µg/mL 20-hydroxyecdysone, 20E) and/or a cyclooxygenase(COX) inhibitor (25 μM indomethacin, INDO; inhibits prostaglandin [PG] biosynthesis) for 24 h (Day 2), 72 h (Day 4), or 120 h (Day 6). We selected a PG biosynthesis inhibitor because PGs act in many aspects of insect biology, such as embryonic development, immunity, and protein phosphorylation. We selected the developmental hormone, 20E, because it also acts in fundamental aspects of insect biology. We identified specific proteins via in silico analysis. Changes in protein expression levels were determined usingliquid chromatography-mass spectrometry (MS) + MS-MS. The largest number of changes in protein expression occurred on Day 2. The combination of 20E plus INDO led to 222 differentially expressed proteins, which documents the deep significance of PGs and 20E in insect biology. 20E and, separately, INDO led to changes in 30 proteins each (pvalue < 0.01; >2X or <0.5X-fold changes). We recorded changes in the expression of 9 or 12 proteins (20E), 10 or 6 proteins (INDO), and 21 or 20 proteins (20E + INDO) on D4 and D6, respectively. While the cell line was established from neuronal tissue, the differentially expressed proteins act in a variety of fundamental cell processes. In this paper, we moved beyond a list of proteins by providing detailed, Gene Ontology term analyses and enrichment, which offers an in-depth understanding of the influence of these treatments on the SfNS2 cells. Because proteins are active components of cell physiology in their roles as enzymes, receptors, elements of signaling transduction pathways, and cellular structures, changes in their expression levels under the influence of signaling molecules provide insights into their function in insect cell physiology.

Investigating the Drug Resistance Mechanisms of Refractory Diffuse Large B-Cell Lymphoma Based on Proteomic Research

Diffuse large B-cell lymphoma (DLBCL) is the most common histological subtype of non-Hodgkin's lymphoma, more than half of the patients of which can achieve complete remission or even be cured with multidrug immunochemotherapy regimen(R-CHOP). However, for patients with relapsed or refractory DLBCL, there is no effective treatment, while the unfavorable prognosis is related to the primary and acquired resistance to existing therapies. Proteomics is the study of the presence and activity of all proteins in cells. In exploring the mechanisms of DLBCL progression, proteomic technology can identify and quantify changed proteins in DLBCL, reveal the relationship between changes in protein expression levels and different stages of lymphoma occurrence and development, and thus provide information on variation in related proteins and protein-related signaling molecules. Based on proteomic technology research, this study is expected to discover new molecular mechanisms, further clarify resistance mechanisms, and find new therapeutic targets for DLBCL, providing more powerful weapons for better prognosis. We first collected paired clinical patient samples that met the experimental requirements and selected biopsy specimens from 3 patients who relapsed within 1 year after systemic treatment, including biopsy specimens at initial diagnosis and relapse. Next, paraffin sections and HE-stains were performed on the samples, and target areas were micro dissected using a laser microscope. The dissected samples were collected in specific EP tubes, lysed ultrasonically by adding lysis buffer to each group of samples, and the protein concentration of the supernatant was determined after centrifugation. Equal amounts of protein from each sample were digested with trypsin and the final concentration was controlled. Analysis was then performed using the EASY-nLC 1200 ultra-high performance liquid phase system and the Q Exactive TM HF-X mass spectrometer. Finally, bioinformatic analysis was performed to analyze differential proteins and find relevant drug targets or markers. The number of identified peptides was 36,107, the number of identified proteins was 6,338, and the number of quantifiable proteins was 6,165. The top five differentially upregulated proteins in the volcano plot of differential proteins were HOPX, TSNAX, PTMA, FTH1, and IFI44, respectively; and the top five downregulated differential proteins were KRT9, KRT1, KRT10, KRT2, and PIP, respectively. Through enrichment analysis of differential proteins, we noticed the ferroptosis pathway as a key pathway, and found that FTH1, TF, and GSH were involved in iron death-related processes, while FTH1 was one of the top five upregulated differential proteins. Iron death is an iron-dependent, non-apoptotic form of cell death discovered in recent years, activation of which can lead to tumor cell death and inhibit tumor growth. Increased expression of the iron-binding subunit FTH1 can suppress activation of iron death and reduce iron death in tumor cells. This suggests that by developing targeted drugs that act on increasing FTH1 expression or iron death inducers which are capable of slowing down the process, it may be possible to address the problem of traditional chemo-resistance in DLBCL and provide new directions for overcoming DLBCL resistance.

