Altered Protein Expression Research Articles

Proteome Reprogramming and Acquired Stress Tolerance in Potato Cells Exposed to Acute or Stepwise Water Deficit.

Water deficit negatively impacts crop productivity and quality. Plants face these challenges by adjusting biological processes and molecular functions according to the intensity and duration of the stress. The cultivated potato (Solanum tuberosum) is considered sensitive to water deficit, thus breeding efforts are needed to enhance its resilience. To capture novel functional information and gene regulatory networks, we carried out mass spectrometry-based proteomics in potato cell suspensions exposed to abrupt or stepwise osmotic stresses. Both forms of stress triggered significant alterations in protein expression, though with divergent response mechanisms. Stress response pathways orchestrated by key proteins enrolled in primary and secondary metabolism, antioxidant processes, transcriptional and translational machinery and chromatin organization were found in adapted cells. Target metabolites and reactive oxygen species levels were quantified to associate functional outcomes with the proteome study. Remarkably, we also showed that adapted cells tolerate an array of diverse conditions, including anoxia, salt and heat stress. Finally, the expression patterns of genes encoding selected differentially expressed proteins were investigated in potato plants subjected to either drought or salt stress. Collectively, our findings reveal the complex cellular strategies of osmotic stress adaptation, identifying new fundamental genes that could enhance potato resilience.

Integrating Metabolomics, Histopathology, and Cardiac Marker Analysis to Assess Valsartan's Efficacy in Mitigating Dasatinib-Induced Cardiac Toxicity in Sprague-Dawley Rats.

Dasatinib (DASA) is associated with cardiotoxic effects, posing risks to patients. Valsartan (VAL) may offer protective benefits against these effects. This study evaluates the impact of DASA, VAL, and their combination on cardiac health. Wistar rats were treated with DASA, VAL, and a combination of VAL and DASA intraperitoneally every other day for 14 days. Body weight and survival rates were monitored. Serum levels of cardiac biomarkers (CPK, LDH, AST) were analyzed. Histopathological and immunohistochemical analyses assessed myocardial architecture and apoptosis-related protein expression. Metabolomic profiling was conducted using GC-MS to identify metabolic changes across treatment groups. The DASA group experienced significant weight loss and a 50% mortality rate, while the combination group had no mortality. Cardiac biomarkers like CPK, LDH, and AST were elevated in the DASA group but significantly reduced in the VAL + DASA group. Histopathological examination showed significant myocardial injury in the DASA group, with improved cardiac tissue morphology in the combination group. Immunohistochemical analysis revealed altered expression of apoptosis-related proteins, including caspase-3 and BCL-2, with improved levels in the combination group compared to DASA alone. Metabolomic profiling identified significant metabolic shifts, with 15 metabolites differentiating the treatment groups, and the VAL + DASA group mitigated the metabolic disturbances caused by DASA. The study suggesting VAL's potential therapeutic role in managing DASA-induced cardiac toxicity. The combination of VAL with DASA not only improved survival rates and reduced cardiac biomarker levels but also preserved myocardial architecture and normalized metabolic profiles. These findings highlight the importance of integrated approaches in evaluating drug efficacy and suggest VAL as a promising candidate for protecting cardiac function in preclinical models of DASA therapy.

NLRP3 activation maintains intestinal epithelial barrier and reduces liver injury in alcoholic liver disease mice.

