Self-folding RCA product into a parallel monolayer DNA nanoribbon and woven into a nano-fence structure by a short bridge strand.

The universal programmed construction of patterned periodic self-assembled nanostructures is a technical challenge in DNA origami nanotechnology but has numerous potential applications in biotechnology and biomedicine. In order to circumvent the dilemma that traditional DNA origami requires a long unusual single-stranded virus DNA as the scaffold and hundreds or even thousands of short strands as staples, we report a method for constructing periodically-self-folded rolling circle amplification products (RPs). The repeating unit is designed to have 3 intra-unit duplexes (inDP1,2,3) and 2 between-unit duplexes (buDP1,2). Based on the complementary pairing of bases, RPs each can self-fold into a periodic grid-patterned ribbon (GR) without the help of any auxiliary oligonucleotide staple. Moreover, by using only an oligonucleotide bridge strand, the GRs are connected together into the larger and denser planar nano-fence-shaped product (FP), which substantially reduces the number of DNA components compared with DNA origami and eliminates the obstacles in the practical application of DNA nanostructures. More interestingly, the FP-based DNA framework can be easily functionalized to offer spatial addressability for the precise positioning of nanoparticles and guest proteins with high spatial resolution, providing a new avenue for the future application of DNA assembled framework nanostructures in biology, material science, nanomedicine and computer science that often requires the ordered organization of functional moieties with nanometer-level and even molecular-level precision.

Electrode Droplet Microarray (eDMA): An Impedance Platform for Label-Free Parallel Monitoring of Cellular Drug Response in Nanoliter Droplets.

Label-free real-time monitoring of cellular behavior using impedance spectroscopy is important for drug development and toxicological assessments. Parallelization and miniaturization of such experiments are essential for increasing throughput and enabling experiments with low abundant stem or primary cells. Traditional methods are not miniaturized and require large volumes of reagents and number of cells, limiting their suitability for cost effective high-throughput screening of cells of limited availability. Here, the fabrication, optimization, and application of a bioelectrical signaling monitoring system - electrode droplet microarray (eDMA) are demonstrated. The eDMA platform is based on preparation of a hydrophilic-superhydrophobic patterns covering an array of individually addressable microelectrodes, which confines cells to individual microelectrodes, allowing for parallel, real-time, and label-free detection of cellular responses to drug treatments in nanoliter droplets. The real-time monitoring of cytotoxic effect of an anticancer drug is demonstrated over 48 h with real-time calculation of the half-inhibitory concentration (IC50) values through impedance spectroscopy. This demonstrates eDMA's ability to dynamically assess responses to various drugs in parallel at any given time point, which is crucial for functional personalized oncology. Specifically, the platform can be employed for monitoring anticancer drug toxicity using limited patient samples, where the miniaturization provided by eDMA is essential.

Biallelic Loss of 7q34 (TRB) and 9p21.3 (CDKN2A/2B) in Adult Ph-Negative Acute T-Lymphoblastic Leukemia.

Tumor cells of acute lymphoblastic leukemia (ALL) may have various genetic abnormalities. Some of them lead to a complete loss of certain genes. Our aim was to reveal biallelic deletions of genes in Ph-negative T-ALL. Chromosomal microarray analysis (CMA) was performed for 47 patients with de novo Ph-negative T-ALL, who received treatment according to RALL-2016m clinical protocol at the National Medical Research Center for Hematology (Moscow, Russia) from 2017 to 2023. Out of forty-seven patients, only three had normal molecular karyotype. The other 44 patients had multiple gains, losses, and copy neutral losses of heterozygosity. Biallelic losses were found in 14 patients (30%). In ten patients (21%), a biallelic deletion of 9p21.3 involved a different number of genes, however CDKN2A gene loss was noted in all ten cases. For seven patients (15%), a biallelic deletion of 7q34 was found, including two genes-PRSS1, PRSS2 located within the T-cell receptor beta (TRB) locus. A clonal rearrangement of the TRB gene was revealed in 6 out of 7 cases with 7q34 biallelic loss. Both biallelic deletions can be considered favorable prognostic factors, with an association with 9p21 being statistically significant (p = 0.01) and a trend for 7q34 (p = 0.12) being observed.

Benchmarking of Approaches for Gene Copy-Number Variation Analysis and Its Utility for Genetic Aberration Detection in High-Grade Serous Ovarian Carcinomas.

