Enhancing cancer immunotherapy with Anti-NKG2D/IL-15(N72D)/Sushi fusion protein: Targeting cytotoxic immune cells and boosting IL-15 efficacy.

BACKGROUND: There are a number of distinct challenges and complexities associated with administering IL-15 for cancer immunotherapy that must be taken into consideration. OBJECTIVE: The purpose of this study was to design a fusion protein for targeting cytotoxic immune cells and enhance IL-15 efficiency. METHODS: A fusokine that contains IL-15(N72D), a Sushi domain, and anti-NKG2D scFv was designed. The fusion protein was in-silico modeled using the Swiss model server, followed by docking and molecular dynamics simulations. The in-vitro purified fusokine was evaluated using dot blot and Western blot. Then, flow cytometry was employed to evaluate biological properties such as proliferation, cytotoxicity, and degranulation. RESULTS: Fusokine and IL-15(N72D)/Sushi, which had molecular weights of about 52 kDa and 26 kDa, respectively, were expressed in CHO-K1 cells. The fusokine binds 69.6 % of the CHO-NKG2D+ cells that express 83.1 % NKG2D. Both the fusokine and the IL-15(N72D)/Sushi significantly stimulate the proliferation of lymphocytes. After 14 days of growth, the vitality of untreated cells decreased to about 17.5 %, but 82.2 % and 56.6 % of cells were still alive when fusokine and IL-15(N72D)/Sushi were present. Furthermore, administration of fusokine was associated with the highest rates of target tumor cell cytotoxicity. Additionally, although it was not statistically significant, fusokine increased the expression of CD107a and granzyme B by 1.25 times and 2.4 times, respectively. CONCLUSION: The fusokine possesses the capability to stimulate the survival and multiplication of lymphocytes, as well as their ability to eliminate tumors. These characteristics have led to its consideration as a potential treatment for immunotherapy.

Unveiling promising breast cancer biomarkers: an integrative approach combining bioinformatics analysis and experimental verification.

BACKGROUND: Breast cancer remains a significant health challenge worldwide, necessitating the identification of reliable biomarkers for early detection, accurate prognosis, and targeted therapy. MATERIALS AND METHODS: Breast cancer RNA expression data from the TCGA database were analyzed to identify differentially expressed genes (DEGs). The top 500 up-regulated DEGs were selected for further investigation using random forest analysis to identify important genes. These genes were evaluated based on their potential as diagnostic biomarkers, their overexpression in breast cancer tissues, and their low median expression in normal female tissues. Various validation methods, including online tools and quantitative Real-Time PCR (qRT-PCR), were used to confirm the potential of the identified genes as breast cancer biomarkers. RESULTS: The study identified four overexpressed genes (CACNG4, PKMYT1, EPYC, and CHRNA6) among 100 genes with higher importance scores. qRT-PCR analysis confirmed the significant upregulation of these genes in breast cancer patients compared to normal samples. CONCLUSIONS: These findings suggest that CACNG4, PKMYT1, EPYC, and CHRNA6 may serve as valuable biomarkers for breast cancer diagnosis, and PKMYT1 may also have prognostic significance. Furthermore, CACNG4, CHRNA6, and PKMYT1 show promise as potential therapeutic targets. These findings have the potential to advance diagnostic methods and therapeutic approaches for breast cancer.

Exploring heterogeneous expression of beta-actin (ACTB) in bladder cancer by producing a monoclonal antibody 6D6.

BACKGROUND: To predict outcomes and identify potential therapeutic targets for cancers, it is critical to find novel specific biomarkers. The objective of this study was to search for and explore novel bladder cancer-associated protein biomarkers. METHODS: A library of monoclonal antibodies (mAbs) against the JAM-ICR cell line was first generated, and clones with high affinity were selected. Hybridomas were screened using bladder cancer (BLCA) cell lines and normal cells. The target of the selected mAb was then characterized through immunoaffinity purification, western blotting, and mass spectrometry analysis. Expression of the target antigen was assessed by flow cytometry and IHC methods. Several databases were also used to evaluate the target antigen in BLCA and other types of cancers. RESULTS: Based on screenings, a 6D6 clone was selected that recognized an isoform of beta-actin (ACTB). Our data showed that ACTB expression on different cell lines was heterogeneous and varied significantly from low to high intensity. 6D6 bound strongly to epithelial cells while showing weak to no reactivity to stromal, endothelial, and smooth muscle cells. There was no association between ACTB intensity and related prognostic factors in BLCA. In silico evaluations revealed a significant correlation between ACTB and overexpressed genes and biomarkers in BLCA. Additionally, the differential expression of ACTB in tumor and healthy tissue as well as its correlation with survival time in a number of cancers were shown. CONCLUSIONS: The heterogeneous expression of ACTB may suggest the potential value of this marker in the diagnosis or prognosis of cancer.