Alcoholic liver disease (ALD) patients with bacterial infections usually exhibit high mortality rates. Infections frequently involve bacteria such as Vibrio vulnificus and Enterococcus faecalis. Nevertheless, the mechanisms predisposing ALD patients to bacterial infections and the role of the NLRP3 inflammasome in the intestinal epithelial barrier in ALD remain unclear. We established ALD mice models of WT, Nlrp3-/- and Gsdmd-/- through chronic alcohol consumption feeding and acute alcohol induction. We compared alterations in gut microbiota, ileitis, and adhesion protein expression, to analyze the role and potential mechanism of NLRP3 in the early onset of ALD. Concurrently, we examined the changes in inflammation and liver damage in the ileum of ALD and healthy mice following foodborne infection with V. vulnificus. Compared with the control group, the expression levels of ZO-1, Claudin-1 and E-cadherin were reduced in the ileum of ALD mice, while those of NLRP3, caspase-1(p20), GSDMD-N and IL-1β were elevated. Nlrp3-/- and Gsdmd-/- ALD mice showed an increased gut bacterial load, decreased ileal expression of E-cadherin, more severe ileitis, pronounced liver damage, steatosis and higher plasma levels of FITC-dextran, D-LA and ZO-1 compared with WT mice. Notably, Nlrp3-/- ALD mice exhibited a higher presence of Deferribacterota and Enterobacteriaceae. Furthermore, ALD mice infected with V. vulnificus infection exhibited no further activation of NLRP3 in the ileum, leading to increased intestinal permeability and bloodstream infections. This study indicated that NLRP3 activation in the ileum of ALD mice stabilizes the inflammation-related gut microbiota, preserves the intestinal epithelial barrier, and diminishes inflammation and liver injury. Furthermore, the compromised immune defence in ALD mice may contribute to their heightened susceptibility to bacterial pathogens. Activation of the NLRP3-GSDMD pathway in the ileum of Alcoholic liver disease (ALD) mice. NLRP3 activation maintains homeostasis of gut microbiota and intestinal epithelial barrier in ALD mice. ALD mice infected with V. vulnificus infection exhibited no further activation of NLRP3 in the ileum, leading to increased intestinal permeability and bloodstream infections.

DJ-1-mediated repression of the RNA-binding protein FMRP is predicted to impact known Alzheimer's disease-related protein networks.

RNA-binding proteins (RBPs) modulate the synaptic proteome and are instrumental in maintaining synaptic homeostasis. Moreover, aberrant expression of an RBP in a disease state would have deleterious downstream effects on synaptic function. While many underlying mechanisms of synaptic dysfunction in Alzheimer's disease (AD) have been proposed, the contribution of RBPs has been relatively unexplored. To investigate alterations in RBP-messenger RNA (mRNA) interactions in AD, and its overall impact on the disease-related proteome. We first utilized RNA-immunoprecipitation to investigate interactions between RBP, DJ-1 (Parkinson's Disease protein 7) and target mRNAs in controls and AD. Surface Sensing of Translation - Proximity Ligation Assay (SUnSET-PLA) and western blotting additionally quantified alterations in mRNA translation and protein expression of DJ-1 targets. Finally, we utilized an unbiased bioinformatic approach that connects AD-related pathways to two RBPs, DJ-1 and FMRP (Fragile X messenger ribonucleoprotein 1). We find that oligomeric DJ-1 in AD donor synapses were less dynamic in their ability to bind and unbind mRNA compared to synapses from cognitively unimpaired, neuropathologically-verified controls. Furthermore, we find that DJ-1 associates with the mRNA coding for FMRP, Fmr1, leading to its reduced synaptic expression in AD. Through the construction of protein-protein interaction networks, aberrant expression of DJ-1 and FMRP are predicted to lead to the upregulation of key AD-related pathways, such as thyroid hormone stimulating pathway, autophagy, and ubiquitin mediated proteolysis. DJ-1 and FMRP are novel targets that may restore established neurobiological mechanisms underlying AD.

Minimally invasive serial collection of cerebrospinal fluid reveals sex-dependent differences in neuroinflammation in a rat model of mild traumatic brain injury

Traumatic brain injuries (TBI) are the seventh leading cause of disability globally with 48.99 million prevalent cases and 7.08 million years lived with diability. Approximately 80 % of TBI patients are diagnosed with mild TBI (mTBI), or concussion, caused by nonpenetrating mechanical trauma to the head or body along with sudden rotational motion of the head. Studies investigating the temporal dynamics of neuroinflammation after mTBI are greatly needed. Without longitudinal studies, translating preclinical studies to clinical studies remains challenging as the difference in timing remains poorly understood. In this study, we describe a method of minimally invasive serial cerebrospinal fluid (CSF) collection that enables longitudinal investigation of CSF inflammation. The method described in this study can easily be adapted by any laboratory prepared for animal studies. Multiplex immunoassay of serially collected and singly collected CSF samples show collection frequency does not alter protein expression in the CSF. Further, sex-dependent differences in TBI have been reported, but remain poorly understood. This study establishes a framework for assessing sex difference in neuroinflammation after a concussion. We showed that results vary based on the framing of the statistical test. However, it is evident that males experience a more robust inflammatory response to a single concussion than females.

Chronic Hypobaric Hypoxia Stimulates Differential Expression of Cognitive Proteins in Hippocampal Tissue.