Objective: The goal of this study was to compare the results of CNV detection by three different methods using 13 paired carcinoma samples, as well as to perform a statistical analysis of the agreement. Methods: CNV was studied using NanoString nCounter v2 Cancer CN Assay (Nanostring), Illumina Infinium CoreExome microarrays (CoreExome microarrays) and digital droplet PCR (ddPCR). Results: There was a good level of agreement (PABAK score > 0.6) between the CoreExome microarrays and the ddPCR results for finding CNVs. There was a moderate level of agreement (PABAK values ≈ 0.3-0.6) between the NanoString Assay results and microarrays or ddPCR. For 83 out of 87 target genes studied (95%), the agreement between the CoreExome microarrays and NanoString nCounter was characterized by PABAK values < 0.75, except for MAGI3, PDGFRA, NKX2-1 and KDR genes (>0.75). The MET, HMGA2, KDR, C8orf4, PAX9, CDK6, and CCND2 genes had the highest agreement among all three approaches. Conclusions: Therefore, to get a better idea of how to genotype an unknown CNV spectrum in tumor or normal tissue samples that are very different molecularly, it makes sense to use at least two CNV detection methods. One of them, like ddPCR, should be able to quantitatively confirm the results of the other.

Expression of Trefoil Factor 1 (TFF1) in Cancer: A Tissue Microarray Study Involving 18,878 Tumors.

Background/Objectives: Trefoil factor 1 (TFF1) plays a role in the mucus barrier. Methods: To evaluate the prevalence of TFF1 expression in cancer, a tissue microarray containing 18,878 samples from 149 tumor types and 608 samples of 76 normal tissue types was analyzed through immunohistochemistry (IHC). Results: TFF1 staining was detectable in 65 of 149 tumor categories. The highest rates of TFF1 positivity were found in mucinous ovarian carcinomas (76.2%), colorectal adenomas and adenocarcinomas (47.1-75%), breast neoplasms (up to 72.9%), bilio-pancreatic adenocarcinomas (42.1-62.5%), gastro-esophageal adenocarcinomas (40.4-50.0%), neuroendocrine neoplasms (up to 45.5%), cervical adenocarcinomas (39.1%), and urothelial neoplasms (up to 24.3%). High TFF1 expression was related to a low grade of malignancy in non-invasive urothelial carcinomas of the bladder (p = 0.0225), low grade of malignancy (p = 0.0003), estrogen and progesterone receptor expression (p < 0.0001), non-triple negativity (p = 0.0005) in invasive breast cancer of no special type, and right-sided tumor location (p = 0.0021) in colorectal adenocarcinomas. Conclusions: TFF1 IHC has only limited utility for the discrimination of different tumor entities given its expression in many tumor entities. The link between TFF1 expression and parameters of malignancy argues for a relevant biological role of TFF1 in cancer. TFF1 may represent a suitable therapeutic target due to its expression in only a few normal cell types.

Array Comparative Genomic Hybridization (aCGH) Results among Patients Referred to Invasive Prenatal Testing after First-Trimester Screening: A Comprehensive Cohort Study.

Introduction: Invasive prenatal testing with chromosomal microarray analysis after first-trimester screening is a relevant option but there is still debate regarding the indications. Therefore, we evaluated the prevalence of numerical chromosomal aberrations detected by classic karyotype and clinically relevant copy number variants (CNVs) in prenatal samples using array comparative genomic hybridization (aCGH) stratified to NT thickness: 4.5 mm, and by the presence/absence of associated structural anomalies detected by ultrasonography. Materials and Methods: Retrospective cohort study carried out at two tertiary Polish centers for prenatal diagnosis (national healthcare system) in central and south regions from January 2018 to December 2021. A total of 1746 prenatal samples were received. Indications for invasive prenatal testing included high risk of Down syndrome in the first-trimester combined test (n = 1484) and advanced maternal age (n = 69), and, in 193 cases, other reasons, such as parental request, family history of congenital defects, and genetic mutation carrier, were given. DNA was extracted directly from amniotic fluid (n = 1582) cells and chorionic villus samples (n = 164), and examined with classic karyotype and aCGH. Results: Of the entire cohort of 1746 fetuses, classical karyotype revealed numerical chromosomal aberrations in 334 fetuses (19.1%), and aCGH detected CNV in 5% (n = 87). The frequency of numerical chromosomal aberrations increased with NT thickness from 5.9% for fetuses with NT < p95th to 43.3% for those with NT > 4.5 mm. The highest rate of numerical aberrations was observed in fetuses with NT > 4.5 mm having at least one structural anomaly (50.2%). CNVs stratified by NT thickness were detected in 2.9%, 2.9%, 3.5%, 4.3%, 12.2%, and 9.0% of fetuses with NT < 95th percentile, 95th percentile-2.9 mm, 3.0-3.4 mm, 3.5-3.9 mm, 4.0-4.5 mm, and >4.5 mm, respectively. After exclusion of fetuses with structural anomalies and numerical aberrations, aCGH revealed CNVs in 2.0% of fetuses with NT < 95th percentile, 1.5% with NTp95-2.9 mm, 1.3% with NT 3.0-3.4 mm, 5.4% with NT 3.5-3.9 mm, 19.0% with NT 4.0-4.5 mm, and 14.8% with NT > 4.5 mm. Conclusions: In conclusion, our study indicates that performing aCGH in samples referred to invasive prenatal testing after first-trimester screening provides additional clinically valuable information over conventional karyotyping, even in cases with normal NT and anatomy.

