Mean Expression Level Research Articles

Multimodal screen identifies noise-regulatory proteins.

Gene-expression noise can influence cell-fate choices across pathology and physiology. However, a crucial question persists: do regulatory proteins or pathways exist that control noise independently of mean expression levels? Our integrative approach, combining single-cell RNA sequencing with proteomics and regulator enrichment analysis, identifies 32 putative noise regulators. SON, a nuclear speckle-associated protein, alters transcriptional noise without changing mean expression levels. Furthermore, SON's noise control can propagate to the protein level. Long-read and total RNA sequencing shows that SON's noise control does not significantly change isoform usage or splicing efficiency. Moreover, SON depletion reduces state switching in pluripotent mouse embryonic stem cells and impacts their fate choice during differentiation. Collectively, we demonstrate a class of proteins that control noise orthogonally to mean expression levels. This work serves as a proof of concept that can identify other functional noise regulators throughout development and disease progression.

The miR-338-3p expression level in pemphigus diagnosis

BACKGROUND: Pemphigus is a group of potentially fatal chronic cutaneous diseases in which blisters appear on the skin and mucous membranes as a result of IgG autoantibodies binding to desmosomes in the epidermis, leading to keratinocytes acantholysis. Currently, methods to monitor disease activity and therapy efficiency using various biomarkers are being investigated. MicroRNA expression, in particular miR-338-3p, has been one of these biomarkers, as changes in miR-338-3p expression may trigger the Th1/Th2 cell imbalance and possibly be involved in the pathogenesis of the disease. AIM: This study aimed to design a protocol to evaluate the level of miR-338-3p expression in peripheral blood mononuclear cells and verify the diagnostic value of miR-338-3p expression in pemphigus. MATERIALS AND METHODS: Experimental prospective comparative study was conducted from February 2023 to February 2024 at the Dermatology Department of Sechenov University. The study included 10 patients with pemphigus in the active stage of the disease, 3 patients in remission, and 9 participants of the control group. The expression of miRNA-338-3p was analyzed by real-time polymerase chain reaction, cDNA was obtained using StemLoop method. The evaluation of miRNA-338-3p expression level was based on its comparison with the expression of U6 gene using 2-ΔΔСt method. RESULTS: The expression level of miR-338-3p was analyzed in 10 patients in the active stage of the disease (5 men, 50%; 5 women, 50%; mean age 46±10.7 years), 3 patients in remission (2 women, 66.7%; 1 man, 33.3%; mean age 57±8 years), 9 control group (8 women, 88.9%; 1 man, 11.1%; mean age 36±16.8 years). The mean expression level of miR-338-3p was 8.64 (SD±5.72) in patients with active disease, 3.38 (SD±1.44) in patients in remission, and 1.48 (SD±1.12) in controls. A statistically significant increase in the expression level of miR-338-3p was found in patients in the active disease stage compared to the control group (p=0.002). A statistically significant correlation was found between the level of miR-338-3p expression and the PDAI index score (p 0.001). CONCLUSION: Based on the data obtained in this study, it can be assumed that microRNAs are important in pemphigus, and miR-338-3p expression in particular may serve as a key element in pemphigus pathogenesis. More detailed study of microRNAs and analysis of expression variability according to clinical data may provide the basis for developing new diagnostic methods and severity scoring, allowing more accurate and less invasive diagnostic methods, as well as monitoring and predicting disease progression.

Exosomal miRNA-21-5p and miRNA-21-3p as key biomarkers of myocardial infarction.

Coronary artery disease (CAD) is a debilitating condition that can lead to myocardial infarction (MI). Exosomal miRNAs (exo-miRNA) can be diagnostic biomarkers for detecting MI. Here, we conduct a study to evaluate the efficacy of exo-miRNA-21-5p/3p for early detection of MI. A total of 135 CAD patients and 150 healthy subjects participated in this study. Additionally, we randomly divided 26 male Wistar rats (12 weeks old) into two groups: control and induced MI. Angiographic images were used to identify patients and healthy individuals of all genders. In the following, serum exosomes were obtained, and exo-miRNA-21-5p/3p was measured by reverse-transcriptase polymerase chain reaction. We observed an upregulation of exo-miRNA-21-5p/3p in CAD patient and MI-induced animal groups compared to controls. Analysis of the ROC curves defined 82% and 88% of the participants' exo-miRNA-21-5p and exo-miRNA-21-3p diagnostic power, respectively, which in the animal model was 92 and 82. This study revealed that the mean expression levels of exo-miRNA-21-5p/3p were significantly increased in CAD patients and animal models of induced MI. Also, these results are associated with the atherogenic lipid profile of CAD patients, which may play an important role in the progression of the disease. Therefore, they can be considered as novel biomarkers.

