ATF4 Research Articles

Silymarin diminishes oleic acid-induced lipid accumulation in HepG2 cells by modulating the expression of endoplasmic reticulum stress markers

Background and objectivesNon-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder which presents with the accumulation of triglycerides in hepatocytes due to imbalances in the uptake, synthesis, export, and oxidation of fatty acids. Silymarin is a flavonolignan derived from milk thistle, a herbal medicine that has been used for centuries to treat liver disease. In this study, the effect of silymarin on genes involved in lipogenesis and endoplasmic reticulum (ER) stress was investigated in the cellular NAFLD model. Materials and methodsHepG2 cells were treated with 0.5 mM oleic acid (OA) in the presence or absence of silymarin for 24 h, and subsequently, lipid accumulation was assessed by staining the cells with Oil red O as well as the measurement of intracellular triglyceride (TG) content with an enzymatic method. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to measure the expression of sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), Sirtuin 1 (SIRT1), TRB3, and ATF4. ResultsLipid accumulation was increased in OA-treated hepatocytes, an effect that was attenuated by silymarin. The expression of FAS, SREBP-1, TRB3, and ATF4 was significantly increased in OA-treated HepG2 cells, while the expression of SIRT1 was decreased. Treatment with silymarin could successfully diminish SREBP-1, FAS, TRB3, and ATF4 gene expression while augmenting the expression of SIRT1. ConclusionsSilymarin not only efficiently reduces lipogenesis but also decreases ER stress, which is a critical parameter in lipogenesis. Therefore, silymarin might be considered an effective supplementary option in the treatment of liver steatosis.

ONLY THE INQUIRING MIND SOLVES PROBLEMS

<h3>Objective</h3> Hyalinizing clear cell carcinoma (HCCC) is a rare low-grade malignancy involving mainly the minor salivary glands of the palate. It usually presents as a painless swelling and follows an indolent behavior. Histologically, it demonstrates nests of monomorphic clear cells and cells with eosinophilic granular cytoplasm. The histologic overlap between different tumor types poses diagnostic challenges, and molecular changes are becoming pivotal for classification. Most HCCCs have a t(12;22)(q13;q12) chromosomal translocation, leading to fusion of the EWSR1 and ATF1 genes. The integration of histology, fluorescence in situ hybridization (FISH), and immunohistochemistry confirms the diagnosis of HCCC, dictating the prognosis and management. Most HCCCs have a good outcome and respond well irrespective of treatment. <h3>Case Summary</h3> A 61-year-old man with a medical history significant for hyperlipidemia and diabetes mellitus presented for virtual (video) evaluation of throat pain when swallowing present for 1 month. Limited evaluation exhibited an oral mass and the patient was evaluated in person the next day. Examination revealed a soft, crater-like mass of 3 cm in diameter involving the soft and hard palate. Incisional biopsy demonstrated an infiltrative tumor composed of nests of monotonous, round to polygonal epithelial cells with variably eosinophilic to clear cytoplasm surrounded by hyalinized stroma and with perineural invasion. Immunohistochemistry demonstrated p40 positivity of neoplastic nonductal cells; FISH analysis was positive for EWSR1 rearrangement in 82% of the nuclei. The integrated pathologic findings confirmed the diagnosis of HCCC. Imaging was negative for involved cervical nodes or distant findings. The patient underwent partial maxillectomy and bilateral neck dissection followed by adjuvant radiation therapy. <h3>Conclusions</h3> HCCC is a rare low-grade salivary gland malignancy with indolent behavior. Morphologic features remain the cornerstone of salivary gland tumor assessment; however, ancillary testing now helps define the diagnosis. FISH for EWSR1 is a paramount marker for several tumors; nonetheless, EWRS1 rearrangement is not specific for 1 entity and FISH interpretation should be intertwined with clinical, morphologic, and immunohistochemical features to confirm the diagnosis, predict good prognosis, and guide subsequent management of HCCC. Although virtual care can be an effective triage tool, there must be a low threshold for subsequent in-person evaluation to provide adequate care.