Objective: We aimed to determine changes in cognitive function resulting from chronic hypobaric hypoxia through proteomic analysis of hippocampal tissue. We screened cognition-related proteins to provide ideas and directions that could help prevent and treat hypoxia-associated cognitive impairment. Methods: We analyzed hippocampal tissues from mice exposed to high altitudes and control mice using 4 D label-free quantitative proteomics. The data were analyzed by protein quantitative analysis, functional annotation, differential protein screening, clustering analyses, and functional classification and enrichment. Differential protein expression was investigated using targeted quantitative omics based on parallel response monitoring. Results: We identified and quantified 20 target proteins in 12 samples, of which 18 were significant validated proteins that were or might be related to cognitive functions. Signaling pathways that were significantly enriched in differentially expressed proteins were pyrimidine metabolism, 5'-Adenosine Triphosphate-activated protein kinase signaling, phospholipase D signaling, purine metabolism, inflammatory mediator regulation of transient receptor potential channels, hedgehog signaling pathways, dilated cardiomyopathy, platelet activation, insulin resistance, mRNA surveillance pathways, drug metabolism-other enzymes, and drug metabolism-cytochrome P450. Conclusion: Chronic hypoxia alters protein expression in murine hippocampal tissues. Eighteen differentially expressed cognition-related proteins might be related to cognitive impairment in mice exposed to chronic high-altitude hypoxia.

An integrated multi-omics analysis identifies novel regulators of circadian rhythm and sleep disruptions under unique light environment in Antarctica.

Light is the dominant zeitgeber for biological clocks, and its regulatory mechanism for sleep-wake activity has been extensively studied. However, the molecular pathways through which the unique Antarctic light environment, with polar days in summer and polar nights in winter, affects human sleep and circadian rhythm remain largely unidentified, although previous studies have observed delayed circadian rhythm and sleep disruptions among expeditioners during polar nights. In this study, we conducted comprehensive dynamic research on the expeditioners residing in Antarctica for over one year. By integrating the phenotypic changes with multi-omics data, we tried to identify the novel candidate regulators and their correlation networks involved in circadian and sleep disorders under abnormal light exposure. We found that during the austral winter, expeditioners exhibited delayed bedtime and getting up time, reduced sleep efficiency, and increased sleep fragmentation. Meanwhile, serum dopamine metabolite levels significantly increased, while serotonin metabolites and antioxidants decreased. These changes were accompanied by altered expression of genes and proteins associated with neural functions, cellular activities, transcriptional regulation, and so on. Through the correlation and causal mediation analysis, we identified several potential pathways modulating human sleep-wake activity, involving genes and proteins related to neural function, glucose metabolism, extracellular matrix homeostasis, and some uncharacterized lncRNAs. Based on the identified causal mediators, LASSO regression analysis further revealed a novel candidate gene, Shisa Family Member 8 (SHISA8), as a potential key regulatory hub in this process. These findings shed light on the probable molecular mechanisms of sleep disorders in Antarctica and suggest SHISA8 as a novel candidate target for medical intervention in sleep disorders under unique light environments.

Decoding the pathological and genomic profile of epithelial ovarian cancer.

Ovarian cancer (OC) is one of the most common cancers in women, with a high mortality rate. Most of published studies have been focused on Caucasian populations, with the need to explore biological features and clinical outcomes of patients from other ethnicities. We described clinical outcome (progression-free survival and overall survival) and biomarkers associated with survival in a cohort of patients with OC from Tunisia. Using immunohistochemistry, we assessed the expression of 14 proteins known to be altered in OC in a cohort of 198 patients. We explored the correlation between protein expression and copy number alteration (CNA) profiles. FIGO stage, menopausal status and mismatch repair deficiency were associated with survival. ERBB2 amplification was correlated with high ERBB2 expression (OR = 69.32, p = 4.03 E-09), and high PDL1 expression was associated to CD274 amplification (OR = 4.97, p = 5.79 E-2). We identified a correlation between survival and exposure to two CNA signatures (MAPK pathway and BRCA-related homologous recombination deficiency). Moreover, Gama-H2AX protein expression was correlated with exposure to a genomic signature associated with homologous recombination deficiency. We observed that OC clinical and pathological characteristics of these patients from Tunisia were similar to those of Caucasian patients. We identified frequent CNA in this population that need to be confirmed in other sets from Africa.