Production of a biosimilar version of aflibercept to improve VEGF blocker cytotoxicity on endothelial cells.

This project aimed to produce a biosimilar version of aflibercept (AFL) and evaluate the effect of the co-treatment of AFL with other vascular endothelial growth factor (VEGF) blocker drugs. For this purpose, the optimized gene was inserted into the pCHO1.0 plasmid and transfected into the CHO-S cell line. The final concentration of biosimilar-AFL for the selected clone was 782 mg/L. Results revealed that the inhibition potential of the biosimilar-AFL on HUVEC cells was significant at 10 and 100 nM concentrations and in a dose-dependent manner. Furthermore, co-treatment of biosimilar-AFL with Everolimus (EVR), Lenvatinib (LEN), and Sorafenib (SOR) could reduce HUVEC cell viability/proliferation, more than when used alone. When LEN and SOR were co-treated with biosimilar-AFL, their cytotoxicity increased 10-fold. The most and least efficient combination was seen when biosimilar-AFL combined with LEN and EVR, respectively. Finally, biosimilar-AFL may improve the efficiency of LEN, EVR, and SOR in reducing the VEGF effect on endothelial cells.

Anti-CD19/CD8 bispecific T cell engager for the potential treatment of B cell malignancies.

The administration of blinatumomab was accompanied by several adverse effects, including activation of regulatory T-cells and cytokine storm. The objective of this study was to produce and evaluate a novel αCD8/CD19 BiTE (αCD8/CD19) with the potency to directly target CD8+T-cells. In-silico studies were utilized for determining proper folding, receptor binding, and structural stability of αCD8/CD19 protein. Western blotting and indirect surface staining were used to evaluate the size accuracy and binding potency of the purified protein. Functionality was assessed for granzyme B production, cytotoxicity, and proliferation. TheαCD8/CD19recombinant protein was produced in the CHO-K1 cell line with a final concentration of 1.94 mg/l. The αCD8/CD19 bound to CD8+and CD19+cell lines and induced significant granzyme B production, cytotoxic activity and proliferation potential in the presence of IL-2 and tumor target cells. The maximum CD8+T-cell biological activity was observed on the 10th day with 10:1 effector-to-target ratio.

Association of IFITM1 Promoter Methylation with Severity of SARS-CoV-2 Infection.

BACKGROUND: During viral infections such as SARS-CoV-2, epigenetic changes within the promoter region of the immune system genes would possibly occur and have an effect on the immune system response as well as disease outcome. We aimed to evaluate and compare the methylation level of the IFITM1 gene promoter in different stages of COVID-19 disease with a healthy control group. METHODS: In this cross-sectional study, 75 COVID-19 patients (25 mild, 25 severe, and 25 critical in addition to 25 age- and gender-matched healthy volunteers) have been included. DNA was extracted from the peripheral white blood cells using a commercial DNA extraction kit. PCR was performed using two types of primers designed for the methylated and unmethylated forms of the IFITM1 gene promoter. RESULTS: The mean age of the patient and healthy volunteer groups was 52.733 ± 13.780 and 49.120 ± 12.490, respectively. Out of a hundred participants, 52 were male. The results demonstrated that severe (p = 0.03, OR 6.729) and critical (p = 0.001, OR 11.156) patients were much more likely to show methylation of the IFITM1 gene in contrast with mild patients. Moreover, IFITM1 methylation was significantly higher in COVID-19 patients in comparison with the healthy volunteer group (p = 0.004, OR 3.17). Furthermore, IFITM1 methylation in male patients with critical status, (p = 0.01) was significantly higher than in male patients with mild status. In addition, IFITM1 methylation of male (p = 0.03) and female (p = 0.01) critical patients was considerably higher compared to males and females of volunteer group. CONCLUSIONS: Increased methylation of the IFITM1 gene in the severe and critical stage of COVID-19 diseases may indicate the role of SARS-CoV-2 infection in increasing methylation of this antiviral gene. This might be involved in suppressing the immune system, promoting SARS-CoV-2 replication and disease outcome.