Transcriptomic analysis provides insights into the growth and maturation of ovarian follicles in the ricefield eel (Monopterus albus)

The ricefield eel is becoming an important aquaculture species in China. However, the regulation of oocyte growth and maturation in ricefield eels is poorly understood. In this study, the development of ricefield eel ovarian follicles was classified into nine stages, namely the primary growth/previtellogenic (PG/PV), early vitellogenic (EV), mid-vitellogenic (MV), late vitellogenic (LV), full-grown (GV0), germinal vesicle migration 1 (GV1), germinal vesicle migration 2 (GV2), germinal vesicle migration 3 (GV3), and germinal vesicle breakdown (GVBD) stages, which are based on follicle morphologies and sizes, vitellogenic states, and the positions of germinal vesicles. Comparative transcriptomic analysis was performed on isolated ovarian follicles from the MV to the GVBD stages. A total of 438,410 high-quality reads were obtained after filtering from six cDNA libraries of ovarian follicles at the MV, LV, GV0, GV1, GV2, and GVBD stages, 92.09% of which were perfectly mapped to the ricefield eel genome. Real-time PCR analysis showed that the expression profiles of 20 representative genes related to oocyte growth and maturation largely paralleled with those inferred from transcriptome data, which indicated the reliability of RNA-Seq data for gene expression analysis in the present study. The estradiol biosynthesis pathway was highly activated in ovarian follicles at the LV stage, which may be related to the stimulation of vitellogenin synthesis and/or meiotic arrest. The high expression of the Vtg receptor gene vldlr in ovarian follicles at MV and LV is consistent with active vitellogenesis at these stages. Thirteen cathepsin-encoding genes were highly expressed in ovarian follicles with different expression profiles during development from MV to GVBD, which implied their differential roles in vitellogenin hydrolysis during oocyte growth and maturation. Many cell cycle-related genes showed the lowest expression at GV0 and then increased expression at GV1 and GV2, which indicated that the resumption of meiosis may be initiated at GV0. The highest expression of the adenyl cyclase genes adcy3a, adcy5, adcy6, adcy6-like, and adcy7 at MV and/or LV, while that of phosphodiesterase genes pde1c, pde4b, pde4b-like, pde4c, pde7a, pde8b, pde10, and of the cyclic AMP-dependent transcription factor atf-3 gene observed at GV0 indicated that cAMP signals may play important roles in oocyte meiotic arrest and resumption in ricefield eels. Parallel expression profiles of urok, mmp2, mmp9, mmp11, mmp14a, mmp14b, mmp17a, mmp19, and mmp25 suggest their potential roles in tissue remodeling during oocyte maturation and ovulation in ricefield eels. Collectively, the data of the present study will aid in further understanding the molecular mechanisms underlying oocyte growth and maturation in ricefield eels as well as in other teleosts.

Identification of differentially expressed genes and signaling pathways with Candida infection by bioinformatics analysis

BackgroundOpportunistic Candida species causes severe infections when the human immune system is weakened, leading to high mortality.MethodsIn our study, bioinformatics analysis was used to study the high-throughput sequencing data of samples infected with four kinds of Candida species. And the hub genes were obtained by statistical analysis.ResultsA total of 547, 422, 415 and 405 differentially expressed genes (DEGs) of Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis groups were obtained, respectively. A total of 216 DEGs were obtained after taking intersections of DEGs from the four groups. A protein–protein interaction (PPI) network was established using these 216 genes. The top 10 hub genes (FOSB, EGR1, JUNB, ATF3, EGR2, NR4A1, NR4A2, DUSP1, BTG2, and EGR3) were acquired through calculation by the cytoHubba plug-in in Cytoscape software. Validated by the sequencing data of peripheral blood, JUNB, ATF3 and EGR2 genes were significant statistical significance.ConclusionsIn conclusion, our study demonstrated the potential pathogenic genes in Candida species and their underlying mechanisms by bioinformatic analysis methods. Further, after statistical validation, JUNB, ATF3 and EGR2 genes were attained, which may be used as potential biomarkers with Candida species infection.

The Timing of Nitrogen Addition Impacts Yeast Genes Expression and the Production of Aroma Compounds During Wine Fermentation.

