Response-related Proteins Research Articles

MUC1 and MUC4 expression are inversely correlated and trigger immunological response and transport pathways in adult-type diffuse gliomas.

Adult-type diffuse gliomas arise from glial or progenitor cells. These tumors are currently classified as astrocytoma isocitrate dehydrogenase (IDH)-mutant or IDH-mutant oligodendroglioma with co-deletion of chromosomal arms 1p and 19q, both of which could be either slow-growing tumors, or glioblastoma (GBM), which is a more aggressive tumor. Despite advances in diagnosis and treatment, the median survival time after GBM diagnosis remains low at approximately 15 months, with a 5-year overall survival (OS) rate of 6.8%. Therefore, new biomarker and therapeutic target discoveries are required to improve prognosis. Mucin 1 (MUC1) and MUC4 are membrane-bound mucins and potential biomarkers of several tumors. However, the role of these mucins in adult gliomas has not been well explored. In this retrospective study, in silico analysis of data from patients with adult-type diffuse glioma revealed differential methylation and expression patterns of MUC1 and MUC4 between GBM and non-GBM groups. In the GBM group, decreased methylation and elevated expression of MUC1 were observed (r=-0.25, p<0.0001), whereas increased methylation and decreased expression of MUC4 were observed (r=-0.13, p=0.1344). Conversely, in the non-GBM group, MUC1 exhibited higher methylation and lower expression (r=-0.27, p<0.0001), whereas MUC4 showed lower methylation and higher expression (r=-0.32, p<0.0001). The expression of these genes influenced OS in adult patients with glioma (p=0.0344), with high MUC1 and low MUC4 expression associated with worse OS. MUC1 and MUC4 expression correlated with that of MUC20 in both GBM (r=0.54) and non-GBM (r=0.53) groups (p<0.0001). Functional enrichment analysis identified the biological roles of MUC1-co-expressed genes as involvement in innate immunity, antigen processing, and proinflammatory responses in both the non-GBM and GBM groups, and integrin-based signaling pathways in the GBM group. MUC4-co-expressed genes are involved in ion transport in GBM patients. Using molecular docking, we observed that MUC1 domains physically interact with immune response-related proteins, such as receptors for advanced glycation end products (RAGE), major histocompatibility complex II (MHC-II), and extracellular matrix receptor integrin alpha 2 (ITGA2). To our knowledge, this is the first retrospective study and in silico analysis demonstrating the relevance and correlation of MUC1 and MUC4 in adult gliomas. These findings elucidate the molecular mechanisms underlying adult-type diffuse glioma progression and highlight MUC1 and MUC4 as potential prognostic markers and therapeutic targets for glioma management.

M1 polarization of hepatic macrophages in cows with subclinical ketosis is an important cause of liver injury.

Subclinical ketosis (SCK) is highly prevalent and easily overlooked, with insidious and slow progression of hepatic injury, often characterized by an imbalance in immune homeostasis. In nonruminants, macrophage polarization plays an important regulatory role in hepatic lipid accumulation, fibrosis and inflammatory processes. Thus, we aimed to investigate the status of hepatic macrophage polarization in SCK cows and to corroborate its association with liver injury and inflammation. Twelve Holstein dairy cows (parity, 2-4) were selected, and liver biopsy and blood were collected on the second week postpartum (10-14 d days in milk). On the basis of serum BHBA concentrations., selected cows were categorized into healthy (n = 6; BHBA <1.0 mM) and SCK (n = 6; 1.2 mM ≤ BHBA < 3.0 mM) groups. Serum biochemical parameters were measured using an automatic biochemical analyzer, which indicated higher serum levels of BHBA and NEFA and an upregulation of liver injury indicators (AST, ALT, TP, GLB) in SCK cows compared with healthy cows. ELISA assays revealed that SCK cows displayed systemic low-grade inflammation, as demonstrated by increased serum levels of HP, SAA, TGF-β, IFN-γ, and IL-1β. Liver biopsies revealed pathological histological alterations, hepatic inflammation, and macrophage polarization status. Oil red staining indicated steatosis, while Sirius red staining demonstrated mild extracellular matrix deposition in the liver of SCK cows. The expression of inflammatory response-related proteins (TLR4, p-NFκB, p-I-κB, NLRP3, and Caspase-1) was elevated in the liver of SCK cows, with the increased mean fluorescence intensity of NFκB further confirming the activation of the inflammatory pathway. Furthermore, the levels of pro-inflammatory factors, TNF-α and IFN-γ, were elevated in the tissue homogenate. Macrophage phenotypic changes in SCK cows were further explored based on the results of liver injury and inflammation. Compared with healthy cows, the protein and mRNA abundance of the macrophage marker CD68 in the liver of SCK cows was higher, along with an increased mean fluorescence intensity of CD68. SCK cows also exhibited reduced mRNA expression of the Kupffer cell marker CLEC4F and elevated chemokine levels (CXCL1 and CCL2). As evidenced by greater protein and mRNA abundance of macrophage M1 polarization markers (iNOS, IL-1β, CD86, IL-6, IL-12b, and CCL3), higher fluorescence intensity of iNOS and CD86, and an increased number of CD68+/CD86+-positive cells observed via immunofluorescence, the macrophage polarization phenotype in the liver of SCK cows was predominantly M1. In contrast, the protein and mRNA abundances of M2 polarization markers (CD206, IL-10, and Arg1) were lower in SCK cows, accompanied by a reduced fluorescence intensity of CD206 and a lower number of CD68+/CD206+-positive cells. Overall, the present study revealed that the number of macrophages in liver is enhanced during subclinical ketosis and is dominated by pro-inflammatory macrophages (M1 macrophages). This could partly explain the increased risk of steatosis, fibrosis, and inflammatory response processes in these cows.

