Fold Change Values Research Articles

P-106 The inhibition of sperm SIRT1 enhances fertilization and modifies subsequent embryonic epigenetic quality

Abstract Study question Is SIRT1 present in sperm and thus involved in fertilization and/or sperm epigenetic code? Do the SIRT1-driven epigenetic marks be transmitted to the progeny? Summary answer SIRT1 localized at the sperm head and influences fertilization and sperm epigenetic code via its deacetylation activity. SIRT1-modulated epigenome is transmitted to zygotes and blastocysts. What is known already The addition or removal of acetyl group from lysine residua (i.e. acetylation) post-translationally modulates proteins through the action of acetylases and deacetylases, respectively. SIRT1 (also Sirtuin 1) is capable to deacetylate a number of proteins, including histones, with roles in male reproduction. Testicular SIRT1 deficit leads to subfertility and increases number of abnormal and immotile spermatozoa that fail to fertilize. However, neither sperm SIRT1 presence nor its function has been investigated despite the significance of acetylation in the events around fertilization. Moreover, the epigenetic mode of action of sperm SIRT1 highlights its relevance for the success of the pre-implantation embryo. Study design, size, duration Mouse sperm of 14-18-weeks-old males of CD-1 x C57Bl6 hybrid strain were isolated from the epididymis. Sperm were incubated under capacitating conditions in presence of sirtinol (5 µM), a selective SIRT1 inhibitor, or the vehicle (0.1% v/v DMSO) for 1 h in 37 °C and 5% CO2. Participants/materials, setting, methods Motility and acrosome reaction was evaluated as previously stated (Ritagliati et al., 2018). Moreover, sperm proteins were extracted into RIPA-buffer and acetylated proteins were depicted by western blot. Finally, in vitro fertilization and embryo culture were performed. Above parameters defining IVF outcomes (i.e., fertilization, cleavage, and blastocyst rate), immunocytochemistry of acetylated histone 4 in the 16th residua of lysine (H4K16) in zygotes and blastocyst was quantified. Main results and the role of chance SIRT1 was localized in the sperm head, apparently at the perinuclear theca and perforatorium. Inhibition of SIRT1 leads to the increase of lysine acetylation (2.28 vs 1, fold change values) and hyperactivation (16.55 ± 0.79 vs 10.84 ± 0.79 %sperm), the enhanced motility that facilitates sperm to penetrate the oocyte, as well as the acrosome reaction (28.69 ± 0.41 vs 17.98 ± 0.41 %sperm). Subsequently, these spermatozoa were able to better fertilize oocytes in vitro (90.5 ± 7.21 vs 66.25 ± 7.21, %). Interestingly, zygotes derived from sirtinol-pre-treated spermatozoa developed to 2-cell embryos at a higher rate than those fertilized by untreated spermatozoa (79.33 ± 7.21 vs 67.00 ± 7.21, %). Moreover, we observed significant hyperacetylation of H4K16 in the male pronuclei of zygotes and embryos derived from sirtinol-treated sperm (8.19 ± 1.02 vs 6.10 ± 2.09 and 45.55 ± 13.96 vs 27.62 ± 13.96, integrated density of the signals, respectively). Limitations, reasons for caution Although acetylation of H4K16 was accumulated in male pronuclei, we do not know which subset of genes was affected. Therefore, we only can assure that SIRT1-driven epigenetic marks are transmitted to the offspring, but their consequences should be further elucidated. For knowledge transfer to humans, a verification study is required. Wider implications of the findings the reduction in testicular SIRT1 expression is an age-related phenomenon. Therefore, our results support SIRT1 as the basis for new interventions against age-related male infertility. Moreover, due to the high reactivity of SIRT1 to its inactivation, SIRT1 becomes a target to pharmacologically enhance sperm fertilization ability in assisted reproductive techniques. Trial registration number Animal procedures were conducted in accordance with Act No. 246/1992 Coll. on the Protection of Animals Against Cruelty under the supervision of the Animal Welfare Committee of the Ministry of Education, Youth and Sports of the Czech Republic, approval ID MSMT-11925/2016-3.

P-376 Restoration of mRNA expression level of endometrial MEIS1, as a co-factor of HOXA10, after laparoscopic salpingectomy of women with hydrosalpinx