An initial characterisation of the Unfolded Protein Response pathway in haematopoietic canine cancer cell lines - a necessary step for the future development of new therapies in dogs with neoplasia.

New and more effective therapies for canine cancer patients are urgently required and this necessitates advanced experimental research. Dogs are good models for studies in comparative oncology; however, canine cancer cell biology research is currently limited by low availability of validated antibody reagents and techniques. This study characterises the expression of key components of the unfolded protein response (UPR) in a panel of haematopoietic canine cancer cell lines using commercially available antibodies, and validates the methods used to study this pathway. The CLBL-1 canine lymphoma cell line and the GL-1 canine leukaemia cell line sourced externally and two counterparts established in house (CNK-89 and CLB70) were used as models of different lymphoma and leukaemia canine cell lines for the study. The human U2OS cell line served as the control. Antibodies were selected for identifying UPR proteins according to known canine cell reactivity and canine-murine and canine-human homology. Endoplasmic reticulum stress was induced with thapsigargin and MG132 in the cell lines. Etoposide was used to induce DNA damage in the cells. The techniques used for this validation analysis were RNA sequencing to observe the expression of UPR components in canine cell lines, Western blot to observe changes of protein expression levels after inducing ER stress in the cells, and flow cytometry in order to study cell death. Substantial variations in both the basic expression and agonist-induced activation of the UPR pathway were observed in canine cancer cell lines, although the biological significance of these differences requires further investigation. These findings will be a starting point for future studies on cancer biology in dogs. They will also contribute to developing novel anticancer therapies for canine patients and may provide new insights into human oncology.

Proteomic profiles of male and female Caligus rogercresseyi: first approach to assess differential protein expression

Caligidosis is a disease that affects salmonids in sea and estuaries of the Chilean coast, with a negative impact in the aquaculture activity, producing great economic damage to salmon farming. This parasitic disease is caused by the marine ectoparasite Caligus rogercresseyi, commonly known as “Caligus”. In recent years, our understanding about the biology of Caligus has been increased by the use of “omics” tools such as genomics and transcriptomics. Indeed, progress in these areas revolutionize our understanding about the physiology of this parasite, in particular, the metabolic process and the invasive mechanisms associated with infestation. However, the knowledge of the changes in protein expression levels during the different stages of the life cycle or under stress conditions of an organism, as well as the differences between sexes, have a very interesting role in the development of specific and effective pharmacological tools against this parasite. Nevertheless, the proteomic information about this parasite is insufficient. In this study, we evaluated differential expression of proteins between males and females C. rogercresseyi not exposed to antiparasitic treatments, using a tandem mass tags (TMT)-based proteomic approach, which allows the simultaneous identification and quantification of protein expression profiles from multiple samples in a single LC-MS/MS analysis. Here, we report the first advance of the proteomic analysis from male and female C. rogercresseyi. By TMT we detect 1666 proteins, of which 1307 were expressed in both males and females. However, within this group of proteins, the differential expression between males and females was detected in 688 proteins. Furthermore, between the proteins expressed exclusively in each sex, 148 proteins were detected exclusively males and 211 proteins in females. Several of the proteins described here have previously been associated with changes in their transcriptional expression levels in response to different antiparasitic drugs. The sex-biased protein expression described in this work makes sense with the different antiparasitic sensitivities observed between males and females of C. rogercresseyi.This work is the first mass identification analysis of proteins in C. rogercresseyi in which we definitively established the existence of a sex-biased protein expression.