Among the different compounds present in the must, nitrogen is an essential nutrient for the management of fermentation kinetics but also plays an important role in the synthesis of fermentative aromas. To address the problems related to nitrogen deficiencies, nitrogen additions during alcoholic fermentation have been implemented. The consequences of such additions on the main reaction are well known. However, their impact on aromas synthesis is still poorly understood. So, the main objective of this study was to determine the impact of nitrogen addition during the stationary phase on both the fermentation kinetics and aroma synthesis. To reach this goal, we used a transdisciplinary approach combining statistical modeling (Box-Behnken design and response surface modeling) and gene expression study (transcriptomic analysis). Our results indicated that nitrogen metabolism, central carbon metabolism (CCM), fermentation kinetics and aroma production were significantly impacted by nitrogen addition. The most remarkable point was the different regulation of the bioconversion of higher alcohols into acetate esters on one hand and of fatty acids into ethyl esters on the other hand. We highlighted that the conversion of higher alcohols into acetate esters was maximum when nitrogen was added at the beginning of the stationary phase. Conversely, the highest conversion of acids into ethyl esters was reached when nitrogen was added close to the end of the stationary phase. Moreover, even if the key element in the production of these two ester families appeared to be the enzymatic activity responsible for their production, rather than the availability of the corresponding precursors, these enzymatic activities were differently regulated. For acetate esters, the regulation occurred at gene level: the ATF2 gene was overexpressed following nitrogen addition during the stationary phase. On the opposite, no induction of gene expression was noted for ethyl esters; it seemed that there was an allosteric regulation.

MiRNA-155 inhibition enhances porcine embryo preimplantation developmental competence by upregulating ZEB2 and downregulating ATF4

The in vitro embryo's competence is lower than in vivo counterparts and miRNAs found to affect the developmental competence, affecting pluripotency, stress level and apoptosis in embryos. We aimed to investigate the effect of miRNA-155 on parthenogenically activated early development porcine embryos at the preimplantation blastocyst stage. We designed miRNA-155 mimics and inhibitors and microinjected them into post-activated oocytes. The embryonic cleavage rate, blastocyst rate, and embryo development quality in terms of stress and apoptosis levels were investigated by confocal microscopy. Furthermore, we selected target genes, analyzed gene interaction and prediction networks, and compared the gene expression level in treatments and controls. miRNA-155 inhibition improved in vitro developmental competence by increasing cell numbers and reducing stress and apoptosis levels. The cleavage rate in the miRNA-155 inhibitor group was significantly higher (P < 0.05) than that in the miRNA-155 mimic group, but not in the control, whereas the blastocyst rate of the miRNA-155 inhibitor group was statistically significantly higher (P < 0.05) than in both control and miRNA-155 mimic groups. The relative gene expression level analysis showed downregulation of mRNAs related to stress and apoptosis, BAX, and the stress-induced autophagy gene ATF4, and TNF-ɑ in the miRNA-155 inhibitor group. Moreover, miRNA-155 inhibition showed upregulation of the relative expression of OCT4, ZEB2, BCL2, and IL-1 mRNA compared to control and mimic groups. However, the miRNA-155 mimic-injected group showed lower cleavage rates and blastocyst development rates than the other two groups. In conclusion, miRNA-155 inhibition in porcine in vitro embryos improved their preimplantation developmental competence and in vitro embryo production.

Effect of surgical damage to spinal nerve on dorsal-root ganglion gene expressions: Comprehensive analysis of differentially expressed genes

BackgroundNeuropathic pain can cause significant physical and economic burden, and there are no effective long-term treatments. We conducted a bioinformatics analysis to identify mechanisms to determine strategies for more effective treatments of neuropathic pain. MethodGSE24982 and GSE63442 microarray datasets were extracted from the Gene Expression Omnibus database to analyze transcriptome differences of neuropathic pain in the dorsal root ganglions (DRGs). We filtered the differentially expressed genes (DEGs) in the two datasets and conducted Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the shared DEGs. The Protein–Protein Interaction network was used to determine the hub genes, which were verified in the GSE30691 dataset. miRDB and miRWalk Databases were used to predict potential miRNA of the selected DEGs. We made the spinal nerve ligation (SNL) rat model and qPCR was used to verify the differential expression of hub genes. ResultsA total of 182 overlapped DEGs were found between GSE24982 and GSE63442 datasets. The GO and KEGG analysis showed that the selected DEGs were enriched in infection, transmembrane transport of ion channels, and synaptic transmission. We identified seven hub genes (Atf3, Aif1, Ctss, Gfap, Scg2, Jun, and Vgf). qPCR verified the expression differences of the hub genes in the DRGs after SNL model. Predicted miRNA targeting each selected hub genes were identified. ConclusionsSeven hub genes related to the pathogenesis of neuropathic pain and potential targeting miRNA were identified, expanding understanding of the mechanism of neuropathic pain and facilitating treatment development.