DEPTOR attenuates asthma progression by suppressing endoplasmic reticulum stress through SOD1

Endoplasmic reticulum (ER) stress has been shown to play a pivotal role in the pathogenesis of asthma. DEPTOR (DEP Domain Containing MTOR Interacting Protein) is an endogenous mTOR inhibitor that participates in various physiological processes such as cell growth, apoptosis, autophagy, and ER homeostasis. However, the role of DEPTOR in the pathogenesis of asthma is still unknown. In this study, an ovalbumin (OVA)-induced mice model and IL-13 induced 16HBE cells were used to evaluate the effect of DEPTOR on asthma. A decreased DEPTOR expression was shown in the lung tissues of OVA-mice and IL-13 induced 16HBE cells. Upregulation of DEPTOR attenuated airway goblet cell hyperplasia, inhibited mucus hypersecretion, decreased the expression of mucin MUC5AC, and suppressed the level of inflammatory factors IL-4 and IL-5, which were all induced by OVA treatment. The increased protein expression of ER stress markers GRP78, CHOP, unfolded protein response (UPR) related proteins, and apoptosis markers in OVA mice were also inhibited by DEPTOR overexpression. In IL-13 induced 16HBE cells, overexpression of DEPTOR decreased IL-4, IL-5, and MUC5AC levels, preventing ER stress response and UPR process. Furthermore, from the proteomics results, we identified that SOD1 (Cu/Zn Superoxide Dismutase 1) may be the downstream factor of DEPTOR. Similar to DEPTOR, upregulation of SOD1 alleviated asthma progression. Rescue experiments showed that SOD1 inhibition abrogates the remission effect of DEPTOR on ER stress in vitro. In conclusion, these data suggested that DEPTOR attenuates asthma progression by suppressing endoplasmic reticulum stress through SOD1.

Impaired 26S proteasome causes learning and memory deficiency and induces neuroinflammation mediated by NF-κB in mice.

A reduction in proteasome activity, loss of synapses and increased neuroinflammation in the brain are hallmarks of aging and many neurodegenerative disorders, including Alzheimer disease (AD); however, whether proteasome dysfunction is causative to neuroinflammation remains less understood. In this study, we investigated the impact of 26S proteasome deficiency on neuroinflammation in the Psmc1 knockout (KO) mice deficient in a 19S proteasome subunit limited to the forebrain region. Our results revealed that impaired 26S proteasome led to reduced learning and memory capability and overt neuroinflammation in the synapses of the Psmc1 KO brain at eight weeks of age. Moreover, pronounced neuroinflammation was also found in the whole brain cortex, which was confirmed by increased levels of several key immune response-related proteins, including Stat1, Trem2 and NF-κB, and by activation of astrocytes and microglia in the KO brain. To validate NF-κB mediating neuroinflammation, we administered a selective NF-κB inhibitor to the KO animals at 5 weeks of age for three weeks, and then, animal behaviors and neuroinflammation were assessed when they reached eight weeks of age. Following the treatment, the KO mice exhibited improved behaviors and reduced neuroinflammation compared to the control animals. These data indicate that impaired 26S proteasome causes AD-like cognitive deficiency and induces neuroinflammation mediated largely by NF-κB. These results may aid development of effective therapeutics and better understanding of the pathogenesis of AD and many other neurodegenerative disorders where impaired proteasome is consistently coupled with neuroinflammation.