Association between the variations in metabolic pathways and oral cancer risk: results from a Pakistani case-control study.

Oral cancer (OC) is a significant global health concern, with Pakistan ranking 5th worldwide in OC incidence. Given the poor prognosis, early detection of at-risk individuals is crucial. Genetic factors, particularly single nucleotide polymorphisms (SNPs) in metabolic genes, may influence OC susceptibility. This study investigated the association between SNPs in CYP1A1, COX2, SOD2, and HIF1a genes and OC risk in the Pakistani population. A prospective study was conducted from October 2019 to March 2022, enrolling 215 newly diagnosed OC patients and 410 controls. Genetic variations were analyzed using High-Resolution Melting (HRM)analysis and Sanger sequencing, with protein expression evaluated by ELISA. No significant associations were found between the studied SNPs and OC risk. However, a non-significant trend was observed for the SOD2 variant (rs4880), where the G allele was associated with a higher OC risk than the A allele (p = 0.20). Elevated COX2 and HIF1α levels (p-values of 0.014 and <0.001, respectively) and reduced SOD2 levels (p < 0.0001) were observed in OC patients, while CYP1A1 levels remained similar in both controls and cases. Although SNPs in CYP1A1, COX2, SOD2, and HIF1α were not significantly associated with OC risk in the Pakistani population, altered protein expression levels of COX2, HIF1α and SOD2 suggest additional regulatory mechanisms. Further investigation into post-transcriptional modifications and epigenetic factors could lead to novel biomarkers and therapeutic targets for OC in Pakistan.

Convergent effects of synthetic glucocorticoid dexamethasone and amyloid beta in human olfactory neurosphere-derived cells.

Stressful life events and glucocorticoid (stress) hormones appear to increase the risk of Alzheimer's disease and hasten its progression, but the reasons for this remain unclear. One potential explanation is that when amyloid β (Aβ) pathology is accumulating in the preclinical disease stage, glucocorticoid receptor signalling during stressful events exacerbates cellular dysfunction caused by Aβ. Alternatively, Aβ may disrupt glucocorticoid receptor signalling. To explore these possibilities, we investigated whether the synthetic glucocorticoid dexamethasone and Aβ have overlapping effects on the cellular proteome and whether Aβ influences canonical glucocorticoid receptor function. Human olfactory neurosphere-derived (ONS) cells, collected from the olfactory mucosa of six adult donors, were treated with soluble Aβ40 or Aβ42 followed by dexamethasone. Proteins were quantified by mass spectrometry. After 32 h treatment, Aβ40 and Aβ42 both induced profound changes in innate immunity-related proteins. After 72 h, Aβ42 formed widespread aggregates and induced few proteomic changes, whereas Aβ40 remained soluble and altered expression of mitochondrial and innate immunity-related proteins. ONS cells revealed overlapping impacts of Aβ40 and dexamethasone, with 23 proteins altered by both treatments. For 16 proteins (including eight mitochondrial proteins) dexamethasone counteracted the effects of Aβ40. For example, caspase 4 and methylmalonate-semialdehyde dehydrogenase were increased by Aβ40 and decreased by dexamethasone. Consistent with this finding, Aβ40 increased, but dexamethasone decreased, ONS cell proliferation. For seven proteins, including superoxide dismutase [Mn] mitochondrial, dexamethasone exacerbated the effects of Aβ40. For some proteins, including complement C3, the effects of dexamethasone differed depending on whether Aβ40 was present or absent. Neither Aβ species influenced glucocorticoid receptor nuclear translocation. Overall, this study revealed that glucocorticoid receptor signalling modifies the intracellular effects of Aß40, counteracting some effects and exacerbating others. It suggests that cellular mechanisms through which glucocorticoid receptor signalling influences Alzheimer's disease risk/progression are complex and determined by the balance of beneficial and detrimental glucocorticoid effects.

Tear fluid reflects the altered protein expressions of Alzheimer's disease patients in proteins involved in protein repair and clearance system or the regulation of cytoskeleton.

