GRP94 Research Articles

Dissociations between APP pathology and metabolic signalling in the neuron‐specific hBACE1 knock‐in PLB4 mouse

AbstractBackgroundPatients with Alzheimer’s Disease (AD) frequently suffer comorbidities such as type 2 diabetes mellitus (T2DM) alongside shared common pathologies e.g. increased inflammation, endoplasmic reticulum (ER) stress, and impaired energy homeostasis (Plucinska et al., 2016, https://doi.org/10.1007/s00125-016-3960-1). The complexity of neurodegenerative and metabolic diseases requires a systems‐based approach that examines the interplay between brain pathology and metabolic regulation. Emerging evidence points towards abnormal cellular energy regulation as a common denominator, investigated here in hBACE1 associated pathologies (Findlay et al., 2015, https://doi.org/10.3389/fncel.2015.00382).MethodGlucose tolerance tests were performed in male PLB4 and PLB‐WT controls (3 – 8 months) to determine diabetic status (Plucinska et al., 2014, https://doi.org/10.1523/JNEUROSCI.0433-14.2014). Protein analysis was employed to characterise beta‐amyloid/APP processing, ER stress, glucose regulatory signalling, and bioenergetic metabolites in brain, liver, muscle or plasma, and gene expression analysis examined hypothalamic alterations in energy regulation.ResultNeuronal hBACE1 expression caused early pathological cleavage of APP, in parallel with increased brain ER stress (p‐eIF2α) and the chaperone binding immunoglobulin protein (BIP). APP processing declined over time, alongside increased ER stress (IRE1α) in brain and liver. Systemic glucose intolerance was evident from 3 months, varied between tissues and ages, and was confined to the periphery. All key markers normalised to control levels at 8 months. Evidence for altered bioenergetics metabolites (LDH‐A and BCKDH) in brain and liver, also affected by ageing, were detected in PLB4 mice. Furthermore, hypothalamus‐specific changes in energy and appetite regulators (NPY and GLP‐1 receptor) were uncovered in the hBACE1 mice.ConclusionhBACE1 resulted in early APP misprocessing alongside ER stress but not post‐insulin receptor signalling changes. Peripheral metabolic and bioenergetic alterations were age‐ and tissue‐specific but did not correlate with APP processing. Further tissue analyses are ongoing to determine cellular consequences of hBACE1 expression on systemic bioenergetics.

Integrative Single-Cell RNA-Seq and Single-Cell ATAC-Seq Analysis of Human Plasma Cell Differentiation