Abstract Study question Do salpingectomy could restore HOXA10 co-factor MEIS1 expression in endometrial tissues of hydrosalpinx patients? Summary answer Laparoscopic salpingectomy could restore endometrial HOXA10-cofactor- MEIS1 expression level, in addition to HOXA10, in hydrosalpinx women. What is known already Hydrosalpinx is defined as a distally blocked and dilated fallopian tube which has been filled with serous fluid. Evidence suggests that hydrosalpinx is associated with adverse effect on endometrial receptivity by abnormal expression of key molecules such as HOXA10 in the pre-implantation endometrium. Previous data showed salpingectomy results in statistically significant increase in endometrial HOXA10 expression. MEIS1 is a three-amino-acid loop extension (TALE) family homeobox gene which has been proven to be a novel coactivator of HOXA10 during human and mouse endometrial decidualization. MEIS1-HOXA10 complex binds to the promoters of the target genes which are beneficial for human stromal cell decidualization. Study design, size, duration This prospective study was conducted from January 2021 to January 2022 to determine whether salpingectomy would reverse HOXA10-cofactor- MEIS1 expression levels after salpingectomy. All women signed the informed consent and did not receive any hormonal medication during the last three months. In this study 10 infertile women with moderate to severe hydrosalpinx proven by hysterosalpingography or laparoscopy undergoing salpingectomy were recruited. Ten healthy fertile age-matched women considered as control group. Participants/materials, setting, methods Ten infertile women, aged 20-40, with sonovisible hydrosalpinx (diameter >10mm), AMH>1.2 ng/dL, BMI (18–28 kg/m2) and regular menstrual cycle indicated by mid-luteal progesterone levels of > 10 ng/mL were included. Ten healthy fertile age-matched women with a history of successful pregnancy considered as control group. Mid–luteal-phase endometrial sampling (by pipelle) was performed at the time of surgery and second sampling was obtained forth-post treatment cycle. RNA extraction and cDNA synthesis were done. Main results and the role of chance Quantitative mRNA expression of MEIS1 was determined by real-time PCR technique. Gene expression data were analyzed based on 2 –ΔΔCT to estimate the relative fold change value. The non-parametric Wilcoxon signed rank test was used for data analysis. P value less than 0.05 was considered statistically significant.There was a significant reduction in the expression levels of MEIS1 gene in the endometrial samples of hydrosalpinx group before surgical removal. The obtained data showed that salpingectomy resulted in a statistically significant increase (about 6-fold) in endometrial MEIS1 expression (P = 0.02). Moreover, there was not significant difference in MEIS1 mRNA expression of hydrosalpinx group after salpingectomy and the control group (P > 0.05). Limitations, reasons for caution Larger sample size for the confirmation of these data is needed. Epigenetic evaluation of hydrosalpinx endometrial tissues especially in HOXA10 promoter is recommended. Wider implications of the findings This study suggests that the restoration of MEIS1 gene expression observed in hydrosalpinx women after salpingectomy can be considered as a molecular mechanism by which salpingectomy results in improvement of pregnancy rate in IVF cycle. Trial registration number not applicable

Transcription Analysis of the Acid Tolerance Mechanism of Pichia kudriavzevii NBRC1279 and NBRC1664

Simultaneous saccharification and fermentation (SSF) has been investigated for the efficient production of ethanol because it has several advantages such as simplifying the manufacturing process, operating easily, and reducing energy input. Previously, using lignocellulosic biomass as source materials, we succeeded in producing ethanol by SSF with Pichia kudriavzevii NBRC1279 and NBRC1664. However, various acids that fermentation inhibitors are also produced by the hydrolysis of lignocellulosic biomass, and the extent to which these acids affect the growth and ethanol productivity of the two strains has not yet been investigated. In this study, to better understand the acid tolerance mechanism of the two strains, a spot assay, growth experiment, and transcriptome analysis were carried out using Saccharomyces cerevisiae BY4742 as a control. When the three strains were cultured in SCD medium containing 15 mM formic acid, 35 mM sulfuric acid, 60 mM hydrochloric acid, 100 mM acetic acid, or 550 mM lactic acid, only P. kudriavzevii NBRC1664 could grow well under all conditions, and it showed the fastest growth rates. The transcriptome analysis showed that “MAPK signaling pathway-yeast” was significantly enriched in P. kudriavzevii NBRC1664 cultured with 60 mM hydrochloric acid, and most genes involved in the high osmolarity glycerol (HOG) pathway were up-regulated. Therefore, the up-regulation of the HOG pathway may be important for adapting to acid stress in P. kudriavzevii. Moreover, the log2-transformed fold change value in the expression level of Gpd1 was 1.3-fold higher in P. kudriavzevii NBRC1664 than in P. kudriavzevii NBRC1279, indicating that high Gpd1 expression may be accountable for the higher acid tolerance of P. kudriavzevii NBRC1664. The transcriptome analysis performed in this study provides preliminary knowledge of the molecular mechanism of acid stress tolerance in P. kudriavzevii. Our data may be useful for future studies on methods to improve the tolerance of P. kudriavzevii to acids produced from lignocellulose hydrolysis.

Transcriptome Analysis of Co-Cultures of THP-1 Human Macrophages with Inactivated Germinated Trichophyton rubrum Conidia.

Although most mycoses are superficial, the dermatophyte Trichophyton rubrum can cause systemic infections in patients with a weakened immune system, resulting in serious and deep lesions. The aim of this study was to analyze the transcriptome of a human monocyte/macrophage cell line (THP-1) co-cultured with inactivated germinated T. rubrum conidia (IGC) in order to characterize deep infection. Analysis of macrophage viability by lactate dehydrogenase quantification showed the activation of the immune system after 24 h of contact with live germinated T. rubrum conidia (LGC). After standardization of the co-culture conditions, the release of the interleukins TNF-α, IL-8, and IL-12 was quantified. The greater release of IL-12 was observed during co-culturing of THP-1 with IGC, while there was no change in the other cytokines. Next-generation sequencing of the response to T. rubrum IGC identified the modulation of 83 genes; of these, 65 were induced and 18 were repressed. The categorization of the modulated genes showed their involvement in signal transduction, cell communication, and immune response pathways. In total, 16 genes were selected for validation and Pearson's correlation coefficient was 0.98, indicating a high correlation between RNA-seq and qPCR. Modulation of the expression of all genes was similar for LGC and IGC co-culture; however, the fold-change values were higher for LGC. Due to the high expression of the IL-32 gene in RNA-seq, we quantified this interleukin and observed an increased release in co-culture with T. rubrum. In conclusion, the macrophages-T. rubrum co-culture model revealed the ability of these cells to modulate the immune response, as demonstrated by the release of proinflammatory cytokines and the RNA-seq gene expression profile. The results obtained permit to identify possible molecular targets that are modulated in macrophages and that could be explored in antifungal therapies involving the activation of the immune system.