Discovery of inflammatory response-related proteins as candidate urinary biomarkers of allergic rhinitis

ObjectivesAllergic rhinitis (AR) is a pervasive condition, affecting a significant global population, often with lifelong implications. The objective of this study is to screen for inflammatory-related differential proteins in the urine of AR patients, aiming to analyze the correlation between these identified proteins and the severity of allergic rhinitis. Design and methodsTo diagnose AR, we utilized the sIgE test to measure the allergenic responses. Based on the total IgE levels, patients were segregated into two categories: AR1 (IgE>125IU/mL) and AR2 (IgE≤125IU/mL). The differential proteins related to inflammation in the urine of patients were screened using TMT labeling combined with LC–MS/MS. Subsequently, these proteins were validated through PRM-based proteomics quantification. ResultsWe found significant increases in FCGR3A, A1AT, KRT18, and IL18 in the AR group (P<0.001). PRM-based quantification results highlighted considerable differences in the concentrations of these four proteins across varying degrees of allergic rhinitis severity. A biomarker panel comprising FCGR3A, A1AT, KRT18, and IL18 was identified, with an area under the curve (AUC) value of 0.993 for the detection of AR. ConclusionsThis study provides the first comprehensive report of combined TMT labeling LC–MS/MS quantitative proteomics and PRM analysis for discovering urinary biomarkers related to inflammatory responses in AR. These findings may be instrumental in evaluating the severity of allergic rhinitis and further our understanding of the molecular mechanisms underlying AR.

Targeted proteomics profiling reveals valuable biomarkers in the diagnosis of primary immune thrombocytopaenia.

The lack of biomarkers for accurate diagnosis and prognosis is a major clinical challenge of primary immune thrombocytopaenia (ITP). Using an Olink proteomics platform with a 92 immune response-related human protein panel, we analysed plasma samples from ITP patients (ITP, n = 40), patients with thrombocytopaenia secondary to other causes (Non-ITP, n = 19) and healthy controls (NC, n = 18), of a discovery cohort as well as a validation cohort (ITP, n = 36; NC, n = 20). A total of 10 differentially expressed proteins (DEPs) were identified in the ITP group compared with the non-ITP and NC groups of the discovery cohort. These include CXCL11, GZMH, ARG1, TGF-β1, ANGPT1, CXCL12, CD40-L, PDGF subunit B, IL4 and TNFSF14. Furthermore, least absolute shrinkage and selection operator regression analysis showed some of these DEPs, such as CXCL11, TGF-β1, ARG1 and GZMH to be significant in differentiating between patients with ITP and healthy controls (validation area under the curve = 0.87). The analysis demonstrated that the ITP group has a specific proteomic profile relative to non-ITP and NC groups. In summary, we report for the first time that Olink precision proteomics can specifically detect up-regulated inflammatory proteins as potential diagnostic biomarkers for ITP.

Oocyte-specific deletion of eukaryotic translation initiation factor 5 causes apoptosis of mouse oocytes within the early-growing follicles by mitochondrial fission defect-reactive oxygen species-DNA damage.