Plasma cells (PC) are highly specialized cells representing the end stage of B cell differentiation. They play an important role in humoral immunity by synthesizing and secreting antibodies protecting the host against infections. We have shown that PC differentiation (PCD) can be reproduced in vitrofrom memory B cells (MBC). MBC differentiate into CD20 low/-CD38 - pre-plasmablasts (prePB), CD20 -CD38 +CD138 - plasmablasts (PB) and CD20 -CD38 +CD138 + PC. The molecular changes occurring during PCD are recapitulated in this in vitrodifferentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drives the early stages of PCD. We combined single-cell (sc) RNA-seq and scATAC-seq to decipher the trajectories involved in PCD. scRNA-seq experiments on the four populations (MBC, prePB, PB and PC) generated during normal PCD revealed a highly specific transcriptomic profile for MBC and PC, and a strong heterogeneity of the prePB and PB. The prePB stage presented the most differentially expressed genes (DEG) with almost 2000 DEG showing that the most important changes take place during this stage. Epigenetic analysis using scATAC-seq technology showed that prePB, PB and PC stages were clearly separated from the MBC highlighting chromatin remodeling induced by B cell activation and differentiation. As observed on the transcriptomic level, the number of differentially accessible peaks was higher in prePB (4660) than in other stages (MBC: 641; PB: 44; PC: 105). Among genes that were commonly identified using scATAC-seq and RNA-seq, we identified several TF such as BATF and BATF3 in prePB. Interestingly, differentially accessible peaks characterizing the prePB stage were enriched in motifs of BATF3, FOS and BATF belonging to the TF AP-1 family and among these differentially accessible peaks, 38% of peaks presented BATF3 motif. Since BATF3 TF was previously identified operating in short impulse manner at prePB stage, BATF3 target genes may represent a key transcriptional node involved in PCD. The integration of the chromatin accessibility and transcriptomic data revealed a more mature population of prePB cells characterized by open chromatin in PC genes without significant expression suggesting that a population of prePB were already committed to become antibody-secreting cells. To focus on the understanding of the processes occurring during the transition from prePB to PB which remains largely unknown, we computationally clustered and ordered cells. Pseudotemporal analyses underlined maturation trajectories in prePB with early prePB characterized by downregulation of B cell markers ( CD19, CD22, CD83, CCR7, CCL17 and CCL22) and B cell TF (SPIB, PAX5, STAT5A, RUNX3 and BATF3) together with upregulation of PC markers, adhesion molecules and growth factor receptors ( CD27, CD38, SLAMF7, BCMA, ITGA4, IL-6R, IL-6ST and INSR). The transition from early prePB to more mature prePB is associated with downregulation of AICDA and PRC2 complex ( EZH2, EED). In the transitional prePB, a first wave of UPR activation is observed, associated with mTORC1 pathway activation. In these prePB, HSAP5, also called binding immunoglobulin protein (BiP) is overexpressed together with ERN1, leading to the activation of the Ire1 pathway, known to splice XBP-1 (sXBP-1) producing a highly active TF. Then, prePB overexpress EGR1, FOS, CXCR4 and TFRC. Mature prePB overexpress KLF2 that participate in bone marrow PC homing. Then, PB overexpress gene involved in conventional UPR and protein secretion associated to immunoglobulin gene expression. Altogether, these data underlined that prePB present already UPR priming through mTORC1 pathway activation to prepare for PC function. XBP1 driven UPR activation will be coordinated in PB related to antibody synthesis increase. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of prePB already undergone epigenetic remodeling related to PC profile together with UPR activation and are committed to differentiate in PC. These results and the supporting data generated with our PCD model provide a resource for the identification of molecular circuits that are crucial for early and mature PC biological function and survival. These data thus provide critical insights into epigenetic- and transcriptional-mediated reprogramming events that sustain PCD.

Efficacy of Protac-Based EZH2 Degrader MS1943 in Lymphoid Malignancies: A Comprehensive Study on the Combined Effects of MS1943 and Ibrutinib

Introduction: This study delves into the therapeutic potential of the Enhancer of zeste homolog 2 (EZH2) degrader MS1943 and the Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib in B-cell lymphomas. The study's primary objective was to compare the efficacy of MS1943, a Proteolysis-targeting chimera (PROTAC)-based degrader, with the FDA-approved EZH2 inhibitor Tazemetostat. The study also aimed to explore the potential synergistic effects of combining these therapies with Ibrutinib, a widely used BTK inhibitor in lymphoma treatment. Materials and Methods: The study employed a range of lymphoma cell lines, including B-cell and T-cell lymphomas, which were treated with MS1943, Tazemetostat, and Ibrutinib, either alone or in combination. The impact of these treatments on cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay at different time points and concentrations. Cell cycle distribution was assessed using flow cytometric analysis, and the expression of downstream effectors of the unfolded protein response (UPR) pathway, including Xbp1, Chop, and Bip, was examined using quantitative polymerase chain reaction (qPCR). Western blot analysis was performed to investigate the association with specific apoptosis-related pathways. Results: The results demonstrated that MS1943 exhibited significantly greater inhibitory effects than Tazemetostat in all lymphoma cell lines tested, particularly in B-cell lymphomas. MS1943 treatment resulted in increased expression of UPR pathway effectors, suggesting a correlation between the observed anti-proliferative effects and the differential expression of these factors. The combination of MS1943 and Ibrutinib demonstrated significant inhibitory effects across all B-cell lymphoma cell lines, with the most prominent cytotoxicity observed after 72 hours of culture. The combination treatment resulted in an increase in the number of cells in the G2/M phase, accompanied by a decrease in the number of cells in the G0/G1 phase. Furthermore, the combination treatment led to a notably higher proportion of apoptotic and necroptotic cells compared to either individual treatment or the combination of Ibrutinib and Tazemetostat. The combination treatment resulted in an increase in the expression of p53 and PUMA, and a significant increase in miR29b expression. Discussion: Our findings suggest that the simultaneous treatment of MS1943 and Ibrutinib in B-cell lymphoma induces apoptosis through the miR29b-mediated p53 upregulated pathway, thereby exhibiting potent anti-tumor effects. The study provides compelling evidence that MS1943 exhibits a pronounced inhibitory effect on B-cell lymphomas compared to Tazemetostat, highlighting its potential as a promising therapeutic agent in the treatment of B-cell lymphomas. The combination of MS1943 and Ibrutinib enhances apoptotic and necroptotic cell death, resulting in a significant reduction in viable cells. This study provides valuable insights into the mechanisms underlying the anti-tumor effects of this drug combination, highlighting the potential of MS1943 as an effective treatment option for lymphoma.