Analyzing Dehalochip: A functional DNA microarray for reductive dichlorination in chloroethene-contaminated sites.

Interpreting high-throughput transcriptomic and metagenomic data from non-model microorganisms presents a challenge due to the significant number of genes with unknown functions and sequences. In this study, we applied an innovative microarray, Dehalochip, for detecting the expression of genes in various microorganisms, particularly focusing on genes involved in chloroethene degradation. Our results demonstrated that this approach can effectively identify dechlorination genes, such as 16S rRNA, tceA, bvcA, and vcrA, in Dehalococcoides mccartyi from samples of groundwater contaminated with chloroethene. Noticeably, the sensitivity and specificity of our Dehalochip are comparable to that of quantitative PCR. However, it stands out as a more viable option for in-situ applications due to its greater capacity to infer potential dechlorination genes. Consequently, we believe our dechlorination microarray offers valuable insights into the role of known microorganisms and their associated functional genes in chloroethene-contaminated environments. This contributes to a deeper understanding of the in-situ reductive dechlorination process.

Assessment of the carcinogenic potential of particulate matter generated from 3D printing devices in Balb/c 3T3-1-1 cells.

Recently, there have been reports of sarcoma occurring in a Korean science teachers who used a 3D printer with acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) filaments for educational purposes. However, limited toxicological research data on 3D printing make it challenging to confirm a causal relationship between 3D printing and cancer. Therefore, occupational accidents involving teachers who have developed sarcoma have not been officially recognized. To address this gap, we aimed to evaluate the carcinogenic potential of particulate matter produced from ABS and PLA filaments commonly used in 3D printing. We created a generator mimicking 3D printing to generate particulate matter, which was used as an experimental material. The collected particulate matter was exposed to an in vitro system to investigate genetic damage, effects on cell transformation, and changes in carcinogenesis-related genes. Various assays, such as the comet assay, cell transformation assays, microarray analysis, and glucose consumption measurement, were employed. Cytotoxicity tests performed to determine the exposure concentration for the comet assay showed that cell viability was 83.6, 62.6, 42.0, and 10.2% for ABS at exposure concentrations of 50, 100, 200, and 400 µg/mL, respectively. PLA showed 91.7, 80.3, 65.1, and 60.8% viability at exposure concentrations of 50, 100, 200, and 400 µg/mL, respectively. Therefore, 50 µg/mL was set as the highest concentration for both ABS and PLA, and 25 and 12.5 µg/mL were set as the medium and low concentrations, respectively. The comet assay showed no changes in genetic damage caused by the particulate matter. Cytotoxicity results performed to establish exposure concentrations in the transformation assay showed that ABS showed cell viability of 88.0, 77.4, 84.7, and 85.5% at concentrations of 1.25, 2.5, 5, and 10 µg/mL, respectively, but few cells survived at concentrations above 20 µg/mL. PLA showed minimal cytotoxicity up to a concentration of 20 µg/ml. Therefore, in the cell transformation assay, a concentration of 10 µg/mL for ABS and 20 µg/mL for PLA was set as the highest exposure concentration, followed by medium and low exposure concentrations with a common ratio of 2. In cell transformation assays, only one transformed focus each was observed for both ABS and PLA particulate matter-exposed cells. The microarray assay revealed changes in gene expression, with a 41.7% change at 10 µg/mL for ABS and an 18.6% change at 20 µg/mL for PLA compared to the positive control group. Analysis of carcinogenesis-related gene expression changes on days 1, 7, and 25 of the promotion phase revealed that in cells exposed to 5 µg/mL of ABS, RBM3 gene expression increased by 3.66, 3.26, and 3.74 times, respectively, while MPP6 gene expression decreased by 0.33, 0.28, and 0.38 times, respectively, compared to the negative control group. Additionally, the measurement of glucose consumption showed that it increased in cells exposed to ABS and PLA particulate matter. Our findings suggest that the carcinogenic potential of ABS- and PLA-derived particulate matter in 3D printing cannot be completely ruled out. Therefore, further research in other test systems and analysis of additional parameters related to carcinogenesis, are deemed necessary to evaluate the carcinogenic risk of 3D printers using these materials.