New biomarkers that improve diagnosis of Alzheimer's disease (AD) are warranted. Tear fluid (TF) containing variety of proteins that reflect pathophysiological changes of systemic diseases makes TF proteins potential biomarker candidates for AD. We investigated the expression levels of TF proteins in persons with mild AD and cognitively healthy controls (CO) to find out if altered proteins may link to the AD pathophysiology. We analyzed the data of the 53 study participants (34 COs, mean age 71 and Mini-Mental State Examination (MMSE) 28.9 ± 1.4 and 19 persons with AD, CDR 0.5-1, mean age 71 and MMSE 23.8 ± 2.8). All went through neurological status examination, cognitive tests, and ophthalmological examination. TF was collected using Schirmer strips. The TF protein content was evaluated via mass spectrometry-based proteomics and label-free quantification. Eleven proteins having a role either in protein repair and clearance system, or regulation of cytoskeleton, showed altered expression in AD group compared to CO group. Seven of them were significantly (p ≤ 0.05) upregulated (Sti1, Twf1, Myl6, Otub1, Pls1 and Caza1) or, downregulated (HSP90) in AD group. Altered expression of all these up- or downregulated proteins may be linked to AD pathophysiology. Thus, our results are encouraging for searching new biomarker candidates for AD. TF is potential biomarker candidate, because TF seems to reflect altered protein levels already in mild AD dementia.

Molecular mechanisms restoring olaparib efficacy through ATR/CHK1 pathway inhibition in olaparib-resistant BRCA1/2MUT ovarian cancer models

Resistance to olaparib inevitably develops in ovarian cancer (OC) patients, highlighting the necessity for effective strategies to improve its efficacy. Here, we established a novel olaparib-resistant patient-derived xenograft model of high-grade serous OC with BRCA1/2 mutations and examined the molecular characteristics of acquired resistance and resensitization to olaparib in treatment-naïve tumors in vivo. Olaparib-resistant xenografts were treated with olaparib, ATR inhibitor (ATRi, ceralasertib), CHK1 inhibitor (CHK1i, MK-8776) or their combinations. Proliferation, apoptosis, ATR/CHK1 activity, PARP signaling, DNA damage response (DDR), epithelial-to-mesenchymal transition (EMT), and MDR1 expression, were examined via RT-qPCR, western blot, and immunohistochemistry. Resistant tumors exhibited defects in PARP and ATR/CHK1 signaling, accompanied by altered expression of proteins involved in DDR and EMT. Olaparib rechallenge combined with ATR/CHK1 inhibitors showed promising synergistic effects on tumor growth inhibition. Mechanistically, combined treatments suppressed tumor proliferation without increasing apoptosis or necrosis, while inducing tumor cell vacuolization indicative of cell death. ATRi combined with olaparib induced or augmented downregulation of ATR, CHK1, PARP1, PARG, BRCA1, γH2AX, and PARylated protein expression, while reversing olaparib-induced upregulation of vimentin, BRCA2, and 53BP1. Our collective findings indicate that ATR/CHK1 pathway inhibition restores the olaparib efficacy in resistant BRCA1/2MUT high-grade serous OC, highlighting promising approach for olaparib rechallenge of non-responsive patients. Uncovered mechanisms might improve our understanding of acquisition and overcoming resistance to olaparib in ovarian cancer.

EXTH-56. INVESTIGATING THE EFFECTS OF INDIRUBINS IN PEDIATRIC DIFFUSE HIGH GRADE GLIOMA MODELS

Abstract Diffuse midline glioma (DMG) is a highly invasive and fatal pediatric brain tumor that arises from glial cells within the pons of the brainstem. Despite focal radiation treatment, DMG continues to have a poor prognosis, with a median survival of less than 10 months. A significant challenge in treating DMG is the blood-brain barrier (BBB), which tightly restricts access to therapeutic agents, preventing drugs from reaching the brain at effective concentrations. Previous studies from the Lawler lab have shown that the indirubin derivative 6-bromoindirubin-3′-acetoxime (BIO-acetoxime/BIA) has anti-invasive properties and can enhance survival in glioblastoma xenograft models. We investigated the effects of BIA on pediatric glioma models using various assays. In an in vitro scratch migration assay, BIA significantly slowed cell migration at a concentration of 1µM in pediatric glioma cells over 72 hours. Additionally, BIA was shown to modulate tumor vasculature and improve drug delivery to tumors by targeting tight junctions in tumor-associated endothelium. BIA treatment increased dextran uptake into 3D BBB models and lowered endothelial cell permeability in vitro by reducing the expression of tight junction proteins, as shown by staining and a decrease in TEER values. Furthermore, BIA’s anti-angiogenic effects were demonstrated through staining, showing significant alterations in angiogenesis-related protein expression. To assess potential synergistic interactions, a drug screen was performed on a panel of pediatric glioma cell lines identifying several FDA-approved drugs that combine well with BIA. Drugs identified in the screen are currently under investigation. Our hypothesis is that BIA could be an effective therapeutic agent for DMG by targeting tumor invasion, angiogenesis, and improving drug potency and delivery through modulation of endothelial cell tight junctions. Future studies will focus on elucidating the underlying mechanisms and developing drug formulations for in vivo studies to assess the translational potential of this approach.