Ascites-derived hsa-miR-181a-5p serves as a prognostic marker for gastric cancer-associated malignant ascites

BackgroundPeritoneal carcinomatosis was the main reason leading to gastric cancer (GC)-related death. We aimed to explore the roles of dysregulated microRNAs (miRNAs) and related immune regulation activities in GC-associated malignant ascites.MethodsGSE126399 were downloaded from GEO database. Differentially expressed miRNAs in GC ascites samples was firstly screened, and critical miRNAs were further investigated by LASSO (least absolute shrinkage and selection operator) logistic regression and random forest (RF) algorithm. Receiver operating characteristic of critical miRNAs was also constructed. Moreover, functional analysis, immune cell infiltration associated with differentially expressed mRNAs were further analyzed. After selecting key modules by weighted gene co-expression network analysis, mRNAs related with survival performance and transcription factor (TF)-miRNA-mRNA network were constructed.ResultsHsa-miR-181b-5p was confirmed as critical differentially expressed miRNAs in GC ascites. Then, the tumor samples were divided into high- and low- expression groups divided by mean expression levels of hsa-miR-181b-5p, and subjects with high hsa-miR-181b-5p levels had better survival outcomes. In total, 197 differentially expressed mRNAs associated with hsa-miR-181b-5p levels were obtained, and these mRNAs were mainly enriched in muscle activity and vascular smooth muscle contraction. Hsa-miR-181b-5 was positively related with activated CD4 T cells and negatively related with eosinophil. 17 mRNAs were selected as mRNAs significantly related with prognosis of GC, such as PDK4 and RAMP1. Finally, 75 TF-miRNA-mRNA relationships were obtained, including 15 TFs, hsa-miR-181b-5p, and five mRNAs.ConclusionOur data suggest that the differentially expressed hsa-miR-181b-5p in ascites samples of GC patients may be a valuable prognostic marker and a potential target for therapeutic intervention, which should be validated in the near future.

Characterization of polarization properties of colorectal cancer using decomposition Mueller matrix method

Rectal cancer is the third most prevalent cancer diagnosed and is traditionally screened using colonoscopy. Meanwhile, polarization characteristic measurement has emerged as an attractive alternative in recent years owing to its deep analysis of the structure of samples and non-invasive nature. Accordingly, this study employed the Mueller matrix decomposition method to measure the effective optical parameters of 100 slices from 20 healthy rectal tissue samples and 100 slices from 20 cancerous rectal tissue samples. The mean values and expression levels of the linear birefringence, circular birefringence, linear dichroism, circular dichroism, and depolarization index properties of the samples were compared using t-tests and ANOVA tests. The experimental results showed that the linear birefringence, orientation angle of linear dichroism, and values of linear dichroism and circular dichroism showed significant differences between the different tissue types. Thus, these parameters may serve as useful biomarkers for the diagnosis of rectal cancer.

Comprehensive expression analysis of hormone-like substances in the subcutaneous adipose tissue of the common bottlenose dolphin Tursiops truncatus

Marine mammals possess a specific subcutaneous fat layer called blubber that not only insulates and stores energy but also secretes bioactive substances. However, our understanding of its role as a secretory organ in cetaceans is incomplete. To exhaustively explore the hormone-like substances produced in dolphin subcutaneous adipose tissue, we performed seasonal blubber biopsies from captive female common bottlenose dolphins (Tursiops truncatus; N = 8, n = 32) and analyzed gene expression via transcriptomics. Analysis of 186 hormone-like substances revealed the expression of 58 substances involved in regulating energy metabolism, tissue growth/differentiation, vascular regulation, immunity, and ion/mineral homeostasis. Adiponectin was the most abundantly expressed gene, followed by angiopoietin protein like 4 and insulin-like growth factor 2. To investigate the endocrine/secretory responses of subcutaneous adipose tissue to the surrounding temperature, we subsequently compared the mean expression levels of the genes during the colder and warmer seasons. In the colder season, molecules associated with appetite suppression, vasodilation, and tissue proliferation were relatively highly expressed. In contrast, warmer seasons enhanced the expression of substances involved in tissue remodeling, immunity, metabolism, and vasoconstriction. These findings suggest that dolphin blubber may function as an active secretory organ involved in the regulation of metabolism, appetite, and tissue reorganization in response to changes in the surrounding environment, providing a basis for elucidating the function of hormone-like substances in group-specific evolved subcutaneous adipose tissue.