Evolution of Invasive and Colonizing Pediatric Staphylococcus aureus Isolates

Abstract Background Staphylococcus aureus is the most common bacterial pathogen isolated in children with invasive bloodstream and musculoskeletal infections. A major barrier to vaccine and novel therapeutic development against S. aureus is its continuous evolution. Many recent and current vaccine candidates target antigens that may have appeared crucial to its pathogenesis, only to see these factors recede from circulating invasive strains. Characterization of clinically relevant S. aureus isolates over time is necessary to find targets for novel therapeutics. Efforts toward new interventions should target factors that remain prevalent in invasive isolates over time. Methods We obtained 131 invasive S. aureus isolates from children admitted to the Monroe Carell Jr. Children's Hospital at Vanderbilt from 2010 to 2022, and 246 colonizing isolates. Whole genome sequences were obtained using the Illumina sequencing platform. Virulence factors and multi-locus sequence type (MLST) were determined using Geneious software and through PubMLST, an open-access curated database. Results There were 10 unique clonal complexes (CC) identified among 320 isolates. CC8 remains the most common invasive clonal complex, though CC8 is significantly less frequent now than in 2010-2012. Diversity of invasive strains has increased substantially, with the emergence of CC5 and CC121 lineages causing invasive disease in children. CC1 was found exclusively in colonization isolates. Virulence factor prevalence has changed significantly over time. Panton-Valentine leucocidin (PVL), enterotoxins K and Q (SEK/SEQ), and the pathogenic arginine catabolic mobile element (ACME), have decreased significantly in invasive S. aureus isolates since 2010, from 53-70% to 21-35% of current isolates. Leukocidin ED (LukED) and surface protein staphylokinase (Sak) are strongly associated with invasive isolates compared to colonization isolates. Conversely, toxic shock toxin (TST) and Staphylococcal enterotoxin B (SEB) were seen more commonly in colonization isolates (12-15%) compared to invasive isolates (5-7%). The genes encoding for gamma hemolysins (HlgA-C), leukocidin AB (LukAB), iron-regulated surface proteins (IsdA, IsdB), staphylococcal binding immunoglobulin protein (sbi), extracellular fibrinogen-binding protein (Efb), and clumping factors A and B (ClfA, ClfB) were identified in all isolates tested. Figure 1 Figure 2 Conclusions Significant shifts have occurred over the past decade in the predominant circulating S. aureus strains and their virulence factor repertoires. The once-dominant CC8 (USA300 clone) has receded, and with that has come a significant reduction in some factors once thought crucial for pathogenesis. These findings also have implications for infection prevention and control practices, as eradication of colonizing strains with low potential for pathogenicity may inadvertently allow for replacement by more virulent strains.

IRE1α/XBP-1 promotes β-catenin signaling activation of airway epithelium in lipopolysaccharide-induced acute lung injury

BackgroundAcute lung injury (ALI), along with the more severe condition--acute respiratory distress syndrome (ARDS), is a major cause of respiratory failure in critically ill patients with high morbidity and mortality. Inositol-requiring protein 1α (IRE1α)/X box protein-1 (XBP1) pathway was proved to regulate lipopolysaccharide (LPS)-induced lung injury and inflammation. Yet, its role on epithelial β-catenin in LPS-induced ALI remains to be elucidated. MethodsLPS-induced models were generated in mice (5 mg/kg) and Beas-2B cells (200 μg/mL). Two selective antagonists of IRE1α (4μ8c and STF-083010) were respectively given to LPS-exposed mice and cultured cells. ResultsUp-regulated expression of endoplasmic reticulum (ER) stress markers immunoglobulin-binding protein (BIP) and spliced X box protein-1(XBP-1s) was detected after LPS exposure. Besides, LPS also led to a down-regulated total β-catenin level in the lung and Beas-2B cells, with decreased membrane distribution as well as increased cytoplasmic and nuclear accumulation, paralleled by extensively up-regulated downstream targets of the Wnt/β-catenin signaling. Treatment with either 4μ8c or STF-083010 not only significantly attenuated LPS-induced lung injury and inflammation, but also recovered β-catenin expression in airway epithelia, preserving the adhesive function of β-catenin while blunting its signaling activity. ConclusionThese results illustrated that IRE1α/XBP1 pathway promoted the activation of airway epithelial β-catenin signaling in LPS-induced ALI.