Automated classification of protein expression levels in immunohistochemistry images to improve the detection of cancer biomarkers

BackgroundThe expression changes of some proteins are associated with cancer progression, and can be used as biomarkers in cancer diagnosis. Automated systems have been frequently applied in the large-scale detection of protein biomarkers and have provided a valuable complement for wet-laboratory experiments. For example, our previous work used an immunohistochemical image-based machine learning classifier of protein subcellular locations to screen biomarker proteins that change locations in colon cancer tissues. The tool could recognize the location of biomarkers but did not consider the effect of protein expression level changes on the screening process.ResultsIn this study, we built an automated classification model that recognizes protein expression levels in immunohistochemical images, and used the protein expression levels in combination with subcellular locations to screen cancer biomarkers. To minimize the effect of non-informative sections on the immunohistochemical images, we employed the representative image patches as input and applied a Wasserstein distance method to determine the number of patches. For the patches and the whole images, we compared the ability of color features, characteristic curve features, and deep convolutional neural network features to distinguish different levels of protein expression and employed deep learning and conventional classification models. Experimental results showed that the best classifier can achieve an accuracy of 73.72% and an F1-score of 0.6343. In the screening of protein biomarkers, the detection accuracy improved from 63.64 to 95.45% upon the incorporation of the protein expression changes.ConclusionsMachine learning can distinguish different protein expression levels and speed up their annotation in the future. Combining information on the expression patterns and subcellular locations of protein can improve the accuracy of automatic cancer biomarker screening. This work could be useful in discovering new cancer biomarkers for clinical diagnosis and research.

Tongluo Yishen Decoction Ameliorates Renal Fibrosis via NLRP3-Mediated Pyroptosis In Vivo and In Vitro.

Purpose: In this study, we investigated the mechanism of Tongluo Yishen (TLYS) decoction in more detail, from the perspective of pyroptosis in the unilateral ureteral ligation (UUO) model and the hypoxia-induced renal tubular epithelial (NRK-52E) cell. Method: The UUO model was used, and after 14 days of TLYS intervention, rats were tested for blood creatinine and urea nitrogen, HE staining was used to observe the pathological changes in the kidney, Masson staining was used to assess the degree of interstitial fibrosis, western blot was used to detect the changes of α-smooth muscle actin (α-SMA) protein expression level, immunohistochemistry and western blot detected the changes in protein expression levels of NOD-like receptor protein 3 inflammasome (NLRP3), gasdermin D (GSDMD), cysteinyl aspartate specific proteinase (caspase-1), interleukin 18 (IL-18) and interleukin 1β (I L-1β). A hypoxia model was created using NRK-52E cell, and after different concentrations of TLYS decoction intervention, the changes in the expression levels of pyroptosis were used with immunofluorescence and western blot methods. Results: TLYS decoction improved renal function, delayed the advancement of renal interstitial fibrosis, and inhibited pyroptosis in UUO rats. Furthermore, we observed that TLYS can mitigate hypoxia-induced NRK-52E cell damage via the suppression of the NLRP3-mediated pyroptosis. Conclusion: TLYS decoction exert renoprotective effects by inhibiting NLRP3-mediated pyroptosis.

Abstract 2755: Transcriptomic changes experienced by activated and expanded NK cells for their use in the treatment of hematological malignancies

Abstract Natural Killer (NK) cells have shown great promise as a viable cell-based cancer immunotherapy. We have developed a protocol that produces expanded and highly activated NK (eNK) cells from healthy human donors that have been shown to be effective alone and in combination with monoclonal antibodies in a variety of hematological malignancies. The eNK cells showed a dramatic increase in cytolytic capacity after their expansion and activation using IL-2, IL-15, and 721.221 feeder cells. In order to elucidate the underlying mechanisms behind the changes, mRNAseq and miRNA arrays were performed on mRNA and miRNA samples, respectively, extracted from several NK expansions, taken at Day 0 (before activation) and Day 20 (after activation and expansion). Bioinformatic analysis was performed to identify differential gene expression changes and their downstream effects. Based on the genes with greatest differential expression, we quantified their corresponding proteins using flow cytometry, western blots, and ELISAs. Results reveal several discrete changes in the transcriptome that are reflected in changes in protein expression levels within the eNK cells. We conclude that the increased cytolytic capacity of eNK cells is due to the synergistic effect of variations in different genes responsible for target recognition, effector function, cell cycle progression and cell survival. These data can be used in the future to produce better eNK cells as treatment for hematological malignancies. Citation Format: Chantal Reina-Ortiz, Pilar Mozas Alonso, Alberto Cebollada, David Ovelleiro, Luis Alberto Anel Bernal. Transcriptomic changes experienced by activated and expanded NK cells for their use in the treatment of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2755.