Effect of genomic and cellular environments on gene expression noise

BackgroundIndividual cells from isogenic populations often display large cell-to-cell differences in gene expression. This “noise” in expression derives from several sources, including the genomic and cellular environment in which a gene resides. Large-scale maps of genomic environments have revealed the effects of epigenetic modifications and transcription factor occupancy on mean expression levels, but leveraging such maps to explain expression noise will require new methods to assay how expression noise changes at locations across the genome.ResultsTo address this gap, we present Single-cell Analysis of Reporter Gene Expression Noise and Transcriptome (SARGENT), a method that simultaneously measures the noisiness of reporter genes integrated throughout the genome and the global mRNA profiles of individual reporter-gene-containing cells. Using SARGENT, we perform the first comprehensive genome-wide survey of how genomic locations impact gene expression noise. We find that the mean and noise of expression correlate with different histone modifications. We quantify the intrinsic and extrinsic components of reporter gene noise and, using the associated mRNA profiles, assign the extrinsic component to differences between the CD24+ “stem-like” substate and the more “differentiated” substate. SARGENT also reveals the effects of transgene integrations on endogenous gene expression, which will help guide the search for “safe-harbor” loci.ConclusionsTaken together, we show that SARGENT is a powerful tool to measure both the mean and noise of gene expression at locations across the genome and that the data generatd by SARGENT reveals important insights into the regulation of gene expression noise genome-wide.

Abstract PO2-14-10: A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model

Abstract Background Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer diagnoses but up to 30% of breast cancer-related deaths. Similarly, Inflammatory breast cancer (IBC) accounts for 2-4% of breast cancer diagnosis but causes 8%–10% of all breast cancer-related deaths. The myeloid cell leukemia-1 (MCL-1) is a member of the BCL-2 family of proteins, which is highly amplified in numerous human cancers and associated with cell immortalization, transformation, and chemoresistance. In fact, MCL-1 is amplified in 55% of TNBC after preoperative chemotherapy. Amongst the MCL-1 inhibitors that have been developed, AZD5991 showed high selectivity and affinity for MCL-1. In myeloma and acute myeloid leukemia cells AZD5991 induces apoptosis and showed anti-tumor effects in in vivo models. In the present study, we evaluated the biological role of the MCL-1 inhibitor AZD5991 in IBC and TNBC in vitro and in vivo models. Materials and methods The expression of the MCL-1 gene was analyzed in the World Consortium IBC patient dataset. Next, we screened a panel of TNBC and IBC cell lines for MCL-1 expression by western blot analysis. The effect of AZD5991 on TNBC and IBC cells was analyzed via cell titer-blue proliferation, clonogenic assays and soft agar assay. To further validate these findings, we used siRNA to knockdown MCL-1 and performed cell proliferation, clonogenic and migration assays. To identify signaling pathways involved in MCL-1 Inhibitor’s effects we performed a proteome profiler human apoptosis array in TNBC cells in the presence of AZD5991. The anti-tumor effect of the MCL-1 inhibitor was evaluated using a SUM-149 (TN-IBC) orthotopic xenograft mouse model. The mice were treated with AZD5991 at doses of 15 and 30mg/kg once weekly via tail vein injection for a duration of 4 weeks. Results The mean expression level of MCL-1 was higher in patients with TNBC than in those with non-TNBC. Further, compared to the patients with ER positive, HER-2 negative tumors those with TNBC showed an increased in the mean expression of MCL-1 gene (p value= 0.05). We screened a panel of TNBC and IBC cell lines in which most of the cell lines showed high levels of MCL-1. The cell proliferation assay showed IC50 ranging from 0.27 – 19.6 μM. The SUM149, SUM190, FCIBC02, MDA-IBC3 and MDA-MB-468 cells showed high sensitivity and showed significant decrease in the proliferation rate while BCX010, KPL4, MDA-MB-231, 4T1.2 and E0771 cells as well as the normal breast cells MCF10A exhibited resistance with no significant decrease in proliferation. The clonogenic and soft agar assays with these sensitive and resistant cell lines showed similar results. Further, these findings were validated via genetic knockdown of MCL-1 which resulted in a significant decrease in cell proliferation, colony formation and migration capability of the TNBC cells. To identify signaling pathways involved in MCL-1 Inhibitor’s phenotypic changes, we performed a proteome profiler human apoptosis array which showed a decreased in BCLx expression, and an increase in expression of BAX and phospho-Rad17 proteins in the AZD5991 treated TN-IBC cells. Finally, in a SUM-149 (TN-IBC) orthotopic xenograft mouse model, mice injected with 30mg/kg showed a significant tumor regression compared to the vehicle-treated mice (vehicle vs 30 mg/kg, p value=0.0006). Conclusions and Future directions Our data demonstrates that AZD5991 inhibits tumorigenesis in an inflammatory breast cancer xenograft model. We are currently validating several significant targets to understand the biological mechanisms related to our candidate drug. Our long-term goal is to develop MCL-1 targeted therapy in combination with standard-of-care treatment for inflammatory and triple-negative breast cancers. Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Michael White, Bisrat Debeb, Emilly Villodre, Xiaoding Hu, Debu Tripathy, Wendy Woodward, Rachel Layman, Geoffrey Bartholomeusz, Chandra Bartholomeusz. A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-10.