Identifying of miRNA-mRNA Regulatory Networks Associated with Acute Kidney Injury by Weighted Gene Co-Expression Network Analysis.

BackgroundAcute kidney injury (AKI) is a clinical emergency characterized by a dramatic decline in renal function and the accumulation of metabolic waste products in the body, with a high morbidity and mortality rate. The pathogenesis of AKI remains unclear and there are no effective treatment options.MethodsWe aimed to identify critical genes involved in the pathogenesis of AKI and construct a miRNA–mRNA regulatory network using gene expression data downloaded from Gene Expression Omnibus (GSE85957) for 38 kidneys of AKI and 19 control rats and cisplatin treated kidneys of 3 AKI and 3 control rats. Data in GSE85957 were processed using weighted gene co-expression network analysis (WGCNA), and biological function analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to analyze the functions associated with critical genes.ResultsTwenty-eight modules in the GSE85957 dataset were identified by WGCNA, of which 103 genes in the orange module and 30 genes in the black module were closely associated with AKI and dose. Biological function analysis of genes in the orange and black modules revealed that skeletal muscle cell differentiation, tissue development and organ development were involved in the pathological changes of AKI. Combining with our experimentally processed AKI rat kidney data, eight genes (Atf3, Egr1, Egr2, Fos, Fosb, Gdf15, Serpine1 and Nr1d1) were identified as potential biomarkers of AKI, and miRNA–mRNA regulatory networks were constructed based on the above eight critical genes. Further tissue validation revealed that Egr1 and Fos were highly expressed in AKI.ConclusionOur study identified potential biomarkers of AKI and constructed an associated miRNA–mRNA regulatory network, which may provide new insights into the molecular pathogenesis of AKI.

Acetaminophen-Induced Nephrotoxicity: Suppression of Apoptosis and Endoplasmic Reticulum Stress Using Boric Acid.

Acetaminophen (APAP) is one of the popular and safe pain medications worldwide. However, due its wide availability, it is frequently implicated in intentional or unintentional overdoses where it can cause severe liver injury and even acute liver failure. Boron is a bioactive trace element, found naturally as boric acid (BA) and borate. In this study, the effects of boric acid on the acute renal toxicity induced by APAP in rats were researched in comparison with N-acetyl cysteine (NAC). In the study, 7 groups were formed and 2g/kg dose of paracetamol per rat was prepared by suspending in 1% Carboxy Methyl Cellulose (CMC) solution of phosphate buffer saline (PBS). Boric acid dissolved in saline was administered to experimental animals by gavage at doses of 50, 100, and 200mg/kg. In this study, ER stress and apoptosis formed by paracetamol-induced nephrotoxicity were investigated. This purpose determined iNOS, PERK, ATF6, NFkB p53, caspases 3, 12, bcl-2, and bcl-xL gene mRNA expression kidney tissue. Also, the levels of kidney injury molecule-1 (KIM-1), Cysteine (Cys), and IL-18 levels, which are mentioned today as kidney damage markers were compared with BUN and creatine levels. The effect of boron on kidney damage was determined by histopathologic. Data were statistically analyzed by using SPSS-20 ANOVA and stated as means and standard deviation. According to the data obtained in our study, we believe that boric acid has a protective effect on the negative effects of paracetamol on the kidney. We believe that our study will provide useful data to the literature on the possibility of a supplement to be used as an active compound in paracetamol for the prophylaxis of boric acid and it can also be converted into a useful product.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

Tetrahydropalmatine attenuates liver fibrosis by suppressing endoplasmic reticulum stress in hepatic stellate cells.