Mutations in several translation initiation factors are closely associated with premature ovarian insufficiency (POI), but the underlying pathogenesis remains largely unknown. We generated eukaryotic translation initiation factor 5 (Eif5) conditional knockout mice aiming to investigate the function of eIF5 during oocyte growth and follicle development. Here, we demonstrated that Eif5 deletion in mouse primordial and growing oocytes both resulted in the apoptosis of oocytes within the early-growing follicles. Further studies revealed that Eif5 deletion in oocytes downregulated the levels of mitochondrial fission-related proteins (p-DRP1, FIS1, MFF and MTFR) and upregulated the levels of the integrated stress response-related proteins (AARS1, SHMT2 and SLC7A1) and genes (Atf4, Ddit3 and Fgf21). Consistent with this, Eif5 deletion in oocytes resulted in mitochondrial dysfunction characterized by elongated form, aggregated distribution beneath the oocyte membrane, decreased adenosine triphosphate content and mtDNA copy numbers, and excessive accumulation of reactive oxygen species (ROS) and mitochondrial superoxide. Meanwhile, Eif5 deletion in oocytes led to a significant increase in the levels of DNAdamageresponse proteins (γH2AX, p-CHK2 and p-p53) and proapoptotic proteins (PUMA and BAX), as well as a significant decrease in the levels of anti-apoptotic protein BCL-xL. These findings indicate that Eif5 deletion in mouse oocytes results in the apoptosis of oocytes within the early-growing follicles via mitochondrial fission defects, excessive ROS accumulation and DNA damage. This study provides new insights into pathogenesis, genetic diagnosis and potential therapeutic targets for POI. Eif5 deletion in oocytes leads to arrest in oocyte growth and follicle development. Eif5 deletion in oocytes impairs the translation of mitochondrial fission-related proteins, followed by mitochondrial dysfunction. Depletion of Eif5 causes oocyte apoptosis via ROS accumulation and DNA damage response pathway.

Proteomics analysis of immune response-related proteins in Guillain-Barré Syndrome (GBS) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP)

The objective is to characterize differentially expressed proteins (DEPs) in Guillain-Barré Syndrome (GBS) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) through high-throughput analysis. Sera from 11 healthy controls (HCs), 21 GBS and 19 CIDP patients were subjected to Olink Proteomics Analysis. In the comparison between CIDP and GBS groups, up-regulation of ITM2A and down-regulation of NTF4 were observed. Comparing GBS with HCs revealed 18 up-regulated and 4 down-regulated proteins. Comparing CIDP with the HCs identified 15 up-regulated and 4 down-regulated proteins. Additionally, the correlation between clinical characteristics and DEPs were uncovered. In conclusion, the DEPs have significant potential to advance our understanding of the pathogenesis in these debilitating neurological disorders.

Premeiotic deletion of Eif2s2 causes oocyte arrest at the early diplotene stage and apoptosis in mice.

Eukaryotic translation initiation factor 2 subunit 2 (EIF2S2), a subunit of the heterotrimeric G protein EIF2, is involved in the initiation of translation. Our findings demonstrate that the depletion of Eif2s2 in premeiotic germ cells causes oocyte arrest at the pachytene and early diplotene stages at 1 day postpartum (dpp) and 5 dpp, respectively, and eventually leads to oocyte apoptosis and failure of primordial follicle formation. Further studies reveal that Eif2s2 deletion downregulates homologous recombination-related and mitochondrial fission-related protein levels, and upregulates the integrated stress response-related proteins and mRNA levels. Consistently, Eif2s2 deletion significantly decreases the expression of dictyate genes and compromises mitochondrial function, characterized by elongated shapes, decreased ATP levels and mtDNA copy number, along with an excessive accumulation of reactive oxygen species (ROS) and mitochondrial superoxide. Furthermore, DNA damage response and proapoptotic protein levels increase, while anti-apoptotic protein levels decrease in Eif2s2-deleted mice. An increase in oocytes with positive cleaved-Caspase-3 and TUNEL signals, alongside reduced Lamin B1 intensity, further indicates oocyte apoptosis. Collectively, Eif2s2 deletion in premeiotic germ cells causes oocyte meiotic arrest at the early diplotene stage by impairing homologous recombination, and eventually leads to oocyte apoptosis mainly through the downregulation of mitochondrial fission-related proteins, ROS accumulation and subsequent DNA damage.

Unfolded protein response markers Grp78 and eIF2alpha are upregulated with increasing alpha-synuclein levels in Lewy body disease.