Molecular characteristics of non-small cell lung cancer tissue based on quantitative indicators of progesterone receptors expression

Background. Progesterone receptors (PR) are regulators of cell proliferation and therefore can be considered as an aim for targeted medications in the treatment of oncological diseases. At the same time, a quantitative assessment of PR expression in the tissue of non-small cell lung cancer (NSCLC), which has not yet been carried out in other studies, will determine the possibility of using PR modulators for the treatment of this disease and identify the potential category of patients most susceptible to these drugs.Purpose. To characterize NSCLC by quantitative indicators of PR expression and to determine the correlation of clinically significant characteristics of patients and clinical and morphological parameters of a NSCLC tumor with the PR expression to assess the possibility of using PR modulators in the treatment of this disease.Methods. The PR expression in 130 surgical samples of NSCLC was quantified using an immunofluorescence method associated with flow cytometry. Primary antibodies to PR (NBP2-46388) and secondary antibodies conjugated with DyLight650 (ab98729) were used.Results. The expression of progesterone receptors was detected in all the studied tumors; an abnormal distribution of the marker expression level was noted (P=0.01). The mean expression level was 55.3±16.2%, and the median was 57% with a range of 70%, which indicates heterogeneity of PR expression in tumors of different patients. There were no statistically significant differences in the level of PR expression depending on the histotype and stage of NSCLC, as well as on the sex of patients. At the same time, the level of expression and the frequency of overexpression of PR (>67%) in tumors in non-smoking patients are higher than in smokers (P⩽0.02).Conclusion. The high frequency of occurrence and level of PR expression in NSCLC indicate the possible effectiveness of the use of their modulators in the treatment of this disease, especially in non-smoking patients.

Regulatory role of miR-125a expression with respect to its target genes LIFR, ERBB2 and STAT3 in the pathogenesis of recurrent pregnancy losses.

Studies have investigated miR-125a for its predictable role in recurrent pregnancy loss (RPL) cases to regulate many biological events required for the maintenance of pregnancy by regulating its confirmed target genes LIFR, ERBB2 and STAT3. The present study included 40 cases of women with at least two RPLs in ≤20 weeks of gestation against 40 healthy multiparous women without a previous history of abortion. Expression analysis of ERBB2, LIFR, STAT3 and miR-125a was conducted by quantitative real-time PCR (qPCR). The expression of miR-125a was significantly lower in the plasma of RPL cases (P = 0.0001) and showed a significantly increased mean expression level in product of conception (2.56-fold, P < 0.0001). Among the target gene of miR-125a, ERBB2 and STAT3 gene expression level was significantly increased (2.58-fold, P = 0.04; 1.87-fold, P = 0.025), respectively in RPL cases while the LIFR gene revealed comparable expression (P = 0.64). Furthermore, expression analysis of ERBB2 gene with respect to its regulatory miR-125a cases depicted a significant association (P = 0.0005). Kaplan-Meier survival analysis revealed cases with low miR-125a expression had significantly shorter time to miscarriages, (log-rank P = 0.02). Also, decreased expression of miR-125a significantly conferred >2-fold increased risk for RPL (HR = 2.34: P < 0.05). The overall conclusion of the study was that altered miR-125a expression may cause deregulation in target genes LIFR, ERBB2 and STAT3 resulting in adverse consequence in the outcome of pregnancy.

Abstract 3749: The evaluation of expression level of Cxcl12 gene in peripheral blood samples of breast cancer patients

Abstract Breast cancer, like many other cancer types, is linked to the activation and overexpression of CXCR4. The overexpression of CXCR4 is associated with factors such as tumor growth, angiogenesis, increased metastasis, and therapeutic resistance. Similarly, CXCL12, a specific ligand for CXCR4, plays a significant role in tumorigenesis processes, including survival, migration, and microenvironmental interactions. The CXCL12/CXCR4 signaling pathway has been found to impact the development and progression of breast cancer, regulating proliferation, motility, and metastasis. Hence, molecules within the CXCL12/CXCR4 signaling pathway hold promise for determining breast cancer prognosis, treatment monitoring, and serving as therapeutic agents. The study, conducted at the Istanbul University Institute of Oncology, obtained ethical approval from the Istanbul Faculty of Medicine Ethics Committee. Genetic materials from 300 breast cancer patients and 150 control subjects underwent standard testing, matched based on crucial parameters. Real-time PCR with the MIC qPCR Cycler was statistically analyzed using GraphPad Prism (p &amp;lt; 0.05). Descriptive statistics were presented for categorical and quantitative variables, while the Kolmogorov-Smirnov test assessed the normal distribution of quantitative variables. Statistical comparisons between groups were performed using the Mann-Whitney U test and the Kruskal-Wallis test. was used for calculations. Notably, among the three genes—LASP1, CXCL12, and CXCR4—LASP1 exhibited the highest mean expression level at 11.64 with a considerable standard deviation of 11.80. In contrast, CXCL12 had a much lower mean expression level at 2.96 and a tighter distribution, and CXCR4 displayed the lowest mean expression level at 0.37 with a standard deviation of 0.74. The analysis of diagnostic groups indicated no significant differences in mean LASP1 and CXCL12 levels, but a significant variance in mean CXCR4 levels (p&amp;lt;0.001). Specifically, CXCR4 expression in group Breast and ovarian cancer was notably lower compared to Breast cancer, Bilateral Breast cancer, and Breast and Other cancer, while group Breast cancer exhibited significantly lower CXCR4 expression compared to groups Bilateral Breast cancer and Breast and Other cancer. However, there was no significant difference in CXCR4 expression between groups of Bilateral Breast cancer and Breast and Other cancer. These findings reveal distinct expression patterns of CXCR4 across diagnostic groups, suggesting potential implications for diagnostic categorization and underlying molecular pathways in breast cancer. Understanding these expression disparities could be crucial in deciphering the molecular intricacies influencing disease progression and treatment responses. Citation Format: Arash Adamnejad Ghafour, Seref Bugra Tuncer. The evaluation of expression level of Cxcl12 gene in peripheral blood samples of breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3749.