Tetrahydropalmatine attenuates liver fibrosis by suppressing endoplasmic reticulum stress in hepatic stellate cells.

The role of the integrated stress response in keratinocyte migration

Background and Hypothesis:&#x0D; Cutaneous wound healing involves: hemostatic, inflammatory, proliferative, and tissue remodeling phases. Re-epithelialization can be modeled in vitro using human keratinocytes and artificial wounds. Previous work showed undifferentiated keratinocytes closing wounds in vitro using individual cell migration (ICM), whilst differentiated keratinocytes utilize collective cell migration (KCCM). Therefore, we hypothesize that ICM in vitro is equivalent to keratinocyte migration during squamous cell carcinoma metastasis in vivo and KCCM is a model for wound re-epithelialization. Furthermore, we hypothesize that the integrated stress response (ISR) is important in ICM and KCCM. The ISR is activated by environmental stresses that protein kinases (GCN2 and PERK) can detect and phosphorylate translation factor, eIF2a. Our goal is to define how the ISR, specifically GCN2 and PERK, influence keratinocyte migration.&#x0D; Methods:&#x0D; We will evaluate in vitro wound healing and kinetic variation in protein expression and cytoskeleton remodeling. We will utilize four keratinocyte cell lines, control human keratinocyte NTERTs, and CRISPR-derived gene knockouts of GCN2, PERK, and ISR effector gene ATF4. Quantitative analysis of wound healing is accomplished using an IncuCyte ZOOM instrument. Protein expression is measured via immunoblots following high density wounding. Cytoskeletal analyses was done by immunofluorescence.&#x0D; Results:&#x0D; Preliminary results show PERK-KO and GCN2-KO cells have reduced expression of F-actin. Immunoblots showed actin-binding protein, phospho-cofilin, at lower levels in PERK-KO and GCN2-KO cells than in NTERT cells. Wound healing assays showed differentiated keratinocytes healing faster than undifferentiated in all cells, except GCN2-KO. GCN2-KO cells healed significantly slower than other differentiated cells and undifferentiated GCN2-KO cells. Wound healing assays showed undifferentiated PERK-KO cells healing slower than other undifferentiated cell lines.&#x0D; Conclusion/Potential Impact:&#x0D; The results indicate PERK and GCN2 could be key components in ICM and CCM respectfully. GCN2 and PERK could thus be potential therapeutic targets to provide cost-effective therapeutics to enhance/inhibit keratinocyte migration.

Effects of dietary arginine on growth, activity of digestive enzymes, GCN2‐ATF4 signalling pathway and nutritional metabolism‐related gene expression of large yellow croaker ( Larimichthys crocea ) larvae

A 30-day feeding experiment was conducted to elucidate the effects of dietary arginine on growth performance, activity of digestive enzymes, GCN2-ATF4 signalling pathway and nutritional metabolism-related gene expression of large yellow croaker larvae (initial weight: 10.56 ± 0.26 mg). Four isonitrogenous and isolipidic diets with different arginine levels (19.5, 31.0, 39.6 and 48.6 g kg−1 of dry matter) were formulated. Results showed that no significant differences in survival, growth and whole-body proximate composition were observed among dietary treatments. The whole-body arginine content of fish larvae was significantly increased with increasing contents of dietary arginine, and the maximum was observed in larvae fed the diet with 48.6 g kg−1 arginine. However, the contents of other amino acids were not significantly affected. Larvae fed the diet with 19.5 g kg−1 arginine showed higher activity of alkaline phosphatase (AKP) in the intestinal segments (IS) and brush border membranes (BBM) than other groups. The expression of GCN2-ATF4 signalling pathway-related genes (atf4, chop and asns) in the visceral mass of larvae was significantly upregulated by dietary 39.6–48.6 g kg−1 arginine supplementation. Dietary 48.6 g kg−1 arginine significantly upregulated the expression of proteolytic genes (murf1 and glud) in the visceral mass of larvae. Furthermore, the highest expression of genes related to fatty acid β-oxidation (cpt1α) and glycolysis (gk and pk) was observed in the 39.6 g kg−1-arginine group. In summary, those results suggested that dietary arginine had no significant influence on the growth performance of large yellow croaker larvae. However, the arginine level significantly affected the arginine content of whole larvae, activity of digestive enzymes, lipid metabolism and glycolysis of larvae. The GCN2-ATF4 signalling pathway was stimulated by high-dose (31.0–48.6 g kg−1) arginine, maybe due to the antagonistic effect of arginine and lysine. These results might indicate that 19.5 g kg−1 dietary arginine (37.6 g kg−1 dietary protein) could meet the growth requirement of large yellow croaker larvae.