240 pH dependence of second immunoglobulin binding protein (Sbi) secretion by S aureus: Implications for atopic dermatitis immunopathogenesis

240 pH dependence of second immunoglobulin binding protein (Sbi) secretion by S aureus: Implications for atopic dermatitis immunopathogenesis

Antioxidants restore store-operated Ca2+ entry in patient-iPSC-derived myotubes with tubular aggregate myopathy-associated Ile484ArgfsX21 STIM1 mutation via upregulation of binding immunoglobulin protein.

Store-operated Ca2+ entry (SOCE) is indispensable for intracellular Ca2+ homeostasis in skeletal muscle, and constitutive activation of SOCE causes tubular aggregate myopathy (TAM). To understand the pathogenesis of TAM, we induced pluripotent stem cells (iPSCs) from a TAM patient with a rare mutation (c.1450_1451insGA; p. Ile484ArgfsX21) in the STIM1 gene. This frameshift mutation produces a truncated STIM1 with a disrupted C-terminal inhibitory domain (CTID) and was reported to diminish SOCE. Myotubes induced from the patient's-iPSCs (TAM myotubes) showed severely impaired SOCE, but antioxidants greatly restored SOCE partly via upregulation of an endoplasmic reticulum (ER) chaperone, BiP (GRP78), in the TAM myotubes. Our observation suggests that antioxidants are promising tools for treatment of TAM caused by reduced SOCE.

Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants.

The GRIN genes encoding N-methyl-D-aspartate receptor (NMDAR) subunits are remarkably intolerant to variation. Many pathogenic NMDAR variants result in their protein misfolding, inefficient assembly, reduced surface expression, and impaired function on neuronal membrane, causing neurological disorders including epilepsy and intellectual disability. Here, we investigated the proteostasis maintenance of NMDARs containing epilepsy-associated variations in the GluN2A subunit, including M705V and A727T. In the transfected HEK293T cells, we showed that the two variants were targeted to the proteasome for degradation and had reduced functional surface expression. We demonstrated that the application of BIX, a known small molecule activator of an HSP70 family chaperone BiP (binding immunoglobulin protein) in the endoplasmic reticulum (ER), dose-dependently enhanced the functional surface expression of the M705V and A727T variants in HEK293T cells. Moreover, BIX (10 μM) increased the surface protein levels of the M705V variant in human iPSC-derived neurons. We revealed that BIX promoted folding, inhibited degradation, and enhanced anterograde trafficking of the M705V variant by modest activation of the IRE1 pathway of the unfolded protein response. Our results suggest that adapting the ER proteostasis network restores the folding, trafficking, and function of pathogenic NMDAR variants, representing a potential treatment for neurological disorders resulting from NMDAR dysfunction.

Nano fraction of pesticide induces genotoxicity and oxidative stress-dependent reticulum stress.

The implementation of nanotechnology in different sectors has generated expectations as a new source of use due to the novel characteristics that it will bring. Particularly, nano pesticides promise to be more sustainable and less harmful to the ecosystem and human health; however, most studies continue to focus on their efficacy in the field, leaving aside the effect on humans. This project aimed to evaluate the genotoxic effect of a nano-encapsulated pesticide on bronchial epithelial cells (NL-20) in vitro and elucidate the mechanism through which they induce damage. The nano fraction (NF) of the pesticide Karate Zeon® 5 CS was characterized and isolated, and the uptake into the cell and the changes induced in the cellular ultrastructure were evaluated. In addition, the primary markers of oxidative stress, reticulum stress, and genotoxicity were assessed using the micronucleus test. A 700 nm fraction with a Z potential of -40 mV was obtained, whose main component is polyurea formaldehyde; this allows the capsules to enter the cell through macropinocytosis and clathrin-mediated endocytosis. Inside, they induce oxidative stress activating a reticulum stress response via the BIP protein and the IRE-1 sensor, triggering an inflammatory response. Likewise, stress reduces cell proliferation, increasinggenotoxic damage through micronuclei; however, this damage is mainly induced by direct contact of the capsules with the nucleus. This pioneering study uses a nanometric encapsulated commercial pesticide to evaluate the molecular mechanism of induced damage. It makes it the first step in analyzing whether these substances represent a contaminant or an emerging solution.