Genotype imputation in human genomic studies.

Imputation is a method that supplies missing information about genetic variants that could not be directly genotyped with DNA microarrays or low-coverage sequencing. Imputation plays a critical role in genome-wide association studies (GWAS). It leads to a significant increase in the number of studied variants, which improves the resolution of the method and enhances the comparability of data obtained in different cohorts and/or by using different technologies, which is important for conducting meta-analyses. When performing imputation, genotype information from the study sample, in which only part of the genetic variants are known, is complemented using the standard (reference) sample, which has more complete genotype data (most often the results of whole-genome sequencing). Imputation has become an integral part of human genomic research due to the benefits it provides and the increasing availability of imputation tools and reference sample data. This review focuses on imputation in human genomic research. The first section of the review provides a description of technologies for obtaining information about human genotypes and characteristics of these types of data. The second section describes the imputation methodology, lists the stages of its implementation and the corresponding programs, provides a description of the most popular reference panels and methods for assessing the quality of imputation. The review concludes with examples of the use of imputation in genomic studies of samples from Russia. This review shows the importance of imputation, provides information on how to carry it out, and systematizes the results of its application using Russian samples.

Chromosomal Abnormalities Detected by Chromosomal Microarray Analysis and Karyotype in Fetuses with Ultrasound Abnormalities.

Objective: Chromosomal microarray analysis (CMA) is a first-line test to assess the genetic etiology of fetal ultrasound abnormalities. The aim of this study was to evaluate the effectiveness of CMA in detecting chromosomal abnormalities in fetuses with ultrasound abnormalities, including structural abnormalities and non-structural abnormalities. Methods: A retrospective study was conducted on 368 fetuses with abnormal ultrasound who received interventional prenatal diagnosis at Meizhou People's Hospital from October 2022 to December 2023. Samples of villi, amniotic fluid, and umbilical cord blood were collected according to different gestational weeks, and karyotype and CMA analyses were performed. The detection rate of chromosomal abnormalities in different ultrasonic abnormalities was analyzed. Results: There were 368 fetuses with abnormal ultrasound, including 114 (31.0%) with structural abnormalities, 225 (61.1%) with non-structural abnormalities, and 29 (7.9%) with structural combined with non-structural abnormalities. The detection rate of aneuploidy and pathogenic (P)/likely pathogenic (LP) copy number variations (CNVs) of CMA in fetuses with structural abnormalities was 5.26% (6/114), the detection rate of karyotype was 2.63% (3/114), and the additional diagnosis rate of CMA was 2.63%. In the fetuses with ultrasonic non-structural abnormalities, the detection rate of karyotype was 6.22% (14/225), the detection rate of aneuploidy and P/LP CNVs in fetuses with ultrasonic structural abnormalities was 9.33% (21/225), and the additional diagnosis rate of CMA was 3.11%. There was no significant difference in chromosome abnormality detection rate of CMA among structural abnormality, non-structural abnormality, and structural abnormality combined with non-structural abnormality groups (5.3%, 9.3%, and 13.8%, p = 0.241), also among multiple ultrasonic abnormality and single ultrasonic abnormality groups (14.8%, and 7.3%, p = 0.105). Conclusion: CMA can significantly improve the detection rate of genetic abnormalities in prenatal diagnosis of ultrasonic abnormal fetuses compared with karyotype analysis. CMA is a more effective tool than karyotyping alone in detecting chromosomal abnormalities in fetuses with ultrasound abnormalities.