Specific Targeting of Recombinant Human Pancreatic Ribonuclease 1 using Gonadotropin-Releasing Hormone Targeting Peptide toward Gonadotropin-Releasing Hormone Receptor-Positive Cancer Cells.

Background: Targeted drug delivery is a novel method to specifically deliver anticancer therapeutics to tumor sites. Gonadotropin-releasing hormone (GnRH) is a decapeptide, and its target binding property has attracted attention as a means of targeted drug delivery. Human pancreatic ribonuclease 1 (hpRNase1) has been shown to exert anticancer properties, when fused to a targeting moiety. The goal of the present study was to add a GnRH targeting peptide to the N-terminus of hpRNase1 to specifically target GnRH receptor (GnRH-R) expressing cells. Methods: This in vitro study was conducted at Shiraz Institute for Cancer Research (Shiraz, Iran) in 2019. The coding sequence of GnRH and hpRNase1 were fused, and the chimeric protein together with non-fused hpRNase1 were produced in E. coli (BL21). The recombinant proteins were purified, and their biological activity was evaluated using MTT and apoptosis assays. Non-parametric Kruskal-Wallis tests with Dunn's post hoc tests were performed to determine the significant differences between the study groups. Results: GnRH-hpRNase1 chimeric protein specifically inhibited the proliferation of PC-3 (P=0.021), LNCaP (P=0.034), and AD-Gn (P=0.041) cells, while the growth of negative cells (AD-293) was not significantly affected (P=0.081). GnRH-hpRNase1 decreased the IC50 values more than non-fused hpRNase1, by approximately 26.5-fold (P=0.036) for PC-3 cells, and exerted its growth inhibitory effects through apoptosis induction. Conclusion: Fusion of GnRH to hpRNase1 structure produced an enzyme, which could specifically target tumor cells. This approach can be used to eliminate tumors that harbor GnRH-R.

Serodiagnosis of human cystic echinococcosis based on recombinant antigens B8/1 and B8/2 of Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis (CE) is a widespread parasitic disease caused by the larval stage of Echinococcus granulosus. Since current methods for the diagnosis of CE are not efficient enough, rapid, and reliable tests are required for the acceleration of CE diagnosis. The present study aimed to produce recombinant B8/1 and B8/2 antigens of E. granulosus and evaluate their sensitivities and specificities separately and simultaneously for the diagnosis of CE. METHODS: The recombinant B8/1 and B8/2 antigens were produced and used in an ELISA system for the diagnosis of CE. The sera specimens including 30 sera from pathologically confirmed CE patients, 30 from other non-CE patients, and 30 from healthy controls, were evaluated by the ELISA, using AgB8/1 and AgB8/2. RESULTS: The results showed a sensitivity of 93.33%, 90%, and 96.7% for AgB8/1, AgB8/2, and their combination, respectively. The specificities were 91.7%, 93.33%, and 93.33% for AgB8/1, AgB8/2, and their combination, respectively. CONCLUSION: Simultaneous usage of AgB8/1 and AgB8/2 increased the test sensitivity for the diagnosis of CE. Furthermore, the specificity of AgB8/1 and AgB8/2 combination was more than AgB8/1 and equal to AgB8/2 alone. The findings revealed that the simultaneous usage of AgB8/1 and AgB8/2 could be a suitable approach for the diagnosis of CE.

In vitro cytotoxic effect of Trastuzumab in combination with Pertuzumab in breast cancer cells is improved by interleukin-2 activated NK cells.

Targeting erb-b2 receptor tyrosine kinase 2 (ERBB2) using the combination of Trastuzumab and Pertuzumab has demonstrated promising results in breast cancer therapy. It has further been revealed that interleukin-2 (IL-2) can activate Natural Killer cells (NK cells) and elevate their cytotoxic potency against tumor cells. In this study, we explored the cytotoxic effect of recombinant human IL-2 in combination with Trastuzumab and Pertuzumab on the ERBB2 positive (SK-BR-3) and negative (MDA-MB-231) breast cancer cell lines. The cytotoxicity level of IL-2 activated NK cells (approximately 75%) were significantly higher than untreated cells (approximately 55%) in the presence of Trastuzumab and Pertuzumab against SK-BR-3 cells, while no difference was observed in the case of MDA-MB-231 cells (about 15%).