Up-regulation of CDR1 and MDR1 efflux pump genes and fluconazole resistance are involved in recurrence in Candida albicans-induced vulvovaginal candidiasis

Recurrent vulvovaginal candidiasis (RVVC) due to fluconazole resistance in Candida albicans isolates causes a wide range of complications. A number of 63 Candida albicans isolates obtained from vulvovaginal candidiasis (VVC) were identified by Internal Transcribed Spacer-Restriction Fragment Length Polymorphism (ITS-RFLP). Antifungal susceptibility testing was performed by broth microdilution method according to the CLSI protocol. The role of CDR1 and MDR1 genes in progress of VVC to RVVC was examined and the activity of virulence-related enzymes was assessed. Candida albicans was diagnosed in 62.4 % cases, of which 22.2 % were confirmed as RVVC. Voriconazole was the most active drug among five tested antifungals. The mean expression level of CDR1 and MDR1 was higher in RVVC isolates compared to multidrug azole-resistant VVC isolates. Our results demonstrated that the expression of CDR1 and MDR1 and the level of phospholipase and proteinase activities could be quite important to induce fluconazole resistance in C. albicans and to progress of VVC to become RVVC in involved patients.

Phylogenetic analysis, biofilm formation, antimicrobial resistance and relationship between these characteristics in Uropathogenic Escherichia coli.

In the present study, we examine the prevalence of phylogenetic groups, O-serogroups, adhesin genes, antimicrobial resistance, the level of gene expression associated with biofilm formation, and the presence of extended-spectrum beta-lactamase (ESBL) in UPEC strains isolated from both pediatric and adult patients. In this cross-sectional study, 156 UPEC isolates were collected from UTI patients. ESBL-producing isolates were detected using the double-disc synergy (DDS) method, and biofilm formation was assessed through a microplate assay. The presence of O-serogroups, adhesion factors and resistance genes, including ESBLs and PMQR genes, was detected by PCR, and isolates were categorized into phylogenetic groups using multiplex PCR. Additionally, the quantitative real-time PCR method was also used to determine the expression level of genes related to biofilm. During the study period, 50.6% (79/156) of the samples were obtained from children, and 49.4% (77/156) were from adults. The highest rate of resistance was to NA (91.7%), while FM (10.9%) had the lowest rate of antibiotic resistance. In addition, 67.9% (106/156) of UPEC isolates were ESBL producers. Most of UPEC isolates belonged to phylogenetic group B2 (37.1%). This study revealed that blaCTX-M and qnrS are widely distributed among UPEC isolates. The mean expression levels of fimA genes were significantly higher in non-biofilm producers than in biofilm producers (p < 0.01). The high antibiotic resistance rates in this study highlight the significance of local resistance monitoring and investigating underlying mechanisms. Our findings indicate the dominance of phylogroup B2 and group D as the prevailing phylogenetic groups. Consequently, it is imperative to investigate the epidemiological aspects and characterize UPEC isolates across diverse regions and time frames.

CoGTEx: Unscaled system-level coexpression estimation from GTEx data forecast novel functional gene partners.