Endoplasmic reticulum stress followed by the unfolded protein response is one of the cellular mechanisms contributing to the progression of α-synuclein pathology in Parkinson's disease and other Lewy body diseases. We aimed to investigate the activation of endoplasmic reticulum stress and its correlation with α-synuclein pathology in human post-mortem brain tissue. We analysed brain tissue from 45 subjects-14 symptomatic patients with Lewy body disease, 19 subjects with incidental Lewy body disease, and 12 healthy controls. The analysed brain regions included the medulla, pons, midbrain, striatum, amygdala and entorhinal, temporal, frontal and occipital cortex. We analysed activation of endoplasmic reticulum stress via levels of the unfolded protein response-related proteins (Grp78, eIF2α) and endoplasmic reticulum stress-regulating neurotrophic factors (MANF, CDNF). We showed that regional levels of two endoplasmic reticulum-localised neurotrophic factors, MANF and CDNF, did not change in response to accumulating α-synuclein pathology. The concentration of MANF negatively correlated with age in specific regions. eIF2α was upregulated in the striatum of Lewy body disease patients and correlated with increased α-synuclein levels. We found the upregulation of chaperone Grp78 in the amygdala and nigral dopaminergic neurons of Lewy body disease patients. Grp78 levels in the amygdala strongly correlated with soluble α-synuclein levels. Our data suggest a strong but regionally specific change in Grp78 and eIF2α levels, which positively correlates with soluble α-synuclein levels. Additionally, MANF levels decreased in dopaminergic neurons in the substantia nigra. Our research suggests that endoplasmic reticulum stress activation is not associated with Lewy pathology but rather with soluble α-synuclein concentration and disease progression.

Kuwanon C inhibits proliferation and induction of apoptosis via the intrinsic pathway in MDA-MB231 and T47D breast cancer cells

Breast cancer ranks as the most prevalent malignancy, presenting persistent therapeutic challenges encompassing issues such as drug resistance, recurrent occurrences, and metastatic progression. Therefore, there is a need for targeted drugs that are less toxic and more effective against breast cancer. Kuwanon C, an isoamylated flavonoid derived from mulberry resources, has shown promise as a potential candidate due to its strong cytotoxicity against cancer cells. The present study focused on investigating the anticancer activity of kuwanon C in two human breast cancer cell lines, MDA-MB231 and T47D cells. MTS assay results indicated a decrease in cell proliferation with increasing concentrations of kuwanon C. Furthermore, kuwanon C upregulated the expression levels of the cyclin-dependent kinase inhibitor p21 and effectively inhibited cell DNA replication and induced DNA damage. Flow cytometry confirmed that kuwanon C induced cell apoptosis and upregulated the expression levels of pro-apoptotic proteins (Bax and c-caspase3). Additionally, it stimulated the production of reactive oxygen species (ROS) in the cells. Transmission electron microscopy and Fluo-4 AM-calcium ion staining experiments provided insights into the endoplasmic reticulum (ER), revealing that kuwanon C induced ER stress. Kuwanon C upregulated the expression levels of unfolded protein response-related proteins (ATF4, GADD34, HSPA5, and DDIT3). Overall, the present findings suggested that kuwanon C exerts a potent inhibitory effect on breast cancer cell proliferation through modulating of the p21, induction of mitochondrial-mediated apoptosis, activation of ER stress and induction of DNA damage. These results position kuwanon C as a potential targeted therapeutic agent for breast cancer.

Comparative Proteomic Analysis of Ridge Gourd Seed (Luffa acutangula (L.) Roxb.) during Artificial Aging.

Seed aging is a complicated process influenced by environmental conditions, impacting biochemical processes in seeds and causing deterioration that results in reduced viability and vigor. In this study, we investigated the seed aging process of ridge gourd, which is one of the most exported commercial seeds in Thailand using sequential window acquisition of all theoretical fragment ion spectra mass spectrometry. A total of 855 proteins were identified among the two groups (0 d/15 d and 0 d/30 d). The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of differentially expressed proteins revealed that in ridge gourd seeds, the aging process altered the abundance of proteins related to the oxidative stress response, nutrient reservoir, and metabolism pathway. The most identified DEPs were mitochondrial proteins, ubiquitin-proteasome system proteins, ribosomal proteins, carbohydrate metabolism-related proteins, and stress response-related proteins. This study also presented the involvement of aconitase and glutathione pathway-associated enzymes in seed aging, with aconitase and total glutathione being determined as possible suggestive biomarkers for aged ridge gourd seeds. This acquired knowledge has the potential to considerably improve growing methods and seed preservation techniques, enhancing seed storage and maintenance.

Pinctada martensii Hydrolysate Modulates the Brain Neuropeptidome and Proteome in Diabetic (db/db) Mice via the Gut-Brain Axis.