S3059 Clear Cell Sarcoma of the Jejunum: Dissecting an Obscure Malignancy

Introduction: Clear Cell Sarcoma (CCS) of the jejunum is an extremely rare and mortiferous presentation of a poorly understood malignancy. CCS is a cancer of the soft tissue and aponeuroses of tendons, affecting young individuals with predilection towards the ankle. It is often confused for malignant melanoma, as it is also derived from neural crest origin and shares a similar histological appearance. Currently, the only definitive treatment is surgical resection. Even so, CCS is known to recur. Due to the sheer modicum of cases, our medical knowledge is limited. Case Description/Methods: We present a case of a 49-year-old gentleman from Cameroon with a past medical history of hypertension and diabetes mellitus type II, who presented to the emergency department with complaints of abdominal pain, nausea, and vomiting for a one-month duration. CT scan of the abdomen and pelvis revealed left-sided small bowel obstruction. The patient underwent diagnostic laparoscopy, which converted to open surgery, with small bowel resection and umbilical hernia repair. A 2.6 cm circumferential obstructing jejunal mass was identified and resected. The margins of resection were negative for malignancy. There was no lymph vascular invasion, and 12 lymph nodes were negative for malignancy as well. Immunohistochemistry stains resulted positive for S100, SOX 10, and EMA. FISH was positive for EWSR1. Recent studies have revealed a chromosomal translocation, t(12;22) (q13;q12), also known as the EWSR1 and ATF1 gene, respectively, which has been widely accepted as pathognomonic for CCS. At 2 years of follow-up, our patient had a drastic improvement in his symptoms, with active surveillance and close follow-up, as CCS has a high recurrence rate. Discussion: This case report aims to describe the clinical presentation and evolution of this unique soft-tissue malignancy, which can be found in the GI tract in extraordinary cases. We hope this rare case of Clear Cell Sarcoma can enhance our knowledge about this obscure cancer that we still have so much to learn.Figure 1.: This 2.6 cm tumor is present in the submucosa, muscularis propria and subserosa and shows nests and cords of patterns by network of delicate vascular septa. Neoplastic cells are plump with epithelioid, clear cell and focal spindle cell appearance. Mitotic figures are about 5/10HPF. Immunostains show that tumor cells are positive for S100, SOX-10, and EMA but negative for HMB-45, Melan A, carcinoma markers (AE1/AE3, CAM5.2, CK7 and CK20), gastrointestinal stromal tumor markers (CKIT, CD34, and DOG1), lymphoid markers (CD3 and CD20), and desmin and ASMA. EWSR1 is positive by FISH studies. A) (Magnification x40, lower power, hematoxylin-eosin) and B) (Magnification x400, high power, hematoxylin-eosin) show epithelioid and clear cell appearance. C) (Magnification x400, antibody for S100) and D) (Magnification x400, antibody for SOX-10) reveal positivity.