Production and characterization of monoclonal antibody against a triple negative breast cancer cell line.

Breast cancer is the most prevalent malignancy among women around the world such that more than 1,400,000 new cases are being diagnosed each year. Despite immense studies over many years on diagnosis and treatment of breast cancer, about 30% of treated patients will relapse and require subsequent therapy. By development of hybridoma technology, murine monoclonal antibodies (MAbs) against several human tumor-associated antigens have been produced and characterized in many laboratories. The purpose of these studies is to generate effective monoclonal antibodies that could be useful in tumor diagnosis and therapy. In this study, splenic lymphocytes of immunized BALB/c mouse with a new established breast cancer cell line (Pari-ICR cell line, established in Shiraz Institute for Cancer Research) were fused with the mouse myeloma cell line SP2/0 in the presence of polyethylene glycol. We generated a panel of monoclonal antibodies against the newly established cell line. The hybrid cultures were screened by flow cytometry. Hybridomas that produced antibody to surface antigens of immunizing cell line but not to Human Gingival Fibroblasts, adipose stem cells, and leucocytes isolated from peripheral blood were selected and cloned by limiting dilution method. The 1E3 clone (IgG2a type) that displayed clonal stability was further analyzed for specificity by flow cytometry. MAb 1E3 showed weak to strong reactivity to other cell lines compared with Pari-ICR cell line. Antigen identification was performed by a workflow consisting of immunoaffinity purification, SDS-PAGE, Western blotting, and mass spectrometry analysis. The target of 1E3 mAb was identified as NCAM1. In conclusion, using the antibody-based strategy we identified NCAM1 as a potential therapeutic target and biomarker for breast cancer.

Improving Pertuzumab production by gene optimization and proper signal peptide selection.

Using proper signal peptide and codon optimization are important factors that must be considered when designing the vector to increase protein expression in Chinese Hamster Ovary (CHO) cells. The aim of the present study is to investigate how to enhance Pertuzumab production through heavy and light chain coding gene optimization and proper signal peptide selection. First, CHO-K1 cells were transiently transfected with whole-antibody-gene-optimized, variable-regions-optimized and non-optimized constructs and then we employed five different signal peptides to improve the secretion efficiency of Pertuzumab. Compared to the native antibody gene, a 3.8 fold increase in Pertuzumab production rate was achieved with the whole heavy and light chain sequence optimization. Although an overall two fold increase in monoclonal antibody production was achieved by human albumin signal peptide compared to the control signal peptide, this overproduction was not statistically significant. Selected signal peptides had no effect on the binding of Pertuzumab to the ErbB2 antigen. The combined data indicate that human albumin signal peptide along with whole antibody sequence optimization can be used to improve Pertuzumab production rates. This sequence was used to produce Pertuzumab producing CHO-K1 stably transfected cells. This result is useful for producing Pertuzumab as a biosimilar drug.

Metformin Exhibits Anti-Inflammatory Effects by Regulating microRNA-451/CXCL16 and B Cell Leukemia/Lymphoma 2 in Patients With Osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is the most common cause of chronic disability in joints among older individuals. The primary goal of OA treatment is pain relief to improve the quality of life. Inflammation and aging are involved in the pathogenesis of pain in OA. In this study, we evaluated the ability of metformin to regulate microRNAs, such as miR-451 and miR-15b, and their target proteins, CXCL16 and B cell leukemia/lymphoma 2 (BCL-2), involved in inflammation and apoptosis. METHODS: In this double-blind placebo-controlled clinical trial, patients were randomly divided into two groups: one receiving metformin and the other receiving a placebo for four months (starting at 0.5 g/day for the first week, increasing to 1 g/day for the second week, and increasing to 1.5 g/day for the remaining period). In addition to evaluating the clinical response using the Knee Injury and Osteoarthritis Outcome Score questionnaire, miR-451 and miR-15b expression levels were detected using real-time polymerase chain reaction. The serum levels of CXCL16 and BCL-2 were evaluated using enzyme-linked immunosorbent assay kits before (time zero) and after treatment (month four). RESULTS: Metformin increased miR-451 expression levels simultaneously with pain reduction, whereas miR-15b expression did not change significantly after four months of treatment. Also, metformin decreased the serum levels of BCL-2 and CXCL16 in patients with OA. CONCLUSION: The effects of metformin in reducing pain can be attributed to many factors, including its anti-inflammatory and antiaging effects. Our findings suggest that metformin may reduce pain and inflammation in patients with OA through the regulation of miR-451/CXCL16 and BCL-2.