Effects of prometryn on cardiac and liver cell lines and mouse tissues

BackgroundPesticides are important biological agents that deter or kill pests. Globally, about 2 million tons of pesticides are being utilized each year which highlights their importance. Previous studies have suggested that high levels of pesticide exposure are associated with increased risk of chronic diseases, including cancers, cardiotoxicity, Parkinson’s disease, diabetes mellitus, neurological deficits, birth defects, and reproductive disorders. Prometryn is a commonly used selective triazine herbicide, which is currently used as an alternative to atrazine‐based products. It is widely applied to agriculture as it has lower acute toxicity than atrazine herbicides, stable chemical properties, and a long period of effectiveness. Although past studies have shown that triazine herbicides have carcinogenic potential in humans, the cytotoxic effects of prometryn on cardiac and hepatic systems are not well investigated or understood. The aim of this study was to determine the effects of prometryn on the heart and liver.MethodsH9c2 cardiac and HepG2 liver carcinoma cells were used to study the in‐vitro effects of prometryn. Cells were treated with varying concentrations of prometryn for 24 h. Cell viability, mitochondrial membrane potential, and reactive oxygen species (ROS) levels were assessed. For animal studies, male mice (10 weeks old) (n=24) were randomized to two treatment groups (control and prometryn). The control group received corn oil while the prometryn treated group were exposed to 185mg/kg b.w. prometryn dissolved in corn oil every 48 hours for 7 days. After a total of 4 doses, mice were euthanized, and the heart and liver tissues were collected. Proteasome and immunoproteasome activity were measured in the heart and liver. Protein expression levels of markers for oxidative stress, apoptosis, and proteasomal degradation such as GSTT2, GSTA1/2, Hsp70, Hsp27, NADPH oxidase, PARP‐1, p53, PSMD12, LMP2, DNP, VU‐1 were detected using immunoblotting. Protein oxidation and polyubiquitination were detected as an indicator of oxidative stress. A paired t‐test was used to assess group differences for protein levels and p values &lt;0.05 were deemed statistically significant.ResultWe found that prometryn had a concentration‐dependent effects on H9c2 and HepG2 cell lines when we assessed cell viability, mitochondria function, and total intracellular reactive oxygen species formation. Increasing the concentration of prometryn increased the ROS formation, decreased cell viability and decreased mitochondrial membrane potential in these cell lines. Furthermore, in animal studies we observed changes in the proteasome and immunoproteasome activities in the liver but not in the heart. Changes in protein expression levels of oxidative stress and proteasomal degradation markers were only detected in the liver.ConclusionThese results indicate that H9c2 and HepG2 cells are sensitive to prometryn in‐vitro. However, prometryn effects on the heart and liver tissue in male mice is significantly different. Overall, our data supports the deduction that prometryn affected mitochondrial function, induced oxidative stress in cells, but also alters the ubiquitin‐proteasome system and increases oxidative stress in mice liver. These results help towards elucidation of the mechanism by which prometryn could cause diseases.

Mitochondrial Damage Response and Fate of Normal Cells Exposed to FLASH Irradiation with Protons.