Coexpression estimations are helpful for analysis of pathways, cofactors, regulators, targets, and human health and disease. Ideally, coexpression estimations should consider as many diverse cell types as possible and consider that available data is not uniform across tissues. Importantly, the coexpression estimations accessible today are performed on a "tissue level", which is based on cell type standardized formulations. Little or no attention is paid to overall gene expression levels. The tissue-level estimation assumes that variance expression levels are more important than mean expression levels. Here, we challenge this assumption by estimating a coexpression calculation at the "system level", which is estimated without standardization by tissue, and show that it provides valuable information. We made available a resource to view, download, and analyze both, tissue- and system-level coexpression estimations from GTEx human data. GTEx v8 expression data was globally normalized, batch-processed, and filtered. Then, PCA, clustering, and tSNE stringent procedures were applied to generate 42 distinct and curated tissue clusters. Coexpression was estimated from these 42 tissue clusters computing the correlation of 33,445 genes by sampling 70 samples per tissue cluster to avoid tissue overrepresentation. This process was repeated 20 times, extracting the minimum value provided as a robust estimation. Three metrics were calculated (Pearson, Spearman, and G-statistic) in two data processing modes, at the system-level (TPM scale) and tissue levels (z-score scale). We first validate our tissue-level estimations compared with other databases. Then, by specific analyses in several examples and literature validations of predictions, we show that system-level coexpression estimation differs from tissue-level estimations and that both contain valuable information reflected in biological pathways. We also show that coexpression estimations are associated to transcriptional regulation. Finally, we present CoGTEx, a valuable resource for viewing and analyzing coexpressed genes in human adult tissues from GTEx v8 data. We introduce our web resource to list, view and explore the coexpressed genes from GTEx data. We conclude that system-level coexpression is a novel and interesting coexpression metric capable of generating plausible predictions and biological hypotheses; and that CoGTEx is a valuable resource to view, compare, and download system- and tissue- level coexpression estimations from GTEx data. The web resource is available at http://bioinformatics.mx/cogtex.

Effect of two different commercial vaccines against bovine respiratory disease on cell-mediated immunity in Holstein cattle.

Bovine respiratory disease (BRD) is a complex illness that impacts the respiratory system of domestic cattle, resulting in significant financial losses for the agriculture industry. Inactivated or modified live (MLV) pathogen vaccines are often used as a management tool to prevent and control BRD effectively. The purpose of this study is to assess the cell-mediated immune response (CMI) induced by two commercially available polyvalent vaccines, namely the MLV (cattle master gold FP) and the inactivated (CATTLEWIN-5K) vaccine. A total of 20 seronegative heifers against 4 BRD viruses, bovine alphaherpisvirus-1 (BoAHV-1), bovine viral diarrhea virus (BVDV BVDV-1: Pesti virus A; BVDV-2: Pesti virus B), bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus-3 (BPIV3) were chosen for this study. The heifers were divided into three groups. The first group (n = 6) received no vaccination and was kept as a control. The second and third groups (seven heifers each) were vaccinated twice with either an MLV or inactivated vaccine. The gene expression level of interleukin-6 (IL-6) and interferon-gamma (INF-γ) was measured using real-time quantitative polymerase chain reaction on the 7th, 14th, 21st, 28th, and 60th days post-vaccination. The results were compared with the control group to study the effectiveness of the vaccines. There was an upregulation in the expression level of IL-6 and INF-γ in both MLV and inactivated vaccinated groups. The level of IL-6 mRNA expression was statistically increased from the 14th and 28th days post-vaccination in MLV and inactivated vaccine groups, respectively. The expression level of INF-γ increased significantly from the 2nd and 4th weeks post-vaccination in the MLV and inactivated vaccine groups, respectively. The mean expression level of IL-6 and INF-γ mRNAs was significantly higher in the MLV vaccine group than in the inactivated vaccine group at each examination time. Both investigated vaccines are efficient in stimulating CMI, particularly with the MLV vaccine showing a higher preponderance in IL-6 and INF-γ.

A pilot study to evaluate the expression of microRNA‑let‑7a in patients with intestinal‑type sinonasal adenocarcinoma.

Despite its histological resemblance to colorectal adenocarcinoma, there is little information about the molecular events involved in the pathogenesis of intestinal-type sinonasal adenocarcinoma (ITAC). The present study investigated the possible role and clinical value of microRNA (miR)-let-7a, a head and neck squamous cell carcinoma-related miR, in a well-characterized and homogeneous cohort of patients with ethmoidal ITAC associated with occupational exposure, treated by primary surgery. miR-let-7a expression levels were analyzed in 23 pairs of ethmoidal ITAC and adjacent normal formalin-fixed paraffin-embedded tissues by reverse transcription-quantitative PCR. The expression was evaluated in tumor and healthy tissues according to: Tumor grade (G) of differentiation and extension, and pTNM stage, and presence/absence of recurrence. Comparisons within and between groups were performed using two-tailed Student's paired t-test and one-way ANOVA with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. miR-let-7a expression in ethmoidal ITAC tissues was significantly lower than that in adjacent normal tissues (P<0.05; mean expression level ± SD, 1.452707±1.4367189 vs. 4.094017±2.7465375). miR expression varied with pT stage. miR-let-7a was downregulated (P<0.05) in advanced stages (pT3-pT4) compared with earlier stages (pT1-pT2). Furthermore, downregulation of miR-let-7a in ITAC was associated with poorly-differentiated (G3) cancer (P<0.05). No other associations were observed between miR-let-7a expression and the other clinicopathological parameters, including disease-free survival. In conclusion, downregulation of miR-let-7a in ITAC was associated with advanced-stage (pT3 and pT4) and poorly-differentiated (G3) disease, suggesting that the mutation of this gene, combined with additional genetic events, could serve a role in ITAC pathogenesis.