GRANULOSA CELLS OF WOMEN WITH PCOS WITH OR WITHOUT INSULIN RESISTANCE DISPLAY SIGNS OF METABOLIC DISTRESS

PCOS is diagnosed based on the presence of oligo/anovulation, hyperandrogenemia/hyperandrogenism, and polycystic ovaries. In addition, many women with PCOS have hyperinsulinemia and insulin resistance (IR), which are associated with significant cellular metabolic abnormalities. In this study, we investigated whether metabolic dysfunction in women with PCOS induces granulosa cell stress and causes activation of endoplasmic reticulum and mitochondrial unfolded protein response (UPRer and UPRmt). Women with PCOS diagnosed based on the Rotterdam criteria were included in the study and divided into two groups: PCOS with insulin resistance (PCOS-IR) when HOMA index was ≥ 2 (n=20) and PCOS with no insulin resistance (PCOS-nIR) when HOMA index was < 2 (n= 20). In addition, a healthy control group (CONT) included women undergoing IVF as oocyte donors (n=20). Granulosa cells (GCs) were collected on the day of oocyte retrieval and total RNA was isolated. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine expression of UPRer genes BIP, ATF4, ATF6, IRE1, CHOP, XBP1 and UPRmt genes HSP60, HSP10, CLPP, HSP40. ANOVA, student’s t-test, and Chi-Square analysis were used for statistical analyses. Women with PCOS-IR and PCOS–nIR were older than the CONT group (30.65 ± 5.25 and 29.94 ± 6.2 vs 25.12 ± 2.26 years, respectively). Body mass index (BMI) of women in the PCOS-IR group was higher (27.64 ± 6.92) compared to both the PCOS-nIR (24.58 ± 3.64; p = 0.032) and CONT (23.76 ± 1.86; p = 0.012). In the GCs of women with PCOS (both -IR and -nIR), UPRer and UPRmt genes were up-regulated compared to the CONT group. Among the genes that regulate UPRmt, chaperone proteins HSP10 and HSP40, involved in the folding of proteins transported into the mitochondria were upregulated in the PCOS-IR (p < 0.01 and p < 0.001) and PCOS-nIR (p < 0.05 and p < 0.05 ) groups compared to CONT, while no difference in the expression of CLPP and HSP60 was found. UPRer-related transcription factors ATF4, IRE1, and XBP1, activated by stress in the endoplasmic reticulum, were significantly increased in GCs of PCOS-IR compared with CONT (p < 0.05 for all), while IRE1 was increased in nIR-PCOS (p < 0.05). The chaperone BIP, involved in the protein folding in the endoplasmic reticulum, was specifically increased in PCOS-IR compared to CONT and PCOS-nIR (p <0.05). No difference was found in ATF6 gene expression. The transcription factor C/EBP homologous protein (CHOP), involved in apoptosis initiation in response to organelle stress, was overexpressed in both PCOS-IR and PCOS-nIR (p < 0.05) compared to CONT. Granulosa cells of women with PCOS with or without insulin resistance display signs of metabolic distress and up-regulation of UPRer and UPRmt genes.

Topoisomerase I inhibition and peripheral nerve injury induce DNA breaks and ATF3-associated axon regeneration in sensory neurons.

SUMMARYAlthough axonal damage induces rapid changes in gene expression in primary sensory neurons, it remains unclear how this process is initiated. The transcription factor ATF3, one of the earliest genes responding to nerve injury, regulates expression of downstream genes that enable axon regeneration. By exploiting ATF3 reporter systems, we identify topoisomerase inhibitors as ATF3 inducers, including camptothecin. Camptothecin increases ATF3 expression and promotes neurite outgrowth in sensory neurons in vitro and enhances axonal regeneration after sciatic nerve crush in vivo. Given the action of topoisomerases in producing DNA breaks, we determine that they do occur immediately after nerve damage at the ATF3 gene locus in injured sensory neurons and are further increased after camptothecin exposure. Formation of DNA breaks in injured sensory neurons and enhancement of it pharmacologically may contribute to the initiation of those transcriptional changes required for peripheral nerve regeneration.

Development of a three-layer consecutive gene delivery system for enhanced bone regeneration

This study developed a three-layer consecutive gene delivery system (T-CGDS) for timely gene delivery into human mesenchymal stem cells (hMSCs). The timing of transcription factor expression is important to effectively induce bone differentiation. Therefore, a three-layered nanocomposite was fabricated using differently sized gold nanoparticles to promote bone regeneration and osteogenic differentiation. The core layer comprised 80 nm gold nanoparticles coupled with ATF4 pDNA. Following coating with heparin-conjugated Pluronic F-127 (HP-F127), 50 nm gold nanoparticles coupled with SP7 pDNA were added to fabricate a bi-layer system. After further coating with HP-F127, 20 nm gold nanoparticles combined with RUNX2 pDNA were added. Consequently, a T-CGDS measuring 350–450 nm was fabricated. Genes were released for more than 8 days, while the size of the T-CGDS decreased over time. When the T-CGDS was applied to hMSCs, the gene in the outer layer (RUNX2) was expressed first, followed by those in the middle (SP7) and core (ATF4) layers. The T-CGDS effectively induced bone differentiation and regeneration in vitro and in vivo. Timely delivery of the ATF4 gene to stem cells via the T-CGDS can greatly assist osteogenic differentiation involved in bone regeneration.