Investigation of gene expression levels in thyroid tissues of rats treated with Wi-Fi electromagnetic wave (2.4–3 GHz Wi-Fi RF-EMF)

In this study, it was aimed to examine the effects of wireless waves applied to rats in the thyroid tissue by evaluating gene expression levels on 12 determined gene regions. 20 healthy 16-week-old Wistar albino female rats weighting 200–220 g were used. In the experiments, two groups, control and experimental groups, were formed, and ten female rats were used in each group. While WI-FI electromagnetic field was applied to the experimental group of these rats, nothing was applied to the control group. Many different gene regions, including beta catenin, beta-actin, GAPDH, L3B, HIF 1-Alpha, GSk-3B, TCF, WNT7a, WNT10a, WNT2, Beclin 1 and Beclin 2, ATG5 and ATG12, were investigated. ACTB and GAPDH gene primers were used as a house keeping gene. The fold change values were calculated statistically from the data obtained from the gene expression results. Compared to the control group, it was observed that the experimental group had significant increases in gene expression levels of many gene regions investigated in the study (P<0.05). Among the study groups, 12 different genes such as beta catenin, beta-actin, GAPDH, L3B, HIF 1-Alpha, GSk-3B, TCF, WNT7a, WNT10a, WNT2, Beclin 1 and Beclin 2, ATG5 and ATG12, which we have determined according to the literature, are known to be related to autophagy and oxidative stress. When the expression levels were investigated, it was determined that the expression coefficients of all the genes studied in the wnt/ß catenin pathway in the experimental group of rats exposed to the Wi-Fi EM field, except for ATG5 and ATG12, were quite high, and there was significant differences between the groups.

Substantiate a read-across hypothesis by using transcriptome data-A case study on volatile diketones.

This case study explores the applicability of transcriptome data to characterize a common mechanism of action within groups of short-chain aliphatic α-, β-, and γ-diketones. Human reference in vivo data indicate that the α-diketone diacetyl induces bronchiolitis obliterans in workers involved in the preparation of microwave popcorn. The other three α-diketones induced inflammatory responses in preclinical in vivo animal studies, whereas beta and gamma diketones in addition caused neuronal effects. We investigated early transcriptional responses in primary human bronchiolar (PBEC) cell cultures after 24h and 72h of air-liquid exposure. Differentially expressed genes (DEGs) were assessed based on transcriptome data generated with the EUToxRisk gene panel of Temp-O-Seq®. For each individual substance, genes were identified displaying a consistent differential expression across dose and exposure duration. The log fold change values of the DEG profiles indicate that α- and β-diketones are more active compared to γ-diketones. α-diketones in particular showed a highly concordant expression pattern, which may serve as a first indication of the shared mode of action. In order to gain a better mechanistic understanding, the resultant DEGs were submitted to a pathway analysis using ConsensusPathDB. The four α-diketones showed very similar results with regard to the number of activated and shared pathways. Overall, the number of signaling pathways decreased from α-to β-to γ-diketones. Additionally, we reconstructed networks of genes that interact with one another and are associated with different adverse outcomes such as fibrosis, inflammation or apoptosis using the TRANSPATH-database. Transcription factor enrichment and upstream analyses with the geneXplain platform revealed highly interacting gene products (called master regulators, MRs) per case study compound. The mapping of the resultant MRs on the reconstructed networks, visualized similar gene regulation with regard to fibrosis, inflammation and apoptosis. This analysis showed that transcriptome data can strengthen the similarity assessment of compounds, which is of particular importance, e.g., in read-across approaches. It is one important step towards grouping of compounds based on biological profiles.

Lipocalin-2 Upregulation in Nasopharyngeal Carcinoma: A Novel Potential Diagnostic Biomarker.

Lipocalin-2 (LCN2) deregulation has been reported in several types of cancer and is implicated in the proliferation, migration, angiogenesis, and progression of tumors. However, its aberrant expression has been rarely studied in nasopharyngeal carcinoma (NPC). In the present study, we investigated the expression of LCN2 in NPC patients. In this descriptive cross-sectional study, 29 NPC and 20 non-cancerous control paraffin pathology blocks were obtained from the seven-year (2011 to 2018) archive of Razi Laboratory in Rasht, Iran. LCN2 mRNA expression was evaluated through quantitative real-time PCR. In addition, immunohistochemistry was performed to evaluate LCN2 expression at the protein level. The fold change value and total immunostaining score (TIS) were applied for quantitative evaluation. The nonparametric Mann-Whitney U test and Fisher's exact test were used through GraphPad Prism 8.3.0 software. P<0.05 was considered statistically significant. Our results revealed that LCN2 mRNA and protein levels in NPC tissues were significantly higher than control tissues (P=0.028 and P=0.002, respectively). At the protein level, 65.51% (19/29) of NPC patients were categorized as having high LCN2 expression (TIS>3) and 34.47% (10/29) as low expression (TIS≤3). While in the control group, 25% (5/20) of subjects represented a high expression of LCN2 (TIS>3), and 75% (15/20) showed no or weak expression (TIS≤3). No significant correlation was found between the overexpression of LCN2 at the protein level and the demographic features of the patients. Our findings suggest that LCN2 might be considered a potential new diagnostic marker for NPC. However, this warrants further studies.