Differential CD38 Expression on Daratumumab-Naïve Vs. Resistant Multiple Myeloma - a Direct Stochastic Optical Reconstruction Microscopy ( dSTORM) Analysis in 53 Patients

Background: The anti-CD38 antibody Daratumumab (Dara) has become the new standard of care for the treatment of newly-diagnosed and relapsed/ refractory multiple myeloma (MM). At the same time, Dara resistance remains poorly understood. Potential resistance mechanisms have been proposed and range from intrinsic downregulation in CD38 dim MM cells to genetic alterations and splicing variants which may affect CD38 epitope expression (Adamia, ASH 2022). Here, we used super-resolution microscopy for high-precision quantification of CD38 in MM and piloted this approach in a large cohort of 53 MM patients with vs. without Dara resistance. Methods: Direct stochastic optical reconstruction microscopy ( dSTORM) is a novel imaging modality based on photoswitching of certain fluorophores coupled to antibodies for molecular representation of surface molecules at unprecedented levels of resolution (&amp;lt;20 nM). To correlate CD38 expression data with clinical outcome, we divided our cohort based on treatment status into two groups: Dara-naïve (n = 31) vs. Dara-resistant (n = 22) patients. Imaging data was complemented by phenotypic assessment using a well-established multi-epitope anti-CD38 antibody (Cytognos), as per standard-of-care. Results: Mean CD38 receptor density as measured with Dara via dSTORM was significantly higher in Dara-naïve (of 48.81 ± 29.65 receptors/µm²) vs. Dara-resistant patients (15.52 ± 3.9 receptors/µm², p = 0.016). This finding was corroborated by multi-epitope phenotypic analysis which revealed mean CD38 receptor expression levels of 32.09 ± 13.14 receptors/µm² in Dara-naïve vs. 21.65 ± 17.73 receptors/µm² in Dara-resistant patients ( p = 0.05). Lower CD38 expression via dSTORM vs. multi-epitope anti-CD38 staining was consistently observed in our cohort and may reflect ongoing blockage of the receptor binding site by Dara in cases with shorter time to last Dara application (median 53 days, range 1-189 days). As there was no correlation with the interval between Dara application and sampling however, splice-site mutations affecting CD38 receptor accessibility as proposed by Adamia et al. may depict a more relevant mechanism of resistance in our Dara-resistant cohort. In a next step, we therefore investigated alternative epitope binding in Dara-resistant patients. To this end, we investigated antibody binding by dSTORM imaging for isatuximab (Isa), an alternative CD38-directed monoclonal antibody that binds to a location in the C-terminal portion of the CD38 extracellular domain which is entirely different from the Dara epitope. Interestingly, there was a trend for increased detection rate with Isa for mean CD38 receptor expression in Dara-resistant patients (22.2 ± 4.6 receptors/µm²) vs. Dara (15.5 ± 3.9 receptors/µm², p = 0.1) and on par with CD38 receptor expression. No data was available to assess Dara binding in Isa pre-exposed patients. Conclusion: Innovative microscopy using dSTORM allows for high-resolution epitope quantification at unprecedented granularity. While this finding enables visual proof of differential epitope binding of Dara vs. Isa on CD38 in a clinically feasible setting, correlation studies to determine the clinical relevance of this observation remain ongoing.

CD38 KO/CD38-CAR Human Primary Natural Killer Cells Enhance Cytotoxicity Against CD38-Expressing Primary Lymphoma, Leukemia, and Myeloma