Prognostic value of PLEKHA4 and its correlation with tumor-infiltrating immune cells in breast cancer: a comprehensive study based on bioinformatics and clinical analysis validation.

Background: Pleckstrin homology containing family A, number 4 (PLEKHA4) plays a role in a number of biological processes in human cells, including cell polarization, growth, and proliferation. However, the relationship between PLEKHA4 expression and survival in breast cancer (BC) remains unclear. The aim of this study is to investigate the potential of PLEKHA4 as a prognostic indicator in BC. Methods: We obtained gene expression profiles of BC and normal tissues from the Tumor Immune Estimation Resource (TIMER), UALCAN web. Immunohistochemistry (IHC) staining was performed to investigate the protein expression and prognostic value of PLEKHA4 in BC patients. The prognostic value was analyzed using Kaplan-Meier curve analysis and Cox regression analysis in R software after downloading The Cancer Genome Atlas (TCGA) databases. The correlations between PLEKHA4 and tumor immune infiltrates were investigated via gene set variation analysis (GSVA). Signaling pathways related to PLEKHA4 expression were identified by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Results: Both bioinformatics and IHC results showed that PLEKHA4 was expressed at low levels in BC tissues compared with the adjacent tissues. Furthermore, the expression of PLEKHA4 was negatively correlated with ages (χ2=6.394, P=0.01), molecular subtype (χ2=15.606, P=0.001), lymph node metastasis (χ2=13.753, P=0.004), tumor-node-metastasis (TNM) stage (χ2=22.616, P<0.001). Kaplan-Meier curves implicated low expression of PLEKHA4 was associated with worse survival of BC patients [hazard ratio (HR) =0.46, P=0.01]. Cox regression models showed that low PLEKHA4 expression could be an independent risk factor for BC (HR =0.911, P=0.006). The results of gene set enrichment analysis (GSEA) showed that cell cycle, Notch signaling pathway, nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway, and Rho GTPases were highly enriched in the low PLEKHA4 expression group, as identified by GO and KEGG. Additionally, in BC, PLEKHA4 expression displayed a positive correlation with the infiltration of natural killer (NK) cells (P<0.001), CD8+ T cells (P<0.001), B cells (P<0.001), neutrophils (P<0.001), and dendritic cells (DCs) (P<0.001). Conclusions: The findings indicate that PLEKHA4 is an independent prognostic biomarker associated with key signaling pathways and immune infiltration in BC. Targeting PLEKHA4 may contribute to improving immunotherapy for BC.

Decreased mitochondrial-related gene expression in adipose tissue after acute sprint exercise in humans: A pilot study.

The aim was to examine the acute effects of sprint exercise (SIT) on global gene expression in subcutaneous adipose tissue (AT) in healthy subjects, to enhance understanding of how SIT influences body weight regulation. The hypothesis was that SIT upregulates genes involved in mitochondrial function and fat metabolism. A total of 15 subjects performed three 30-s all-out sprints (SIT). Samples were collected from AT, skeletal muscle (SM) and blood (brachial artery and a subcutaneous AT vein) up to 15 min after the last sprint. Results showed that markers of oxidative stress, such as the purines hypoxanthine, xanthine and uric acid, increased markedly by SIT in both the artery and the AT vein. Purines also increased in AT and SM tissue. Differential gene expression analysis indicated a decrease in signaling for mitochondrial-related pathways, including oxidative phosphorylation, electron transport, ATP synthesis, and heat production by uncoupling proteins, as well as mitochondrial fatty acid beta oxidation. This downregulation of genes related to oxidative metabolism suggests an early-stage inhibition of the mitochondria, potentially as a protective mechanism against SIT-induced oxidative stress.

Atorvastatin-Loaded Dissolving Microarray Patches for Long-Acting Microdepot Delivery: Comparison of Nanoparticle and Microparticle Drug Formulations.