71P MicroRNA (miRNA) a putative biomarker to better define the molecular apocrine breast cancer (MABC) subtype

Breast cancer (BC), is a heterogeneous disease, divided into molecular subtypes based on gene expression and clinical outcomes. MABC is a poorly diagnosed subtype, characterized by estrogen (ER), progesterone receptor (PR) negativity, and HER-2-amplification in most cases; yet it overexpresses androgen receptor (AR) and activates its pathway. Its improper diagnosis demands the adoption of complementary tools to refine classification; miRNA, key players in regulating cell behavior, AR, and tumorigenic processes of BC hold promise in defining and studying the behavior of MABC. 539 breast cancer microarray data were downloaded from The Cancer Genome Atlas (TCGA-BRCA). Cases with available miRNA data were retained and classified using citbcmst R package (PMID: 30289602). Differential expression analysis identified deregulated miRNAs using DEseq2. A validation set of 131 ER-neg samples was constituted and MABC tumors were characterized apart of triple-negative (TN)BC by the previously described molecular signature (AR, FOXA1 and AR-related genes, PMID: 23663520) on fresh tissue sections. Using miRCURY LNATM miRNA PCR assay, miRNA profiling was done for a panel of 6 differentially expressed miRNAs. Finally, expression of in silico predicted target genes of these miRNAs was assessed by RT-qPCR in MABC and TNBC cell lines after transfection with miRNA mimics. TCGA data analysis indicated MABC as a separate entity based on gene signature. MiRNA-seq data analysis depicted a set of 6 significantly deregulated miRNA with absolute value of log2 fold change> 1 and P-adjusted value <0.05 between MABC and TNBC. We validated on 78 MABC (27 HER2-/ 51 HER2+), and 53 TN samples, by miRNA profiling, significant upregulation of miR-2115-3p and miR-187-3p in MABC compared to TNBC. Preliminary data, showed that the overexpression of these miRNAs significantly downregulates in silico predicted target genes (FGF2, HMGCS1, and ADAM12) implicated in carcinogenesis differently in each of the models used. MABC has a unique signature of miRNA compared to TNBC. The identified miRNAs regulate a set of genes implicated in tumorigenic processes and can potentially serve as biomarkers to effectively diagnose, prognose and treat MABC

Transcriptomic analysis of the honey bee (Apis mellifera) queen brain reveals that gene expression is affected by pesticide exposure during development.

Honey bees (Apis mellifera) play a pivotal role in agricultural production worldwide, primarily through the provision of pollination services. But despite their importance, honey bee health continues to be threatened by many factors, including parasitization by the mite Varroa destructor, poor queen quality, and pesticide exposure. Accumulation of pesticides in the hive's comb matrix over time inevitably leads to the exposure of developing brood, including queens, to wax contaminated with multiple compounds. Here, we characterized the brain transcriptome of queens that were reared in wax contaminated with pesticides commonly found in commercial beekeeping operations including either (a) a combination of 204,000 ppb of tau-fluvalinate and 91,900 ppb of coumaphos ("FC" group), (b) a combination of 9,800 ppb of chlorpyrifos and 53,700 ppb of chlorothalonil ("CC" group), or (c) 43,000 ppb of amitraz ("A" group). Control queens were reared in pesticide-free wax. Adult queens were allowed to mate naturally before being dissected. RNA isolated from brain tissue from three individuals per treatment group was sequenced using three technical replicates per queen. Using a cutoff log2 fold-change value of 1.5, we identified 247 differentially expressed genes (DEGs) in the FC group, 244 in the CC treatment group, and 668 in the A group, when comparing each group to the control. This is the first study to examine the sublethal effects of pesticides commonly found in wax (particularly amitraz) on the queen's brain transcriptome. Future studies should further explore the relationship between our molecular findings and the queen's behavior and physiology.