A comprehensive meta-analysis of transcriptome data to identify signature genes associated with pancreatic ductal adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) has a five-year survival rate of less than 5%. Absence of symptoms at primary tumor stages, as well as high aggressiveness of the tumor can lead to high mortality in cancer patients. Most patients are recognized at the advanced or metastatic stage without surgical symptom, because of the lack of reliable early diagnostic biomarkers. The objective of this work was to identify potential cancer biomarkers by integrating transcriptome data. METHODS: Several transcriptomic datasets comprising of 11 microarrays were retrieved from the GEO database. After pre-processing, a meta-analysis was applied to identify differentially expressed genes (DEGs) between tumor and nontumor samples for datasets. Next, co-expression analysis, functional enrichment and survival analyses were used to determine the functional properties of DEGs and identify potential prognostic biomarkers. In addition, some regulatory factors involved in PDAC including transcription factors (TFs), protein kinases (PKs), and miRNAs were identified. RESULTS: After applying meta-analysis, 1074 DEGs including 539 down- and 535 up-regulated genes were identified. Pathway enrichment analyzes using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that DEGs were significantly enriched in the HIF-1 signaling pathway and focal adhesion. The results also showed that some of the DEGs were assigned to TFs that belonged to 23 conserved families. Sixty-four PKs were identified among the DEGs that showed the CAMK family was the most abundant group. Moreover, investigation of corresponding upstream regions of DEGs identified 11 conserved sequence motifs. Furthermore, weighted gene co-expression network analysis (WGCNA) identified 8 modules, more of them were significantly enriched in Ras signaling, p53 signaling, MAPK signaling pathways. In addition, several hubs in modules were identified, including EMP1, EVL, ELP5, DEF8, MTERF4, GLUP1, CAPN1, IGF1R, HSD17B14, TOM1L2 and RAB11FIP3. According to survival analysis, it was identified that the expression levels of two genes, EMP1 and RAB11FIP3 are related to prognosis. CONCLUSION: We identified several genes critical for PDAC based on meta-analysis and system biology approach. These genes may serve as potential targets for the treatment and prognosis of PDAC.

Anti-PD-L1 chimeric antigen receptor natural killer cell: Characterization and functional analysis.

Like their natural counterparts, chimeric antigen receptor-engineered cells are prone to suppression by inhibitory signals, such as PD-L1, expressed by tumors or suppressor cells in the tumor microenvironment. Consequently, they become impaired, resulting in immune cell exhaustion, tumor progression, and resistance to other therapies. In this study, we developed an anti-PD-L1-CAR NK cell with efficient activity and a notable PD-L1-specific response toward tumor cell lines. The degranulation assay demonstrated that CD107a frequencies between the PD-L1med and PD-L1high groups and between Herceptin-treated and non-treated groups were not statistically different. Further investigation into NK cell characterization, considering different markers such as CD57, KIR2D, and CD25, revealed that the majority of the population are activated expanding NK cells. At the same time, immune checkpoint inhibitors, including PD-1, PD-L1, and LAG-3, showed increased levels following activation and expansion. Regarding the efficient functional activity of PD-L1-CAR NK cells and the instinctive receptor balance-based response of NK cells, this observation could point to the inhibition of NK cell overactivation or even higher cytotoxicity and cytokine production rather than exhaustion, especially in the case of healthy NK cells. These findings can contribute to a better understanding of the potential and challenges of using primary NK cells for CAR-NK cell therapy.

Effects of COVID-19 on Sleep Services Use and Its Recovery.