Natural killer (NK) cells have strong cancer immunotherapeutic potential that can be further augmented by engineering with a chimeric antigen receptor (CAR). CD38, a transmembrane glycoprotein involved in signal transduction and cellular adhesion, is highly expressed on the surface of the hematologic malignancies multiple myeloma (MM), acute myeloid leukemia (AML), Burkitt Lymphoma (BL) and T-cell acute lymphoblastic leukemia (T-ALL). FDA approved CD38 monoclonal antibodies (mAb) daratumumab and isatuximab are used to treat MM in patients, and have been tested to treat other CD38 expressing malignancies. NK cells play an imperative role in the efficacy of these antibodies and have natural antitumor activity against these cancers. However, because NK cells also highly express CD38, anti-CD38 mAb can cause NK cell fratricide, limiting their efficacy. Here, we generated multiple CD38 scFv based on isatuximab sequences and generated fratricide-resistant primary CD38-CAR NK cells using CRISPR/Cas9 genome editing and AAV6 gene delivery. Using this approach, we inserted the CD38-CAR gene into the CD38 locus to efficiently generate simultaneous CD38 KO and CD38-CAR KI with a mean expression level of %60. The CAR-expressing NK cells could be expanded on mbIL21-expressing feeder cells to a clinically relevant number of cells with no decline in CAR-expression level during two weeks of expansion confirming the lack of CAR mediated fratricide. We evaluated transduction efficiency and expression of CD38-CAR, and compared the cytotoxic function of two constructs with CD8 transmembrane (TM)/stalk and 41BB signaling domains and reverse heavy and light chains against AML, MM, T-ALL and BL immortalized cell lines and primary patient samples. We enhanced CD38 expression in target cancer cells by pre-treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A that pushes cells to maturation, consequently upregulating CD38. The sequential treatment of target cells with 10nM ATRA for 48 hours enhanced the cytotoxic activity of the CD38 KO/CD38-CAR-NK cells against primary MM, BL and AML samples. This would be particularly important for patients with low expression of CD38 on their cancers. The enhanced anti-tumor activity of the CAR was also studied and observed by CyTOF. Additionally, to better understand the therapeutic potential of CD38-CAR NK cells compared to CD38-CAR T cells we generated CD38 KO/CD38-CAR T cells using the same engineering approach and comparing their cytotoxic function. We showed that both CD38-CAR-T and CD38-CAR-NK cells have comparable anti-tumor activity compare to their paired non-edited wild type cells. Finally, we investigated the need for a CD38 KO to develop a fratricide-resistant therapy by comparing the cytolytic and metabolic functions of CD38 +/AAVS1 KO and CD38 KO CD38-CAR NK cells. We conclusively report a CD38-CAR NK cell therapy with enhanced metabolism and cytotoxicity toward a variety of hematologic malignancies, which can be further augmented by combination treatment with ATRA to target CD38-low malignancies.

Deep-learning-assisted Sort-Seq enables high-throughput profiling of gene expression characteristics with high precision.

Owing to the nondeterministic and nonlinear nature of gene expression, the steady-state intracellular protein abundance of a clonal population forms a distribution. The characteristics of this distribution, including expression strength and noise, are closely related to cellular behavior. However, quantitative description of these characteristics has so far relied on arrayed methods, which are time-consuming and labor-intensive. To address this issue, we propose a deep-learning-assisted Sort-Seq approach (dSort-Seq) in this work, enabling high-throughput profiling of expression properties with high precision. We demonstrated the validity of dSort-Seq for large-scale assaying of the dose-response relationships of biosensors. In addition, we comprehensively investigated the contribution of transcription and translation to noise production in Escherichia coli, from which we found that the expression noise is strongly coupled with the mean expression level. We also found that the transcriptional interference caused by overlapping RpoD-binding sites contributes to noise production, which suggested the existence of a simple and feasible noise control strategy in E. coli.

Pathobiological alterations affecting the distinct clinical courses of pediatric versus adult COVID-19 syndrome.

The clinical presentation of pediatric coronavirus disease 2019 (COVID-19) is associated with a milder disease course than the adult COVID-19 syndrome. The disease course of COVID-19 has three clinicobiological phases: initiation, propagation, and complication. This study aimed to assess the pathobiological alterations affecting the distinct clinical courses of COVID-19 in pediatric age groups versus the adult population. We hypothesized that critical biogenomic marker expressions drive the mild clinical presentations of pediatric COVID-19. Blood samples were obtained from 72 patients with COVID-19 hospitalized at Ankara City Hospital between March and July 2021. Peripheral blood mononuclear cells were isolated using Ficoll-Paque and density-gradient sedimentation. The groups were compared using a t-test and limma analyses. Mean standardized gene expression levels were used to hierarchically cluster genes employing Euclidean Gene Cluster 3.0. The expression levels of identified genes were determined using reverse transcription-polymerase chain reaction. This study found that ANPEP gene expression was significantly downregulated in the pediatric group (p < 0.05, FC: 1.57) and IGF2R gene expression was significantly upregulated in the adult group (p < 0.05, FC: 2.98). The study results indicated that the expression of critical biogenomic markers, such as the first-phase (ACE2 and ANPEP) and second-phase (EGFR and IGF2R) receptor genes, was crucial in the genesis of mild clinical presentations of pediatric COVID-19. ANPEP gene expression was lower in pediatric COVID-19. The interrelationship between the ANPEP and ACE2 genes may prevent the progression of COVID-19 from initiation to the propagating phase in pediatric patients. High IGF2R gene expression could potentially contribute to a protective effect and may be a contributing factor for the mild clinical course observed in pediatric patients.