Abstract LB260: Assessing the suitability of our exosomal small ncRNAs to serve as biomarkers for breast cancer &amp; elucidating the relationship of exosomal ncRNAs and cancer cells from t-47d and hc1143 cell lines

Abstract Background: Exosomes are known to play the role of cargo, carrying biologically important materials such as non-coding RNAs to their recipient cells. Small non-coding RNAs such as miRNA have been shown to exert strong effects on tumorigenesis. The liquid biopsy, which utilizes small RNAs from plasma exosomes, is therefore a promising tool for precision oncology. The purpose of this study is to discover exosomal miRNAs that serve as detection markers of breast cancer. Our hypothesis are 1) if exosomes play the role of cargo, then there should be an abundance of upregulated ncRNAs in exosomes that are also upregulated in the cancer cells themselves. 2) It is likely that the miRNAs in exosomes that are highly expressed have critical roles in cancer cell activity. We performed small RNA-seq and examined the ncRNAs inside cancer cells and exosomal ncRNAs secreted by cancer cells into the culture media. Methods: Two cancer cell lines T-47D and HCC1143, and primary Human Mammary Epithelial Cells (HMEC) were used. We prepared 5 samples of cellular RNA extracted from T-47D and HCC1143 and HMEC. We also extracted 8 samples of exosomal RNA from the media; which included those used for each cell culture and those never used for culture with or without FBS. Exosomes in FBS were depleted using Exosome Depletion Kits. The FASTQ files were uploaded onto QIAGEN GeneGlobe for analysis. The data was primarily normalized using geNorm. Results: Exosomal ncRNAs from both cancer cells were compared to exosomal ncRNAs from normal cells. A total of 879 ncRNAs were detected, 108 of which were significantly upregulated and 104 of which were downregulated in cancer cells. hsa-miR-196a-5p is a ncRNA that had a fold-change of 84.08. Prior studies suggest that this miRNA is associated with MIR196A2 methylation and affects the malignancy of many types of cancers. Comparing cancer exosomal ncRNAs vs. ncRNAs inside cancer cells, a total of 879 ncRNAs were found with a FDR q-value &amp;lt;0.05 and cut-off of 2 fold change. 251 ncRNAs were significantly upregulated in the exosomes. hsa-miR-1-3p had a fold change value of 4025.68 and an associated p-value of 0.0002. High levels of hsa-miR-1-3p are known to be associated with large tumors and is strongly correlated to a lower breast cancer survival rate. Conclusions: The preliminary results indicate that there were significant differences between exosomal ncRNAs from normal cells and exosomal ncRNAs from cancer cells. The data suggests that exosomal ncRNAs are a promising source of prognostic biomarkers for breast cancer as ncRNAs with a high fold change are repeatedly validated to have a significant impact on breast cancer progression. The data supports our hypothesis that the upregulation of ncRNAs in cancer cells &amp; exosomes is linked to cancer activity. The results will be scrutinized further, as other avenues for data analysis will be explored. The next step in our experiment will be to check the cancer survival rates with the TCGA database and attempt to validate our hypothesis through further cell culture experiments. Citation Format: Ken D. Kobayashi, Mayumi Jijiwa, Masaki Nasu, Youping Deng. Assessing the suitability of our exosomal small ncRNAs to serve as biomarkers for breast cancer &amp; elucidating the relationship of exosomal ncRNAs and cancer cells from t-47d and hc1143 cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB260.

Abstract 5249: Polyphenon E effects MXD1 and RGS4 gene expression in PC-3 prostate cancer cells

Abstract Polyphenon E (Poly E) is a standardized, caffeine-free green tea extract with defined polyphenol content. Oral Poly E administration confers chemoprotective activity against prostate cancer (PCa) progression in the TRAMP model of human PCa. Poly E has limited activity against human PCa, but only in those previously diagnosed with atypical small acinar proliferation or high-grade prostate intraepithelial neoplasia. The molecular mechanisms underpinning Poly E chemopreventive activities against PCa are not fully understood. We hypothesized Poly E treatment of PCa cells induces gene expression changes, which could involve molecular mechanisms underpinning the limited Poly E chemoprevention activity against PCa. PC-3 cells were cultured in complete growth media supplemented with varied Poly E concentrations (0, 100, 200, or 300 mg/L) for 24 hr. RNA was isolated for comparative DNA microarray (0 vs. 200 mg/L Poly E) and subsequent qRT-PCR analyses for validation. Microarray data for 54,613 genes were filtered for &amp;gt; 2-fold expression level changes, with 8,319 increased and 6,176 genes decreased. Seven genes (CASP8, CBLB, CCNB1, HDAC4, MXD1, RGCC, RGS4) involved in key cellular signaling pathways and with &amp;gt; 2-fold expression level changes, as well as a control gene (RB1), were selected for qRT-PCR analyses. These eight genes are listed with corresponding fold-change gene expression values for each microarray probe set (Table 1). The qRT-PCR analyses identified two genes with significantly increased expression, MXD1 (13.98-fold; p=0.0003) and RGS4 (21.98-fold; p=0.0011), between poly E treatment levels. The significantly increased MXD1 and RGS4 gene expression in Poly E-treated PC-3 cells, and the Poly E-dose dependence of MXD1 gene expression increases, could implicate MXD1 and RGS4 in the PCa chemopreventive activity of Poly E. Future experimental directions include cell cycle profile and Annexin V analyses, immunoblotting analyses, and including other established PCa cell lines in these studies. Microarray Data from Poly E-treated PC-3 Cells Affymetrix Probe ID Gene symbol Log2-Fold Change 213373_s_at CASP8 -2.23 1553306_at CASP8 -3.20 208348_s_at CBLB 19.01 209682_at CBLB 3.83 214710_s_at CCNB1 -2.77 228729_at CCNB1 -3.47 1554322_a_at HDAC4 -2.47 204225_at HDAC4 -3.23 228813_at HDAC4 -4.45 206877_at MXD1 13.56 228846_at MXD1 7.35 226275_at MXD1 1.91 211540_s_at RB1 1.85 203132_at RB1 -1.43 218723_s_at RGCC 23.12 239827_at RGCC 1.22 204338_s_at RGS4 20.54 204337_at RGS4 6.12 204339_s_at RGS4 3.69 Citation Format: L. Michael Carastro, Ethan J. Vallebuona, Ricardo Cordova, Ashley N. Gannon, Corrine M. Costello, Ricardo A. Declet-Bauzo, Seung Joon Kim, Nagi Kumar, Jong Y. Park. Polyphenon E effects MXD1 and RGS4 gene expression in PC-3 prostate cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5249.