TMIC-40. TARGETING GLIOMA TUMOR MICROENVIRONMENT BY SPECIFIC NEUTRALIZATION OF EXTRACELLULAR NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (ENAMPT) USING ALT-100, A NOVEL HUMANIZED MONOCLONAL ANTIBODY

Abstract BACKGROUND Intracellular nicotinamide phosphoribosyltransferase (iNAMPT is the rate-limiting enzyme of NAD salvage pathway and is preferentially used by glioma cells for regulating energy metabolism and homeostasis. When secreted, extracellular NAMPT (eNAMPT) exerts cytokine-like functions and is associated with poor prognosis in cancer patients. We have previously reported an essential role of iNAMPT for glioma cell proliferation and survival. Here, we examined whether eNAMPT influences glioma biology and tumor microenvironment by neutralizing eNAMPT using ALT-100, a novel humanized monoclonal antibody. METHODS Native levels of eNAMPT in blood samples of patients with primary/recurrent gliomas were determined using ELISA under an IRB approved protocol. Next, the effects of ALT-100 on glioma microenvironment were determined by treating viable human glioma tissue slices with ALT-100 and assessing downstream effects on RNA and protein using RNASeq, reverse phase protein array and spatial transcriptomics. Lastly, the in vivo effect of ALT-100-mediated eNAMPT neutralization on survival was determined in a mouse intracranial glioma model. RESULTS Measurable levels of eNAMPT were noted in all patient samples with a range of (3.7-71.2 ng/ml). Preliminary results showed a correlation of eNAMPT levels with grade of tumor. Human organotypic glioma slice cultures treated with ALT-100, showed a profound alteration of gene and protein expression particularly related to key energy metabolism and proliferation pathways and the PD-1/PD-L1 axis. Additionally, results of spatial profiling of ALT-100 treated human organotypic glioma slices will be presented. Survival and response data from ALT-100 treatment in ongoing in vivo experiment will also be reported. CONCLUSIONS This is the first report of ALT-100-mediated eNAMPT neutralization in gliomas and demonstrates profound effects on the tumor and its microenvironment. Given that ALT-100 is in phase 2 trials (NCT05938036) against ARDS, data from our studies can potentially accelerate translating ALT-100-mediated eNAMPT neutralization to treat gliomas.

Integrated proteomic and metabolomic analysis reveals the potential therapeutic mechanism of Quanduzhong capsule in rats with spontaneous hypertension and knee osteoarthritis

Quanduzhong capsule (QDZ), derived from Eucommia ulmoides Oliv. has been traditionally used in Chinese medicine for its beneficial effects on musculoskeletal health. Its clinical application has extended to conditions such as spontaneous hypertension combined with knee osteoarthritis (SKOA). However, the specific mechanisms by which QDZ alleviates symptoms and improves outcomes in this complex condition remain to be fully elucidated. This study aims to evaluate the therapeutic potential of QDZ in treating SKOA. By performing serum proteomics and metabolomics, we seek to explore the related biological pathways and elucidate the mechanisms underlying QDZ's effects on SKOA. Serum samples from control, spontaneous hypertension (SHR), SKOA, and SKOA treated with QDZ groups were analyzed using data-independent acquisition-based proteomics to identify differentially expressed proteins. Serum levels of angiotensin II, norepinephrine, endothelin-1, classical pro-inflammatory factors such as macrophage colony-stimulating factor, tumor necrosis factor-alpha, and interleukin-1 beta were measured. Additionally, serum metabolomics was performed to examine the changes in metabolite profiles. Correlation analysis was conducted to link changed proteins and metabolites with key pathways affected by QDZ. Proteomics analysis revealed significant alterations in serum protein expression between control, SHR, and SKOA groups, with changes in pathways related to immune regulation, and vascular function. KEGG enrichment analysis highlighted pathways such as endocytosis, synaptic vesicle cycling, and immune responses were enriched in SKOA group compared with control group. QDZ treatment significantly modulated above pathways and reduced inflammatory and cardiovascular markers which were upregulated in SKOA group. Metabolomics analysis showed that QDZ reversed SKOA-induced changes in amino acid and organic acid metabolism, affecting pathways including valine, leucine, and isoleucine metabolism, as well as the TCA cycle. Correlation analysis revealed significant relationships between key proteins and metabolites, underscoring the integrated role of immune and metabolic pathways in QDZ's effects. Our results indicate QDZ has a significant therapeutic potential for SKOA by modulating both protein and metabolite profiles associated with inflammation, vascular dysfunction, and metabolic imbalance. Our findings provide insights into the mechanisms through which QDZ exerts its effects and support its use as a promising treatment for SKOA. This study highlights the impact of QDZ on proteomic and metabolomic alterations, offering a basis for its broader application in treating SKOA.