Radiation therapy (RT) plays an important role in cancer treatment. The clinical efficacy of radiation therapy is, however, limited by normal tissue toxicity in areas surrounding the irradiated tumor. Compared to conventional radiation therapy (CONV-RT) in which doses are typically delivered at dose rates between 0.03-0.05 Gy/s, there is evidence that radiation delivered at dose rates of orders of magnitude higher (known as FLASH-RT), dramatically reduces the adverse side effects in normal tissues while achieving similar tumor control. The present study focused on normal cell response and tested the hypothesis that proton-FLASH irradiation preserves mitochondria function of normal cells through the induction of phosphorylated Drp1. Normal human lung fibroblasts (IMR90) were irradiated under ambient oxygen concentration (21%) with protons (LET = 10 keV/µm) delivered at dose rates of either 0.33 Gy/s or 100 Gy/s. Mitochondrial dynamics, functions, cell growth and changes in protein expression levels were investigated. Compared to lower dose-rate proton irradiation, FLASH-RT prevented mitochondria damage characterized by morphological changes, functional changes (membrane potential, mtDNA copy number and oxidative enzyme levels) and oxyradical production. After CONV-RT, the phosphorylated form of Dynamin-1-like protein (p-Drp1) underwent dephosphorylation and aggregated into the mitochondria resulting in mitochondria fission and subsequent cell death. In contrast, p-Drp1 protein level did not significantly change after delivery of similar FLASH doses. Compared with CONV irradiation, FLASH irradiation using protons induces minimal mitochondria damage; our results highlight a possible contribution of Drp1-mediated mitochondrial homeostasis in this potential novel cancer treatment modality.

Automatic Segmentation of Magnetic Resonance Images of Severe Patients with Advanced Liver Cancer and the Molecular Mechanism of Emodin-Induced Apoptosis of HepG2 Cells under the Deep Learning.

To improve the accuracy of clinical diagnosis of severe patients with advanced liver cancer and enhance the effect of chemotherapy treatment, the U-Net model was optimized by introducing the batch normalization (BN) layer and the dropout layer, and the segmentation training and verification of the optimized model were realized by the magnetic resonance (MR) image data. Subsequently, HepG2 cells were taken as the research objects and treated with 0, 10, 20, 40, 60, 80, and 100 μmol/L emodin (EMO), respectively. The methyl thiazolyl tetrazolium (MTT) method was used to explore the changes in cell viability, the acridine orange (AO)/ethidium bromide (EB) and 4′,6-diamidino-2-phenylindole (DAPI) were used for staining, the Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) (Annexin V-FITC/PI) was adopted to detect the apoptosis after EMO treatment, and the Western blot (WB) method was used with the purpose of exploring the changes in protein expression levels of PARP, Bcl-2, and p53 in the cells after treatment. It was found that compared with the original U-Net model, the introduction of the BN layer and the dropout layer can improve the robustness of the U-Net model, and the optimized U-Net model had the highest dice similarity coefficient (DSC) (98.45%) and mean average precision (MAP) (0.88) for the liver tumor segmentation.

Expression and possible role of Smad3 in postnecrotizing enterocolitis stricture.

To investigate the expression of Smad3 (mothers against decapentaplegic homolog 3) protein in postnecrotizing enterocolitis stricture and its possible mechanism of action. We used immunohistochemistry to detect the expression characteristics of Smad3 and nuclear factor kappa B (NF-κB) proteins in human postnecrotizing enterocolitis stricture. We cultured IEC-6 (crypt epithelial cells of rat small intestine) in vitro and inhibited the expression of Smad3 using siRNA technique. Quantitative PCR, western blotting, and ELISA were used to detect the changes in transforming growth factor-β1 (TGF-β1), NF-κB, tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), and zonula occludens-1 (ZO-1) messenger RNA (mRNA) and protein expressions in IEC-6 cells. CCK8 kit and Transwell cellular migration were used to detect cell proliferation and migration. Changes in epithelial-mesenchymal transition (EMT) markers (E-cadherin and vimentin) in IEC-6 cells were detected by immunofluorescence technique. The results showed that Smad3 protein and NF-κB protein were overexpressed in narrow intestinal tissues and that Smad3 protein expression was positively correlated with NF-κB protein expression. After inhibiting the expression of Smad3 in IEC-6 cells, the mRNA expressions of NF-κB, TGF-β1, ZO-1, and VEGF decreased, whereas the mRNA expression of TNF-α did not significantly change. TGF-β1, NF-κB, and TNF-α protein expressions in IEC-6 cells decreased, whereas ZO-1 and intracellular VEGF protein expressions increased. IEC-6 cell proliferation and migration capacity decreased. There was no significant change in protein expression levels of EMT markers E-cadherin and vimentin and also extracellular VEGF protein expression. We suspect that the high expression of Smad3 protein in postnecrotizing enterocolitis stricture may promote the occurrence and development of secondary intestinal stenosis. The mechanism may be related to the regulation of TGF-β1, NF-κB, TNF-α, ZO-1, and VEGF mRNA and protein expression. This may also be related to the ability of Smad3 to promote epithelial cell proliferation and migration.