The increasing popularity of prolonged-release dosage forms, owing to their ability to provide continuous drug release after administration, has significantly improved patient compliance and overall quality of life. However, achieving prolonged release beyond 24 h frequently requires the use of invasive methods, including injections or implants, which may prove challenging for people suffering from needle phobia. This study introduces atorvastatin (ATR) microparticles (MPs) or nanocrystal (NCs) dissolving microarray patches (D-MAPs) as a noninvasive alternative for intradermal drug delivery over a two-week period for the management of hyperlipidemia. The MP-loaded D-MAPs exhibited an average drug loading of 5.15 ± 0.4 mg of ATR per patch, surpassing the 2.4 ± 0.11 mg/patch observed with NC-loaded D-MAPs. Skin deposition studies demonstrated the superior performance of MP D-MAPs, which delivered 2.0 ± 0.33 mg of ATR per 0.75 cm2 patch within 24 h, representing 38.76% of the initial amount of drug loaded. In contrast, NC D-MAPs delivered approximately 0.89 ± 0.12 mg of ATR per 0.75 cm2 patch at 24 h, equivalent to 38.42 ± 5.13% of the initial ATR loaded. Due to their favorable results, MP D-MAPs were chosen for an in vivo study using Sprague-Dawley rats. The findings demonstrated the capacity of D-MAPs to deliver and attain therapeutically relevant ATR concentrations (>20 ng/mL) for 14 days after a single 24-h application. This study is the first to successfully demonstrate the long-acting transdermal delivery of ATR using MP-loaded D-MAPs after a 24-h single-dose application. The innovative D-MAP system, particularly when loaded with MP, arises as a promising, minimally invasive, long-acting substitute for ATR delivery. This technology has the potential to improve patient compliance and therapeutic outcomes while also significantly advancing the field of transdermal drug delivery.

Optimizing cancer classification: a hybrid RDO-XGBoost approach for feature selection and predictive insights.

The identification of relevant biomarkers from high-dimensional cancer data remains a significant challenge due to the complexity and heterogeneity inherent in various cancer types. Conventional feature selection methods often struggle to effectively navigate the vast solution space while maintaining high predictive accuracy. In response to these challenges, we introduce a novel feature selection approach that integrates Random Drift Optimization (RDO) with XGBoost, specifically designed to enhance the performance of cancer classification tasks. Our proposed framework not only improves classification accuracy but also offers valuable insights into the underlying biological mechanisms driving cancer progression. Through comprehensive experiments conducted on real-world cancer datasets, including Central Nervous System (CNS), Leukemia, Breast, and Ovarian cancers, we demonstrate the efficacy of our method in identifying a smaller subset of unique and relevant genes. This selection results in significantly improved classification efficiency and accuracy. When compared with popular classifiers such as Support Vector Machine, K-Nearest Neighbor, and Naive Bayes, our approach consistently outperforms these models in terms of both accuracy and F-measure metrics. For instance, our framework achieved an accuracy of 97.24% in the CNS dataset, 99.14% in Leukemia, 95.21% in Ovarian, and 87.62% in Breast cancer, showcasing its robustness and effectiveness across different types of cancer data. These results underline the potential of our RDO-XGBoost framework as a promising solution for feature selection in cancer data analysis, offering enhanced predictive performance and valuable biological insights.

PD-1 expression in tumor infiltrating lymphocytes as a prognostic marker in early-stage non-small cell lung cancer.

Introduction: Programmed death ligand - 1 (PD-L1) expression is a well-established predictive biomarker for immunotherapy in non-small cell lung cancer (NSCLC). Programmed death - 1 (PD-1) serves as the target protein to PD-L1 and their interaction serves as a crucial pathway for immune evasion. This study aimed to investigate the expression pattern of PD-1 on Tumor-infiltrating lymphocytes (TILs) in early-stage NSCLC, and its potential role as prognostic biomarker. Materials & methods: PD-1 was evaluated in 474 surgical resected early-stage NSCLC specimens, using Tissue microarray and immunohistochemical staining. Expression was scored as negative (<1%) or positive. Positive PD-1 expression was further divided into low (<10%) and high (≥10%). None of the patients had received treatment with PD-1/PD-L1 inhibitors. Results: PD-1 expression ≥1% in TILs was observed in 83.5% of cases and was associated with pT stage (p=0.02), grade 3 (p=0.004), and adenocarcinoma subtype (p=0.05). Individuals with high PD-1 expression (≥10%) experienced reduced 10-year overall survival (Log-Rank test = 0.005). In addition, high PD-1 expression emerged as an independent factor associated with reduced survival on multivariate analysis (HR: 1.328 (95% CI: 1.074-1.641). Conclusions: Patients with early-stage NSCLC who exhibited PD-1 expression of ≥10% on TILs had an unfavorable 10-year OS rate. These findings indicate that elevated PD-1 expression on TILs can be associated with immune evasion during the early stages of malignancy evolution in the NSCLC setting and further research is required to further delineate the role of PD-1/PD-L1 pathway on tumor immune senescence. These results underline the potential role of PD-1/PD-L1 inhibitors in the treatment of early-stage NSCLC.