Expression and clinical significance of ENC1 in gastrointestinal tumors: Bioinformatics analysis based on a public gene database.

To investigate the expression characteristics of ectodermal-neural cortex 1 (ENC1) in gastrointestinal tumors and its potential value in judging the prognosis of patient survival. RNA sequence (RNA-seq) data and patient survival data related to stomach adenocarcinoma (STAD) and colon adenocarcinoma (COAD) in gastric cancer and colon cancer from The Cancer Genome Atlas (TCGA) were downloaded for expression difference analysis and Cox survival regression analysis. A Kaplan-Meier (KM) survival curve was plotted to analyze the tumor invasion level of patients with different ENC1 expression levels, and the main influencing pathways of ENC1 were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein network analysis. The data of 405 STAD samples and 494 COAD clinical samples of TCGA were analyzed, and it was found that the expression of ENC1 in tumor tissues of patients with both types of cancer was significantly higher than that in normal tissues, with Log2 fold change values of 1.97 and 2.06, respectively, P<0.001. Cox analysis revealed that the high expression of ENC1 was not significantly correlated with the prognosis and survival time of patients with gastric cancer and patients with colon cancer: overall survival (OS) hazard ratio (HR): 1.039, 95% confidence interval (CI): 0.890-1.213, P=0.627 for gastric cancer, OS HR: 0.886, 95% CI: 0.702-1.111, P=0.306 for colon cancer. KEGG pathway enrichment analysis of gene ENC1 revealed that ENC1 was mainly involved in neuroactive ligand-receptor interaction. The high expression of ENC1 was associated with various immune cells and different T cells such as basophils, CD4+ memory T cells, CD4+ TEM, and MV endothelial cells in gastric and colon cancers. The results of ENC1 protein interaction network analysis suggested that ENC1 may be involved in regulating neurite formation and neural crest cell differentiation. ENC1 expression is elevated in both gastric and colon cancers, and ENC1 is associated with various immune cells and different T cells such as basophils, CD4+ memory T cells, CD4+ TEM, and MV endothelial cells in both gastric and colon cancers, but ENC1 does not affect the survival and prognosis of patients.

Mass spectrometry-based proteomic profiling of sonicate fluid differentiates Staphylococcus aureus periprosthetic joint infection from non-infectious failure: A pilot study.

This pilot study aimed to use proteomic profiling of sonicate fluid samples to compare host response during Staphylococcus aureus-associated periprosthetic joint infection (PJI) and non-infected arthroplasty failure (NIAF) and identify potential novel biomarkers differentiating the two. In this pilot study, eight sonicate fluid samples (four from NIAF and four from S. aureus PJI) were studied. Samples were reduced, alkylated, and trypsinized overnight, followed by analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a high-resolution Orbitrap Eclipse mass spectrometer. MaxQuant software suite was used for protein identification, filtering, and label-free quantitation. Principal component analysis of the identified proteins clearly separated S. aureus PJI and NIAF samples. Overall, 810 proteins were identified based on their detection in at least three out of four samples from each group; 35 statistically significant differentially abundant proteins (DAPs) were found (two-sample t-test p-values ≤0.05 and log2 fold-change values ≥2 or ≤-2). Gene ontology pathway analysis found that microbial defense responses, specifically those related to neutrophil activation, to be increased in S. aureus PJI compared to NIAF samples. Proteomic profiling of sonicate fluid using LC-MS/MS differentiated S. aureus PJI and NIAF in this pilot study. Further work is needed using a larger sample size and including non-S. aureus PJI and a diversty of NIAF-types.

Comprehensive Study of Components and Antimicrobial Properties of Essential Oil Extracted from Carum carvi L. Seeds.

Carum carvi L. belongs to the Apiaceae family and is widely used as a vegetable, food spice, preservative, and herbal medicine. This study investigated the impact of essential oil extracted from Carum carvi L. seeds (CEO) on methicillin-resistant Staphylococcus aureus (MRSA) and its possible action mechanism. The dominant chemical components of CEO determined by GC-MS were carvone and limonene. It was observed that CEO had a considerable inhibitory effect against the growth of planktonic bacteria and biofilm in MRSA cells. Untargeted metabolomics based on GC-Q-TOF-MS was used to analyze the possible mechanism of the interaction of MRSA with CEO. It was determined that there were 63 different metabolites based on fold change values greater than 1.5 or less than 1.5, p < 0.05, VIP > 1, which demonstrated amino acid metabolism in MRSA was significantly affected by CEO. In conclusion, CEO has a potent antimicrobial property and has promising potential for use in food and drugs.