Intracellular delivery of a phospholamban-targeting aptamer using cardiomyocyte-internalizing aptamers

The sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a)–phospholamban (PLN) system within the sarcoplasmic reticulum is crucial for regulating intracellular Ca2+ cycling in ventricular cardiomyocytes. Given that impaired Ca2+ cycling is associated with heart failure, modulating SERCA2a activity represents a promising therapeutic strategy. Previously, we engineered an RNA aptamer (Apt30) that binds to PLN, thereby activating SERCA2a by alleviating PLN's inhibitory effect. However, Apt30 alone cannot reach intracellular PLN, necessitating the development of a mechanism for its specific internalization into cardiomyocytes.Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we isolated RNA aptamers capable of internalizing into cardiomyocytes. These aptamers demonstrated sub-micromolar EC50 values for cardiomyocyte internalization and exhibited significantly reduced activity against various non-myocardial cells, highlighting their specificity for cardiomyocytes. Moreover, some of these cardiomyocyte-internalizing aptamers could be linked to Apt30 as a single RNA strand without compromising their internalization efficacy. Supplementing the culture medium with these hybrid aptamers enhanced Ca2+ transients and contractile function in rat cardiomyocytes. These findings provide critical insights for developing novel therapeutics directly acting on PLN in cardiomyocytes, potentially compensating for the disadvantages of conventional methods that involve viral vector-mediated intracellular transduction or alterations in endogenous protein expression.

Progressive amyloid-β accumulation in the brain leads to altered protein expressions in the liver and kidneys of APP knock-in mice

Impaired hepatic and renal function influence Alzheimer's disease (AD) progression; however, whether AD progression affects these important organ functions remains unclear. Here, we investigated the impact of AD progression, characterized by brain amyloid-beta (Aβ) accumulation, on liver and kidney function of AppNL-G-F/NL-G-F (APP-KI) mice using quantitative proteomics. SWATH-based quantitative proteomics revealed changes in mitochondrial, drug metabolism, and pharmacokinetic-related proteins in mouse liver and kidneys during the early (2-month-old) and intermediate (5-month-old) stages of Aβ accumulation. Notably, in 5-month-old APP-KI mouse liver, 25 phase I/II metabolizing enzymes (8 CYPs, 7 UGTs, 7 CESs, and 3 SLCs) and five transporters (2 ABCs and 3 SLCs) were significantly altered; specifically, Ugt1a9 and Slc33a1 protein abundances increased, whereas Ugt1a1 and Abcc3 protein abundances decreased. In the kidneys, 13 phase I/II metabolizing enzymes and 10 ABC-SLC transporters were altered, including Ugt1a6, Ugt1a7, Slc22a7, and Abcb1a. Additionally, plasma proteins, such as albumin and alpha-1-acid glycoprotein, which are critical for drug binding and distribution, were also altered. These results underscore the significant role of progressive brain Aβ accumulation in modifying hepatic and renal protein abundances, potentially influencing drug metabolism and disposition in AD. Our findings provide novel insights into the complex relationship between AD progression and organ dysfunction.