Agenesis of the Ductus Venosus and Its Association With Genetic Abnormalities.

OBJECTIVE: To investigate the association of agenesis of the ductus venosus (ADV) with genetic abnormalities using genetic studies-Chromosomal Microarray Analysis (CMA) and Exome Sequencing (ES). DESIGN: Retrospective study of all fetuses diagnosed with ADV between January 2013 and December 2022 in a tertiary center. RESULTS: ADV was diagnosed in 33 fetuses. The diagnosis was made at a mean gestational age of 21.2 ± 8.4 weeks. Conventional karyotype was applied in a single fetus (3.0%), CMA was applied in 21 fetuses (66.7%), and five fetuses (22.8%) were additionally tested with ES. ADV was isolated in eight fetuses (24%), whereas in 25 (76%) it was associated with abnormal ultrasound findings, including increased nuchal translucency (NT), intrauterine growth restriction (IUGR) and variable structural malformations, mostly cardiac (42%) followed by central nervous system (CNS) and skeletal malformations (24%). Genetic abnormalities were found in six fetuses out of 22 investigated (27%), of which 3 were detected by ES, 3 by CMA and 1 by conventional karyotype. A higher incidence of genetic aberrations was evident among ADVs associated with abnormal ultrasound findings. Genetic abnormalities were indicative of Prader Willi/Angelman syndrome, Noonan syndrome, CASK related disorder, 16q24.3 microdeletion syndrome and Trisomy 21. CONCLUSION: ADV associated with abnormal ultrasound findings is commonly correlated with genetic abnormalities and consequently unfavorable pregnancy outcomes. Our study emphasizes the value of genetic studies chiefly among cases associated with abnormal ultrasound findings, enabling early diagnosis of fetal pathologies associated with ADV, and providing better parental counseling.

Microarray-based evaluation of selected recombinant timothy grass allergens expressed in E. Coli and N. Benthamiana.

BACKGROUND: Timothy grass (Phleum pratense) is a significant source of allergens, and recombinant allergens are increasingly used for diagnostic purposes. However, the performance of different recombinant allergen production systems in diagnostic assays needs further investigation to optimize their use in clinical settings. OBJECTIVE: The main objective of this study was to analyze and compare the diagnostic performance of recombinant timothy grass allergens produced in E. coli and N. benthamiana using a custom-made microarray chip. METHODS: Recombinant timothy grass allergens Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 11, and Phl p 12 were produced in E. coli and/or N. benthamiana. A total of 113 patient serum samples were tested to evaluate the diagnostic sensitivity, specificity, inter-assay variability, and correlation of allergen-specific IgE detection compared to commercial multiplex tests (ALEX and ISAC). Additionally, the prevalence of sIgE to these allergens was assessed. RESULTS: Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 11 showed high or very high positive correlation in immunoreactivity with other commercial multiplex tests. Notably, Phl p 11 fused with maltose-binding protein (MBP) demonstrated high diagnostic specificity and sensitivity, with a 0.3 arbitrary cut-off value. However, a high intra-assay variation was observed. The study also assessed specific IgE prevalence to timothy grass allergens within the tested patient cohort. CONCLUSIONS: Recombinant allergens from both E. coli and N. benthamiana demonstrated strong diagnostic potential on the microarray platform, with Phl p 11 (MBP-fused) showing particularly high performance. High intra-assay variation highlights the need for further optimization in allergen formulation and microarray storage conditions. These results highlight the potential of recombinant allergens for diagnostic applications, despite challenges with allergen stability in microarray formats. Specific IgE prevalence to timothy allergens revealed a sensitization profile consistent with findings from multiple studies.

The cytotoxic activities of the major diterpene extracted from Salvia multicaulis (Bardakosh) are mediated by the regulation of heat-shock response and fatty acid metabolism pathways in human leukemia cells.