Pinctada martensii hydrolysate (PMH) has been proved to have the effect of ameliorating disorders of glucose and lipid metabolism in db/db mice, but the mechanism of its hyperglycemia effect is still unclear. Bacterial communities in fecal samples from a normal control group, a diabetic control group, and a PMH-treated diabetes mellitus type 2 (T2DM) group were analyzed by 16S gene sequencing. Nano LC-MS/MS was used to analyze mice neuropeptides and proteomes. The 16S rDNA sequencing results showed that PMH modulated the structure and composition of the gut microbiota and improved the structure and composition of Firmicutes and Bacteroidetes at the phylum level and Desulfovibrionaceae and Erysipelatoclostridiaceae at the family level. Furthermore, the expressions of functional proteins of the central nervous system, immune response-related protein, and proteins related to fatty acid oxidation in the brain disrupted by an abnormal diet were recovered by PMH. PMH regulates the brain neuropeptidome and proteome and further regulates blood glucose in diabetic mice through the gut-brain axis. PMH may be used as a prebiotic agent to attenuate T2DM, and target-specific microbial species may have unique therapeutic promise for metabolic diseases.

Development of NULISAseq™ 250-Plex Inflammation Panel for Immune Profiling of Autoimmune Diseases

Abstract Comprehensive profiling of cytokines and chemokines in blood can provide deeper insights into the mechanisms underlying this highly complex and heterogeneous group of diseases. However, many of these proteins are present at very low concentrations in plasma, below the limit of detection of current immunoassays. We recently developed a novel automated multiplex immunoassay technology, NULISA™, capable of attomolar-level sensitivity and high levels of multiplexing. Here we report the development of a 250-plex inflammation-focused panel targeting a broad range of cytokines/chemokines and other important inflammation and immune response-related proteins. We conducted a pilot study to assess the utility of this panel for autoimmune disease research. We analyzed plasma samples from patients with rheumatoid arthritis (RA), Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), and ulcerative colitis (UC), and compared to healthy donors. Linear model analysis identified differentially abundant proteins associated with diseases including many low-abundance targets with important roles in autoimmune diseases, such as IL4, IL5, IL20, IL17A, IL17F, IL33, and IL2RB. In summary, with the ultrahigh sensitivity and the most comprehensive inflammatory cytokine/chemokine panel, the NULISA™ Platform promises to be a powerful discovery tool for autoimmune disease research, which may lead to new diagnostic biomarkers and therapeutic targets.

Abstract 1854: Plasma proteomic biomarkers identify non-responders and reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade

Abstract Most patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses, stressing a critical need to identify early non-invasive biomarkers of response. Circulating biomarkers provide easy access for serial monitoring and can provide insight on the mechanisms of response to ICB. We performed plasma proteomics (~3000 proteins) on 250 metastatic melanoma patients before and on ICB treatment and performed analysis to identify response- and time point-associated peripheral protein biomarkers. We next generated patient-matched peripheral blood lymphocyte (PBL) surface proteomic data and tumor sample bulk transcriptomic data to build a multimodal dataset. We leveraged these data to train machine learning models for unsupervised analysis and to classify treatment responses and learned features predictive of survival and patient endotypes. Using univariate linear models to predict protein abundance based on time point, response and the interaction term for time point and response, we identified 343 time point-associated proteins, 141 response-associated proteins, and one interaction-associated protein. We further investigated response-related proteins by fitting multivariate logistic regression and cox proportional hazards regression models to further investigate the ability of these proteins to predict response, progression-free survival (PFS), and overall survival (OS) and identify those proteins that are most important in predicting response. We calculated covarying modules of proteins that were found to be either response-associated or time point-associated to discover underlying biological networks at different treatment time points and disease response states. Additionally, we leveraged these modules to endotype patients and identified trends in protein modules across patient subsets, including modules related to biological processes such as immune infiltration, apoptosis, metaplasia and cell adhesion. To understand the contribution of different compartments on plasma proteomic abundance, we studied correlations of plasma protein abundance with PBL surface protein abundance and tumor bulk mRNA-sequencing expression. Finally, we integrated our data modalities and fit various statistical models to evaluate the relative importance of plasma protein biomarkers in predicting response compared to PBL surface proteins and tumor bulk mRNA-sequencing expression. Together, these data represent one of the deepest peripheral biomarker studies using paired samples in melanoma patients treated with anti-PD1 therapy. Citation Format: Samuel J. Wright, Deepika Yeramosu, Milan Parikh, Marijana Rucevic, Ngan Nguyen, Russell W. Jenkins, Keith T. Flaherty, Nir Hacohen, Genevieve M. Boland, Arnav Mehta. Plasma proteomic biomarkers identify non-responders and reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade [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 1854.