Proteome of Clothianidin Exposed Honey Bees Reveals a Possible Mechanism Behind Impairment of Sucrose Responsiveness

ABSTRACTNeonicotinoids (NNIs) are the most commonly used insecticides in the world and clothianidin, a type of NNI, is commonly found in honey bee collected pollen. Clothianidin has several negative effects on honey bee health and behaviour, but whether and how it might impact learning and memory remains unclear. Therefore, we exposed honey bee workers to a field‐realistic oral dose of clothianidin for 7 days to assess their sugar responsiveness, and olfactory learning and memory using the proboscis extension response paradigm. Sugar responsiveness impacts important colony‐level traits, such as onset of foraging, that help maintain colony homeostasis as well as impacts learning and memory. We then measured how clothianidin alters protein expression in the brain in an effort to understand the mechanism(s) of clothianidin's effects. Clothianidin‐exposed bees had impaired sugar responsiveness, but no effect was seen on learning and memory. We identified 5069 protein groups and showed that the exposed bees had fewer detectable proteins. The exposed bees also had fewer mitochondrial respiration proteins, as well as reduced levels of nicotinic acetylcholine receptor α7 (nAChRα7) subunit (one of the targets of clothianidin) and reduced levels of acetylcholinesterase and acetylcholinesterase‐2 levels. In the exposed bees, the levels of nAChRα7 subunit positively correlated with sugar responsiveness, providing evidence for a possible mechanical explanation.

Novel bi-allelic DNAH3 variants cause oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) is a widespread cause of male infertility. One of the usual clinical manifestations of OAT is multiple morphological abnormalities of the sperm flagella (MMAF), which are frequently associated with mutations and defects in the dynein family. However, the relationship between the newly identified Dynein Axonemal Heavy Chain 3 (DNAH3) mutation and oligonasthenospermia in humans has not yet been established. Whole exome sequencing, pathogenicity analysis, and species conservation analysis of mutation sites were conducted on two patients from different unrelated families with DNAH3 mutations. We identified representative mutation sites and predicted the protein structure following these mutations. The sperm characteristics of the two patients with DNAH3 mutations were verified using Papanicolaou staining, scanning electron microscopy, and transmission electron microscopy. Additionally, mRNA and protein levels were assessed through RT-qPCR and Western blotting. The biallelic mutations in the first progenitor included a heterozygous deletion and insertion, c.6535_6536 delinsAC (to infect mutation (p.Asp2179Thr), and stop codon premutation, c.3249G > A (p.Trp1083Ter). In Family II, the patient (P2) harbored a DNAH3 heterozygous missense mutation, c. 10439G> A(p.Arg3480Gln), along with a stop codon premutation, (c.10260G > A; p.Trp3420Ter). Patients with premature termination of transcription or translation due to DNAH3 mutations exhibit OAT phenotypes, including fibrous sheath dysplasia and multiple tail malformations. We identified the representative sites after mutation, predicted the protein structure, and assessed changes in the protein levels of DNAH3 and related genes following mutations. Notably,a significant reduction in DNAH3 protein expression was validated in these patients. We may explore in the future how DNAH3 affects sperm motility and quality through regulatory mechanisms involving protein structural changes. Novel biallelic mutations in DNAH3, especially those resulting in a premature stop codon, may alter protein expression, structure, and active site, leading to spermatogenic failure and potentially inducing OAT. The discovery of new mutations in DNAH3 may be the key to the diagnosis and treatment of OAT.

Novel Perspectives in the Management of Colorectal Cancer: Mechanistic Investigations Into the Reversal of Drug Resistance via Active Constituents Derived From Herbal Medicine.

The high incidence and mortality rate of colorectal cancer have become a significant global health burden. Chemotherapy has been the traditional treatment for colorectal cancer and has demonstrated promising antitumor effects, leading to significant improvements in patient survival. However, the development of chemoresistance poses a major challenge during chemotherapy in colorectal cancer, significantly impeding treatment efficacy and affecting patient prognosis. Despite the development of a variety of novel anticolorectal cancer chemotherapy agents, their effectiveness and side effects vary, possibly due to the complex mechanisms of resistance in colorectal cancer. Abnormal drug metabolism or protein targets are the most direct causes of resistance. Further studies have revealed that these resistance mechanisms involve biochemical processes such as altered protein expression, autophagy, and epithelial-mesenchymal transitions. Herbal active ingredients offer an alternative treatment option and have shown promise in reversing colorectal cancer drug resistance. This paper aims to summarize the role of various biochemical processes and key protein targets in the occurrence and maintenance of resistance mechanisms in colorectal cancer. Additionally, it elaborates on the mechanisms of action of herbal active ingredients in reversing colorectal cancer drug resistance. The article also discusses the limitations and opportunities in developing novel anticolorectal cancer drugs based on herbal medicine.