Purpose: The COVID-19 pandemic affected the utilization of various healthcare services differentially. Sleep testing services utilization (STU), including Home Sleep Apnea Testing (HSAT) and Polysomnography (PSG), were uniquely affected. We assessed the effects of the pandemic on STU and its recovery using the Veterans Health Administration (VHA) data. Patients and Methods: A retrospective cohort study from the VHA between 01/2019 and 10/2023 of veterans with age ≥ 50. We extracted STU data using Current Procedural Terminology codes for five periods based on STU and vaccination status: pre-pandemic (Pre-Pan), pandemic sleep test moratorium (Pan-Mor), and pandemic pre-vaccination (Pan-Pre-Vax), vaccination (Pan-Vax), and postvaccination (Pan-Post-Vax). We compared STU between intervals (Pre-Pan as the reference). Results: Among 261,371 veterans (63.7±9.6 years, BMI 31.9±6.0 kg/m², 80% male), PSG utilization decreased significantly during Pan-Mor (-56%), Pan-Pre-Vax (-61%), Pan-Vax (-42%), and Pan-Post-Vax (-36%) periods all compared to Pre-Pan. HSAT utilization decreased significantly during the Pan-Mor (-59%) and Pan-Pre-Vax (-9%) phases compared to the Pre-Pan and subsequently increased during Pan-Vax (+6%) and Pan-Post-Vax (-1%) periods. Over 70% of STU transitioned to HSAT, and its usage surged five months after the vaccine Introduction. Conclusion: Sleep testing services utilization recovered differentially during the pandemic (PSG vs HSAT), including a surge in HSAT utilization post-vaccination.

Breast cancer immunotherapy using scFv antibody-based approaches, a systematic review.

Breast cancer is considered as the most common malignancy in women and the second leading cause of death related to cancer. Recombinant DNA technologies accelerated the development of antibody-based cancer therapy, which is effective in a broad range of cancers. The objective of the present study was to perform a systematic review on breast cancer immunotherapy using single-chain fragment variable (scFv) antibody formats. Searches were performed up to March 2023 using PubMed, Scopus, and Web of Science (ISI) databases. Three reviewers independently assessed study eligibility, data extraction, and evaluated the methodological quality of included primary studies. Different immunotherapy approaches have been identified and the most common approaches were scFv-conjugates, followed by simple scFvs and chimeric antigen receptor (CAR) therapy, respectively. Among breast cancer antigens, HER superfamily, CD family, and EpCAM were applied as the most important breast cancer immunotherapy targets. The present study shed more lights on scFv-based breast cancer immunotherapy approaches.

Age modifies the association between severe sleep apnea and all-cause mortality.

PURPOSE: While sleep apnea (SA) gets more prevalent with advancing age, the impact of age on the association between SA and health outcomes is not well known. We assessed the association between the severity of SA and all-cause mortality in different age groups using large longitudinal data. METHOD: We applied a Natural Language Processing pipeline to extract the apnea-hypopnea index (AHI) from the physicians' interpretation of sleep studies performed at the Veteran Health Administration (FY 1999-2022). We categorized the participants as no SA (n-SA, AHI< 5) and severe SA (s-SA, AHI≥30). We grouped the cohort based on age: Young≤40; Middle-aged:40-65; and Older adults≥65; and calculated the odds ratio (aOR) of mortality adjusted for age, sex, race, ethnicity, BMI, and Charlson-Comorbidity Index (CCI) using n-SA as the reference. RESULTS: We identified 146,148 participants (age 52.23 ± 15.02; BMI 32.11 ± 6.05; male 86.7 %; White 66 %). Prevalence of s-SA increased with age. All-cause mortality was lower in s-SA compared to n-SA in the entire cohort (aOR,0.56; 95%CI: 0.54,0.58). Comparing s-SA to n-SA, the all-cause mortality rates (Young 1.86 % vs 1.49 %; Middle-aged 12.07 % vs 13.34 %; and Older adults 26.35 % vs 40.18 %) and the aOR diminished as the age increased (Young: 1.11, 95%CI: 0.93-1.32; Middle-aged: 0.64, 95%CI: 0.61-0.67; and Older adults: 0.44, 95%CI: 0.41-0.46). CONCLUSION: The prevalence of severe SA increased while the odds of all-cause mortality compared to n-SA diminished with age. SA may exert less harmful effects on the aged population. A causality analysis is warranted to assess the relationship between SA, aging, and all-cause mortality.

Age modulates the predictive value of self-reported sleepiness for all-cause mortality risk: insights from a comprehensive national database of veterans.