Effect of daylong exposure to indoor overheating on autophagy and the cellular stress response in older adults.

To protect vulnerable populations during heat waves, public health agencies recommend maintaining indoor air temperature below ∼24-28 °C. While we recently demonstrated that maintaining indoor temperatures≤26 °C mitigates the development of hyperthermia and cardiovascular strain in older adults, the cellular consequences of prolonged indoor heat stress are poorly understood. We therefore evaluated the cellular stress response in 16 adults (six females) aged 66-78 years during 8 h rest in ambient conditions simulating homes maintained at 22 °C (control) and 26 °C (indoor temperature upper limit proposed by health agencies), as well as non-air-conditioned domiciles during hot weather and heat waves (31 and 36 °C, respectively; all 45% relative humidity). Western blot analysis was used to assess changes in proteins associated with the cellular stress response (autophagy, apoptosis, acute inflammation, and heat shock proteins) in peripheral blood mononuclear cells harvested prior to and following exposure. Following 8 h exposure, no cellular stress response-related proteins differed significantly between the 26 and 22 °C conditions (all, P≥0.056). By contrast, autophagy-related proteins were elevated following exposure to 31 °C (p62: 1.5-fold; P=0.003) and 36 °C (LC3-II, LC3-II/I, p62; all≥2.0-fold; P≤0.002) compared to 22 °C. These responses were accompanied by elevations in apoptotic signaling in the 31 and 36 °C conditions (cleaved-caspase-3: 1.8-fold and 3.7-fold, respectively; P≤0.002). Furthermore, HSP90 was significantly reduced in the 36 °C compared to 22 °C condition (0.7-fold; P=0.014). Our findings show that older adults experience considerable cellular stress during prolonged exposure to elevated ambient temperatures and support recommendations to maintain indoor temperatures≤26 °C to prevent physiological strain in heat-vulnerable persons.

Serum Olink Proteomics-Based Identification of Protein Biomarkers Associated with the Immune Response in Ischemic Stroke.

The immune response is considered essential for pathology of ischemic stroke (IS), but it remains unclear which immune response-related proteins exhibit altered expression in IS patients. Here, we used Olink proteomics to examine the expression levels of 92 immune response-related proteins in the sera of IS patients (n = 88) and controls (n = 88), and we found that 59 of these proteins were differentially expressed. Feature variables were screened from the differentially expressed proteins by the least absolute shrinkage and selection operator (LASSO) and the random forest and by determining whether their proteins had an area under the curve (AUC) greater than 0.8. Ultimately, we identified six potential protein biomarkers of IS, namely, MASP1, STC1, HCLS1, CLEC4D, PTH1R, and PIK3AP1, and established a logistic regression model that used these proteins to diagnose IS. The AUCs of the models in the internal validation and the test set were 0.962 (95% confidence interval (CI): 0.895-1.000) and 0.954 (95% CI: 0.884-1.000), respectively, and the same protein detection method was performed in an external independent validation set (AUC: 0.857 (95% CI: 0.801-0.913)). These proteins may play a role in immune regulation via the C-type lectin receptor signaling pathway, the PI3K-AKT signaling pathway, and the B-cell receptor signaling pathway.

Smoking Induces a Decline in Semen Quality and the Activation of Stress Response Pathways in Sperm.