BACKGROUND: Leukemia is one of the most lethal cancers worldwide and represents the sixth-leading cause of cancer deaths. The results of leukemia treatment have not been as positive as desired, and recurrence is common. PURPOSE: Thus, there is an urgent requirement for the development of new therapeutic drugs. Salvia multicaulis (Bardakosh) is a widespread species that contains multiple phytochemical components with anti-cancer activities. METHODS: We isolated and characterized the major diterpene candesalvone B methyl ester from S. multicaulis and investigated its action as a cytotoxic agent towards sensitive and drug-resistant leukemia cells by the resazurin reduction assay. Additionally, the targeted genes and the affected molecular mechanisms attributed to the potent cytotoxic activities were discovered by transcriptome-wide mRNA expression profiling. The targets predicted to be regulated by candesalvone B methyl ester in each cell line were confirmed by qRT-PCR, molecular docking, microscale thermophoresis, and western blotting. Moreover, cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: Candesalvone B methyl ester was cytotoxic with IC50 values of 20.95 ± 0.15 µM against CCRF-CEM cells and 4.13 ± 0.10 µM against multidrug-resistant CEM/ADR5000 leukemia cells. The pathway enrichment analysis disclosed that candesalvone B methyl ester could regulate the heat-shock response signaling pathway via targeting heat shock factor 1 (HSF1) in CCRF-CEM cells and ELOVL fatty acid elongase 5 (ELOVL5) controls the fatty acid metabolism pathway in CEM/ADR5000 cells. Microscale thermophoresis showed the binding of candesalvone B methyl ester with HSF1 and ELOVL5, confirming the results of molecular docking analysis. Down-regulation of both HSF1 and ELOVL5 by candesalvone B methyl ester as detected by both western blotting and RT-qPCR was related to the reversal of drug resistance in the leukemia cells. Furthermore, candesalvone B methyl ester increased the arrest in the sub-G1 phase of the cell cycle in a dose-dependent manner from 1.3 % to 32.3 % with concomitant induction of apoptosis up to 29.0 % in CCRF-CEM leukemic cells upon inhibition of HSF1. CONCLUSION: Candesalvone B methyl ester isolated from S. multicaulis exerted cytotoxicity by affecting apoptosis, cell division, and modulation of expression levels of genes contributing to the heat stress signaling and fatty acid metabolism pathways that could relieve drug resistance of leukemia cells.

Analytical Performance of a Multiplexed Microarray Assay for Rapid Identification and Quantification of a Multivalent mRNA Vaccine.

mRNA vaccines were highly effective in response to the COVID-19 pandemic, making them an attractive platform to address cancers and other infectious diseases. Many new mRNA vaccines in development are multivalent, which represents a difficulty for the standard assays commonly used to characterize the critical quality attributes of monovalent formulations. Here, we present a multiplexed analytical tool with nucleic acid microarray technology using the VaxArray platform that measures the identity and quantity of mono- and multivalent mixtures of naked mRNA and mRNA encapsulated in lipid nanoparticle formulations in under 2 h without any additional preparation steps, such as extraction or RT-PCR. Using a quadrivalent mixture of encapsulated mRNA constructs that encode for four unique proteins in a vaccine formulation, the VaxArray mRNA assay was demonstrated to be highly specific for each mRNA with sensitivity < 1 µg/mL. The quantification of individual mRNAs within the lipid nanoparticle mixture resulted in a precision of ≤10% RSD and an accuracy of 100 ± 9%.

Hepatitis C virus infection and Parkinson's disease: insights from a joint sex-stratified BioOptimatics meta-analysis.

Hepatitis C virus (HCV) infection poses a significant public health challenge and often leads to long-term health complications and even death. Parkinson's disease (PD) is a progressive neurodegenerative disorder with a proposed viral etiology. HCV infection and PD have been previously suggested to be related. This work aimed to identify potential biomarkers and pathways that may play a role in the joint development of PD and HCV infection. Using BioOptimatics-bioinformatics driven by mathematical global optimization-, 22 publicly available microarray and RNAseq datasets for both diseases were analyzed, focusing on sex-specific differences. Our results revealed that 19 genes, including MT1H, MYOM2, and RPL18, exhibited significant changes in expression in both diseases. Pathway and network analyses stratified by sex indicated that these gene expression changes were enriched in processes related to immune response regulation in females and immune cell activation in males. These findings suggest a potential link between HCV infection and PD, highlighting the importance of further investigation into the underlying mechanisms and potential therapeutic targets involved.