Longitudinal transcriptomic analysis of mouse sciatic nerve reveals pathways associated with age-related muscle pathology.

Sarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve, which governs lower limb muscles, in aging mice. Sciatic nerve and gastrocnemius muscle were obtained from female C57BL/6JN mice aged 5, 18, 21 and 24months old (n=6 per age group). Sciatic nerve RNA was extracted and underwent RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) were validated using quantitative reverse transcription PCR (qRT-PCR). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups (adjusted P-value<0.05, likelihood ratio test [LRT]) was performed. Pathological skeletal muscle aging was confirmed between 21 and 24months by a combination of molecular and pathological biomarkers. Myofiber denervation was confirmed with qRT-PCR of Chrnd, Chrng, Myog, Runx1 and Gadd45ɑ in gastrocnemius muscle. Changes in muscle mass, cross-sectional myofiber size and percentage of fibres with centralized nuclei were analysed in a separate cohort of mice from the same colony (n=4-6 per age group). We detected 51 significant DEGs in sciatic nerve of 18-month-old mice compared with 5-month-old mice (absolute value of fold change>2; false discovery rate [FDR]<0.05). Up-regulated DEGs included Dbp (log2 fold change [LFC]=2.63, FDR<0.001) and Lmod2 (LFC=7.52, FDR=0.001). Down-regulated DEGs included Cdh6 (LFC=-21.38, FDR<0.001) and Gbp1 (LFC=-21.78, FDR<0.001). We validated RNA-seq findings with qRT-PCR of various up- and down-regulated genes including Dbp and Cdh6. Up-regulated genes (FDR<0.1) were associated with the AMP-activated protein kinase signalling pathway (FDR=0.02) and circadian rhythm (FDR=0.02), whereas down-regulated DEGs were associated with biosynthesis and metabolic pathways (FDR<0.05). We identified seven significant clusters of genes (FDR<0.05, LRT) with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in age-related changes in skeletal muscles and/or sarcopenia initiation including extracellular matrix organization and an immune response (FDR<0.05). Gene expression changes in mouse peripheral nerve were detected prior to disturbances in myofiber innervation and sarcopenia onset. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies are warranted to confirm the disease modifying and/or biomarker potential of the key changes we report here.

Quantitative N-Glycomic and N-Glycoproteomic Profiling of Peach [Prunus persica (L.) Batsch] during Fruit Ripening.

Being part of the human diet, peach is an important fruit consumed worldwide. In the present study, a systematic first insight into the N-glycosylation of peach fruit during ripening was provided. First, N-glycome by reactive matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated that 6 of 24 N-glycans of peach were differentially expressed. Second, a comparative N-glycoproteome was characterized via 18O-tagged N-glycosylation site labeling followed by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS). Totally 1464 N-glycosites on 881 N-glycoproteins were identified, among which 291 N-glycosites on 237 N-glycoproteins were expressed differentially with a fold change value of 1.5 or 0.67. The enrichment analysis of GO and KEGG revealed that four pathways including other glycan degradation, phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and protein processing in endoplasmic reticulum were mainly enriched, in which several important N-glycoproteins with dynamic change during fruit ripening were further screened out. Our findings on a large scale for N-glycosylation analysis of peach fruit during ripening may provide new molecular insights for comprehending N-glycoprotein functions, which should be of great interest to both glycobiologists and analytical chemists.

Residual phenotypic susceptibility to doravirine in multidrug-resistant HIV-1 from subjects enrolled in the PRESTIGIO Registry

ObjectivesDoravirine shows a rather distinct resistance profile within the nonnucleoside reverse transcriptase inhibitor (NNRTI) class. This study aimed to evaluate the phenotypic susceptibility to doravirine, rilpivirine and etravirine in a panel of multidrug-resistant (MDR) HIV-1 isolates collected from people living with HIV (PLWH) enrolled in the PRESTIGIO Registry. MethodsRecombinant viruses expressing PLWH-derived protease, reverse transcriptase coding regions were generated from plasma samples at virological failure with documented resistance to protease inhibitors, nucleoside reverse transcriptase inhibitors, NNRTIs and integrase strand transfer inhibitors. In vitro susceptibility was assessed through a phenotypic assay measuring fold-change values with respect to the reference NL4-3 virus. Genotypic susceptibility was computed by the Stanford HIVdb algorithm 8.9-1. ResultsPlasma samples were collected from 22 PLWH: 20 (91%) were male, median age 55 years (IQR 50–58), time since HIV-1 diagnosis 27 years (23–31) and time on antiretroviral treatment 23 years (22–26). Median doravirine, etravirine and rilpivirine fold-change values were 9.8 (2.9–40.4), 42.9 (3.1–100.0) and 100.0 (17.9–100.0), respectively. According to the fold-change cut-offs, full susceptibility was observed in five (23%), four (18%) and one (5%) cases with doravirine, etravirine and rilpivirine, respectively. Irrespective of the presence of specific doravirine mutations, higher numbers of NNRTI mutations correlated with higher fold-change values for doravirine. By comparing the distribution of fold-change values with the Stanford HIVdb predicted susceptibility, a significant correlation was detected for doravirine and rilpivirine but not etravirine. ConclusionDespite extensive cross-resistance among NNRTIs, doravirine can be a valid option in a proportion of PLWH with MDR HIV-1. Doravirine activity appeared to be inferred with fair accuracy by the HIVdb algorithm.