STUDY OBJECTIVES: Excessive daytime sleepiness is prevalent and overwhelmingly stems from disturbed sleep. We hypothesized that age modulates the association between excessive daytime sleepiness and increased all-cause mortality. METHODS: We utilized the Veterans' Health Administration data from 1999-2022. We enrolled participants with sleep related International Classification of Diseases 9/10 codes or sleep services. A natural language processing pipeline was developed and validated to extract the Epworth Sleepiness Scale (ESS) as a self-reported tool to measure excessive daytime sleepiness from physician progress notes. The natural language processing's accuracy was assessed through manual annotation of 470 notes. Participants were categorized into normal-ESS (ESS 0-10) and high-ESS (ESS 11-24). We created 3 age groups: < 50 years, 50 to < 65 years, and ≥ 65 years. The adjusted odds ratio of mortality was calculated for age, body mass index, sex, race, ethnicity, and the Charlson Comorbidity Index, using normal-ESS as the reference. Subsequently, we conducted age stratified analysis. RESULTS: The first ESS records were extracted from 423,087 veterans with a mean age of 54.8 (± 14.6), mean body mass index of 32.6 (± 6.2), and 90.5% male. The adjusted odds ratio across all ages was 17% higher (1.15, 1.19) in the high-ESS category. The adjusted odds ratio s only became statistically significant for individuals aged ≥ 50 years in the high-ESS compared to the normal-ESS category (< 50 years: 1.02 [0.96, 1.08], 50 to < 65 years 1.13[1.10, 1.16]; ≥ 65 years: 1.25 [1.21, 1.28]). CONCLUSIONS: High-ESS predicted increased mortality only in participants aged 50 and older. Further research is required to identify this differential behavior in relation to age. CITATION: Maghsoudi A, Azarian M, Sharafkhaneh A, et al. Age modulates the predictive value of self-reported sleepiness for all-cause mortality risk: insights from a comprehensive national database of veterans. J Clin Sleep Med. 2024;20(11):1785-1792.

Immunological mechanisms of the nucleocapsid protein in COVID-19.

The emergence of corona virus disease 2019 (COVID-19), resulting from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has left an indelible mark on a global scale, causing countless infections and fatalities. This investigation delves into the role of the SARS-CoV-2 nucleocapsid (N) protein within the HEK293 cells, shedding light on its influence over apoptosis, interferon signaling, and cytokines production. The N gene was amplified, inserted into the pAdTrack-CMV vector, and then transfected to the HEK293 cells. Changes in the expression of IRF3, IRF7, IFN-ß, BAK, BAX, and BCL-2 genes were evaluated. The levels of proinflammatory cytokines of IL-6, IL-12, IL-1ß, and TNF-α were also determined. The N protein exhibited an anti-apoptotic effect by modulating critical genes associated with apoptosis, including BAK, BAX, and BCL-2. This effect potentially prolonged the survival of infected cells. The N protein also played a role in immune evasion by suppressing the interferon pathway, evidenced by the downregulation of essential interferon regulatory factors of IRF3 and IRF7, and IFN-ß expression. The N protein expression led to a substantial increase in the production of proinflammatory cytokines of IL-6, IL-12, IL-1ß, and TNF-α. The N protein emerged as a versatile factor and was exerted over apoptosis, interferon signaling, and cytokine production. These findings carry potential implications for the development of targeted therapies to combat COVID-19 and mitigate its global health impact.

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans.

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression of aflibercept in a plant-based system with targeted N-glycosylation profiles. Nicotiana benthamiana-based glycoengineering resulted in the production of aflibercept variants carrying designed carbohydrates, namely, N-glycans with terminal GlcNAc and sialic acid residues, herein referred to as AFLIGnGn and AFLISia, respectively. Both variants were transiently expressed in unusually high amounts (2 g/kg fresh leaf material) in leaves and properly assembled to dimers. Mass spectrometric site-specific glycosylation analyses of purified aflibercept showed the presence of two to four glycoforms in a consistent manner. We also demonstrate incomplete occupancy of some glycosites. Both AFLIGnGn and AFLISia displayed similar binding potency to VEGF165, with a tendency of lower binding to variants with increased sialylation. Collectively, we show the expression of functionally active aflibercept in significant amounts with controlled glycosylation. The results provide the basis for further studies in order to generate optimized products in the best-case scenario.