Male infertility is a prevalent concern affecting couples worldwide. While genetic factors, hormonal imbalances, and reproductive system defects play significant roles, emerging evidence suggests that lifestyle choices also profoundly impact male fertility. This study aimed to explore the effects of several lifestyle factors, including tobacco and alcohol consumption, physical activity, and dietary habits, on semen quality parameters and molecular biomarkers. Thirty healthy male volunteers were recruited in the Urology service at Hospital Infante D. Pedro, Aveiro, Portugal. Participants completed lifestyle questionnaires and provided semen samples, which were analyzed according to the World Health Organization criteria by experienced technicians. We also analyzed the expression levels of antioxidant enzymes and heat-shock response-related proteins to explore the activation of signaling pathways involved in stress response within sperm cells. Our results revealed that tobacco consumption reduced semen volume and total sperm count. Although the changes in the percentage of total motility and normal morphology in the smokers' group did not reach statistical significance, a slight decrease was observed. Moreover, we identified for the first time a significant association between tobacco consumption and increased levels of heat shock protein 27 (HSP27) and phosphorylated HSP27 (p-HSP27) in sperm cells, indicating the potential detrimental effects of tobacco on the reproductive system. This study highlights that lifestyle factors reduce semen quality, possibly by inducing stress in sperm, raising awareness about the effects of these risk factors among populations at risk of male infertility.

Multimodal Imaging-Guided Strategy for Developing 177Lu-Labeled Metal-Organic Framework Nanomedicine with Potential in Cancer Therapy.

Nano-metal-organic frameworks (nano-MOFs) labeled with radionuclides have shown great potential in the anticancer field. In this work, we proposed to combine fluorescence imaging (FI) with nuclear imaging to systematically evaluate the tumor inhibition of new nanomedicines from living cancer cells to the whole body, guiding the design and application of a high-performance anticancer radiopharmaceutical to glioma. An Fe-based nano-MOF vector, MIL-101(Fe)/PEG-FA, was decorated with fluorescent sulfo-cyanine7 (Cy7) to investigate the binding affinity of the targeting nanocarriers toward glioma cells in vitro, as well as possible administration modes for in vivo cancer therapy. Then, lutetium-177 (177Lu)-labeled MIL-101(Fe)/PEG-FA was prepared for high-sensitive imaging and targeted radiotherapy of glioma in vivo. It has been demonstrated that the obtained 177Lu-labeled MIL-101(Fe)/PEG-FA can work as a complementary probe to rectify the cancer binding affinity of the prepared nanocarrier given by fluorescence imaging, providing more precise biodistribution information. Besides, 177Lu-labeled MIL-101(Fe)/PEG-FA has excellent antitumor effect, leading to cell proliferation inhibition, upregulation of intracellular reactive oxygen species, tumor growth suppression, and immune response-related protein and cytokine upregulation. This work reveals that optical imaging and nuclear imaging can work complementarily as multimodal imaging in the design and evaluation of anticancer nanomedicine, offering a MIL-101(Fe)/PEG-FA-based pharmaceutical with potential in tumor endoradiotherapy.

Methyl lucidone inhibits airway inflammatory response by reducing TAK1 activity in human bronchial epithelial NCI–H292 cells

BackgroundMethyl lucidone (ML), a methyl derivative of lucidone, has anti-inflammatory properties. However, the molecular mechanisms that reduce the inflammatory effect of ML in human lung epithelial cells remain unkown. This study aimed to elucidate the molecular mechanisms underlying the anti-inflammatory effect of ML. MethodsFour compounds (ML, methyl linderone, kanakugiol, and linderone) from Lindera erythrocarpa Makino were evaluated for their ability to reduce MUC5AC secretion levels in phorbol-12-myristate-13-acetate (PMA)-stimulated NCI–H292 cells using ELISA. The expression and secretion levels of inflammatory response-related proteins were analyzed using quantitative reverse transcription-PCR, ELISA, and western blotting. To determine whether ML directly regulates TGF-β-activated kinase 1 (TAK1), we performed an in vitro kinase assay. ResultsML treatment effectively reduced the levels of inflammatory cytokines, including interleukin-1β and TNF-α, increased by stimulation. Furthermore, ML downregulated the pathway cascade of both IκB kinase (IKK)/NF-κB and p38 mitogen-activated protein (MAP) kinase/CREB by inhibiting the upstream kinase TAK1. An in vitro kinase analysis confirmed that ML treatment significantly reduced the kinase activity of TAK1. ConclusionML pretreatment repressed the PMA-stimulated inflammation reaction by reducing the TAK1-mediated IKK/NF-κB and p38 MAP kinase/CREB signaling. These findings suggest that ML may improve respiratory health and can be used as a dietary supplement or functional food to prevent inflammatory lung diseases.