Calcium and calcium-related proteins in endometrial cancer: opportunities for pharmacological intervention.

Intracellular calcium ions are key second messengers and play an important role in malignant transformation and cancer progression. Estrogen can evoke intracellular calcium increases through membrane-initiated effects and activate subsequent kinase cascades within minutes in normal and cancerous epithelial cells. Ca2+-related proteins are expressed in normal epithelial cells or endometrial cancer cells, some of which are upregulated by estrogen. Both estrogen-induced transient calcium increases and long-term changes in protein expression levels may be involved in regulating cancer initiation, progression and metastasis. Calcium channel blockers are reported to regulate both the rapid estrogen-induced intracellular Ca2+ increase and cell proliferation, apoptosis and migration, thus having the potential for pharmacological modulators to be repurposed for the treatment of endometrial cancer.

Citrus flavanone metabolites protect pancreatic β-cells against cholesterol stress through a multi-proteomic mechanism.

Citrus flavanones may improve oxidative stress and insulin resistance induced by western diets. However, there is a paucity of studies investigating the change in protein expression levels. This study evaluated the protection and the mechanisms of action of citrus flavanone metabolites, hesperetin 7-glucuronide (H7G) and 3-(4'-hydroxyphenyl) propanoic acid (PA), on pancreatic β-cell function under oxidative stress induced by cholesterol using the global proteomics approach. Cholesterol induced changes in the global proteomic profile in the pancreatic β-cell line Min6. On the other hand, proteomics analysis identified 254 proteins differentially expressed with H7G and 352 with PA treatments, most of them were opposite to the changes induced by cholesterol. Bioinformatics analysis showed that these proteins are implicated in cell functions like cell signaling (insulin signaling, p30MAPK signaling, and others), metabolism (glucokinase and glutathione metabolisms), and inflammation pathways (TNF-α and NF-κB pathways). Also, the results of molecular docking suggest that H7G and PA could bind to putative transcription factors (PPAR-γ, STAT-3, CREB1, NF-κB, NFYA) and cell signaling proteins (IKK, RAS, Pi3K, ERK), which results in changes in protein expression observed. Altogether, these data suggest that the treatment with H7G and PA protects pancreatic β-cells against stress induced by cholesterol through multi-proteomic mechanisms of action.

Differential protein expression of mitogen-activated protein kinases in erythrocytes and liver of lamprey Lampetra fluviatilis on the course of prespawning starvation

The study was designed to identify the types of mitogen-activated protein kinases (MAPKs) in erythrocytes and liver tissues of river lamprey Lampetra fluviatilis and monitor the changes in protein expression levels of found enzymes on the course of prespawning starvation (from November to the end of May). Immunoreactivity of the native and phosphorylated forms of ERK1/2, JNK and p38 was examined in the cytosolic and membrane cell fractions. Both lamprey erythrocytes and liver were found to highly express ERK1/2 and JNK, whereas only trace amounts of p38 were revealed in hepatic tissues. ERK1/2 was identified in cytosolic and membrane fractions, whereas JNK and p38 were predominantly cytosolic enzymes. Total cellular amounts of ERK1/2 and phospho-ERK1/2 in both erythrocytes and liver tissues appeared to be relatively stable on the course of prespawning starvation. However, before spawning ERK1/2 translocated from cytosol to membranes, with partial decline of its cytoplasmic expression being compensated by increases in membrane-bound pool. Immunoreactivity of cytoplasmic JNK, phospho-JNK and p38 were stable from November to March, but sharply decreased before spawning exhibiting almost negligible levels in May, which suggests the depletion of their cellular fractions. Most probably, ERK1/2 plays more important role in mediating adaptive responses of erythrocytes and liver tissues to conditions of natural starvation and maintenance of cell viability before spawning and death of animals in May.