Three novel piperidones exhibit tumor-selective cytotoxicity on leukemia cells via protein degradation and stress-mediated mechanisms

BackgroundCancer is an ongoing worldwide health problem. Although chemotherapy remains the mainstay therapy for cancer, it is not always effective and has detrimental side effects. Here, we present piperidone compounds P3, P4, and P5 that selectively target cancer cells via protein- and stress-mediated mechanisms.MethodsWe assessed typical apoptotic markers including phosphatidylserine externalization, caspase-3 activation, and DNA fragmentation through flow cytometry. Then, specific markers of the intrinsic pathway of apoptosis including the depolarization of the mitochondria and the generation of reactive oxygen species (ROS) were investigated. Finally, we utilized western blot techniques, RT-qPCR, and observed the cell cycle profile after compound treatment to evaluate the possible behavior of these compounds as proteasome inhibitors. For statistical analyses, we employed the one-way ANOVA followed by Bonferroni post hoc test.ResultsP3, P4, and P5 induce cytotoxic effects towards tumorigenic cells, as opposed to non-cancerous cells, at the low micromolar range. Compound treatment leads to the activation of the intrinsic pathway of apoptosis. The accumulation of poly-ubiquitinated proteins and the pro-apoptotic protein Noxa, both typically observed after proteasome inhibition, occurs after P3, P4, and P5 treatment. The stress-related genes PMAIP1, ATF3, CHAC1, MYC, and HMOX-1 were differentially regulated to contribute to the cytotoxic activity of P3–P5. Finally, compound P5 causes cell cycle arrest at the G2/M phase.ConclusionTaken together, compounds P3, P4, and P5 exhibit strong potential as anticancer drug candidates as shown by strong cytotoxic potential, activation of the intrinsic pathway of apoptosis, and show typical proteasome inhibitor characteristics.

Isoginkgetin leads to decreased protein synthesis and activates an ATF4-dependent transcriptional response

Isoginkgetin (IGG) is a small molecule inhibitor of pre-mRNA splicing. Failure to accurately remove introns could lead to the production of aberrant mRNAs and proteins. The cellular responses to splicing stress are not well defined. Here, we used oligonucleotide microarrays to assess genome wide changes in gene expression associated with exposure to IGG. Two of the 3 enriched pathways identified using PANTHER analysis of differentially expressed transcripts are linked to the ATF4 transcription factor. We confirmed that ATF4 was selectively translated and upregulated in response IGG despite an almost complete block to total protein synthesis. Importantly, partial disruption of the ATF4 gene using CRISPR-mediated gene editing prevented IGG-induced changes in gene expression. Remarkably, another spliceosome inhibitor, pladienolide B, did not inhibit translation, activate ATF4 or increase ATF4-dependent gene expression. Taken together, IGG activates ATF4 and an ATF4-dependent transcriptional response but these effects are not common to all spliceosome inhibitors.

Increased unfolded protein responses caused by MED17 mutations.

Mediator (MED) is a key regulator of protein-coding gene expression, and mutations in MED subunits are associated with a broad spectrum of diseases. Because mutations in MED17 result in autosomal recessive disorders, including microcephaly, intellectual disability, epilepsy, and ataxia, which are barely reported, with only three case reports to date, genotype-phenotype association should be elucidated. Here, we investigated the impact of MED17 mutations on cellular responses and found increased unfolded protein responses (UPRs) in fibroblasts derived from Japanese patients with MED17 mutations. The expression of the UPR genes CHOP and ATF4 was upregulated, and the phosphorylation of eIF2a was basally increased in patients' cells. Based on our findings, we propose that increased UPRs caused by MED17 mutations might contribute to the clinical phenotype.