Gene Expression of TNF-α among Iraqi COVID-19 Patients with a Different Severity Status

Background: Coronavirus disease 2019 (COVID-19) individuals with varied severity group are affected by the cytokine storm brought on by SARS-CoV2 infection, which is a significant cause of acute respiratory distress syndrome. Objective: The goal of the current study was to examine tumor necrosis factor (TNF-α) gene expression in COVID-19 at various severity levels. Materials and Methods: The study includes 140 divided into 105 COVID-19-positive patients (35 for each mild, moderate, and severe group) and 35 COVID-19-negative healthy people as control. COVID-19 positive patients had 46 males and 59 females, while COVID-19-negative healthy people included 16 males and 19 females. The separation of peripheral blood mononuclear cells (PBMC) was achieved using Ficoll, and then Ribonucleic acid was extracted and converted to cDNA and the gene expression using glyceraldehyde-3-phosphate dehydrogenase as the housekeeping gene. Results: The results revealed non-significant differences at P &lt; 0.05 in age among different COVID-19 groups and control (F-ratio value is 0.54257 and P-value is 0.65397). The results revealed over-expression of TNF-α gene among COVID-19 patients and the relative quantification (fold change) (mean ± standard deviation) values were 6.542 ± 7.29, 5.740 ± 6.41, 7.306 ± 8.85, and 6.580 ± 6.47 for all, mild, moderate, and severe COVID-19 patients, respectively. One-way analysis of variance test relative quantification (fold change) TNF-α (mean ± standard deviation) for mild, moderate, and severe groups revealed non-significant at P &lt; 0.05, the F-ratio value is 0.39889 and the P-value is 0.672109. Conclusion: The present study concludes upregulation of TNF-α gene in PBMC of COVID-19-positive patients without significant differences among different severity groups.

Identification of Enolase 1 as a Potential Target for Magnaporthe oryzae: Integrated Proteomic and Molecular Dynamics Simulation

Rice blast is an essential factor affecting rice yield and quality, which is caused by Magnaporthe oryzae (M. oryzae). Isobavachalcone (IBC) is a botanical fungicide derived from the seed extract of the Leguminosae plant Psoralea corylifolia L. and has shown an excellent rice blast control effect in field applications. To explore the potential targets of rice blast control, the analysis of the differentially expressed proteins (DEPs) between the liquid culture medium of mycelium treated by 10 mg/L of IBC for 2 h and the control group indicated that Enolase 1 (ENO1) was the most significantly down-regulated DEP with a fold change value of 0.305. In vitro experiments showed that after treating liquid culture mycelium with 10 mg/L of IBC for 0.5, 1, 2, 4, and 8 h, the enzymatic activity of ENO1 in the IBC experimental groups was 0.97, 0.75, 0.52, 0.44, and 0.39 times as much as in the control groups, respectively. To further explore the molecular interaction and binding mode between IBC and ENO1, the three-dimensional structure of ENO1 was established based on homology modeling. Molecular docking and molecular dynamics simulation showed that IBC had a pi-pi stacking effect with the residue TYR_365, a hydrogen bond interaction with the residue ARG_393, and hydrophobic interactions with non-polar residues ALA_361, LYS_362, and VAL_371 of ENO1. These findings indicated that ENO1 is a potential target of M. oryzae, which would pave the way for screening novel effective fungicides targeting ENO1.

Six potential biomarkers for bladder cancer: key proteins in cell-cycle division and apoptosis pathways

BackgroundThe bladder cancer (BC) pathology is caused by both exogenous environmental and endogenous molecular factors. Several genes have been implicated, but the molecular pathogenesis of BC and its subtypes remains debatable. The bioinformatic analysis evaluates high numbers of proteins in a single study, increasing the opportunity to identify possible biomarkers for disorders.MethodsThe aim of this study is to identify biomarkers for the identification of BC using several bioinformatic analytical tools and methods. BC and normal samples were compared for each probeset with T test in GSE13507 and GSE37817 datasets, and statistical probesets were verified with GSE52519 and E-MTAB-1940 datasets. Differential gene expression, hierarchical clustering, gene ontology enrichment analysis, and heuristic online phenotype prediction algorithm methods were utilized. Statistically significant proteins were assessed in the Human Protein Atlas database. GSE13507 (6271 probesets) and GSE37817 (3267 probesets) data were significant after the extraction of probesets without gene annotation information. Common probesets in both datasets (2888) were further narrowed by analyzing the first 100 upregulated and downregulated probesets in BC samples.ResultsAmong the total 400 probesets, 68 were significant for both datasets with similar fold-change values (Pearson r: 0.995). Protein-protein interaction networks demonstrated strong interactions between CCNB1, BUB1B, and AURKB. The HPA database revealed similar protein expression levels for CKAP2L, AURKB, APIP, and LGALS3 both for BC and control samples.ConclusionThis study disclosed six candidate biomarkers for the early diagnosis of BC. It is suggested that these candidate proteins be investigated in a wet lab to identify their functions in BC pathology and possible treatment approaches.