Pentapeptide PYRAE triggers ER stress-mediated apoptosis of breast cancer cells in mice by targeting RHBDF1-BiP interaction.

Reinforced cellular responses to endoplasmic reticulum (ER) stress are caused by a variety of pathological conditions including cancers. Human rhomboid family-1 protein (RHBDF1), a multiple transmembrane protein located mainly on the ER, has been shown to promote cancer development, while the binding immunoglobulin protein (BiP) is a key regulator of cellular unfolded protein response (UPR) for the maintenance of ER protein homeostasis. In this study, we investigated the role of RHBDF1 in maintaining ER protein homeostasis in breast cancer cells. We showed that deleting or silencing RHBDF1 in breast cancer cell lines MCF-7 and MDA-MB-231 caused marked aggregation of unfolded proteins in proximity to the ER. We demonstrated that RHBDF1 directly interacted with BiP, and this interaction had a stabilizing effect on the BiP protein. Based on the primary structural motifs of RHBDF1 involved in BiP binding, we found a pentapeptide (PE5) targeted BiP and inhibited BiP ATPase activity. SPR assay revealed a binding affinity of PE5 toward BiP (Kd = 57.7 μM). PE5 (50, 100, 200 μM) dose-dependently promoted ER protein aggregation and ER stress-mediated cell apoptosis in MCF-7 and MDA-MB-231 cells. In mouse 4T1 breast cancer xenograft model, injection of PE5 (10 mg/kg, s.c., every 2 days for 2 weeks) significantly inhibited the tumor growth with markedly increased ER stress and apoptosis-related proteins in tumor tissues. Our results suggest that the ability of RHBDF1 to maintain BiP protein stability is critical to ER protein homeostasis in breast cancer cells, and that the pentapeptide PE5 may serve as a scaffold for the development of a new class of anti-BiP inhibitors.

ER stress modulated Klotho restoration: A prophylactic therapeutic strategy against acute kidney injury-diabetes comorbidity

Klotho is a renoprotective factor that is at the forefront of research as a potential therapeutic agent and biomarker of acute kidney injury (AKI). Endoplasmic reticulum (ER) stress and Klotho downregulation are the critical hallmarks of AKI progression. Importantly, the crosstalk between ER and Klotho is still elusive in AKI under diabetic condition. Therefore, this study aimed to elucidate the affiliation between ER stress and Klotho regulation by using the ischemia-reperfusion renal injury (IRI) model based on male Wistar rats and the hypoxia-reperfusion injury (HRI) using NRK52E cells. Study outcomes demonstrated that the expression of AKI biomarkers: plasma creatinine, neutrophil gelatinase-associated lipocalin, kidney-injury molecule 1, and ER stress markers such as binding immunoglobulin binding protein (BiP), R/PKR-like ER kinase (PERK), and eukaryotic initiation factor-2 (eIF2α), were observed during AKI. Increased ER stress was associated with apoptosis induction as depicted by increased levels of Poly (ADP-ribose) polymerase (PARP) and caspase-7 and decreased tubular Klotho expression. Under diabetic settings, ER stress and apoptosis were exacerbated by additional Klotho downregulation. Treatment with Tauroursodeoxycholic acid (TUDCA) inhibited the ER stress, apoptosis, restored endogenous Klotho levels and ameliorated AKI under diabetic and non-diabetic conditions. ER stress and Klotho appear to be shared factors involved in the pathogenesis of AKI-diabetes comorbidity and targeting them could prove a novel therapeutic approach.

The stability of FKBP9 maintained by BiP is crucial for glioma progression

FK506-binding protein 9 (FKBP9) is involved in tumor malignancy by resistance to endoplasmic reticulum (ER) stress, and the up-regulation of FKBP9 is associated with patients' poor prognosis. The current knowledge of the molecular mechanisms is still limited. One previous study showed that FKBP9 could confer glioblastoma cell resistance to ER stress through ASK1-p38 signaling. However, the upstream regulatory mechanism of FKBP9 expression is still indistinct. In this study, we identified the FKBP9 binding proteins using co-immunoprecipitation followed by mass spectrometry. Results showed that FKBP9 interacted with the binding immunoglobulin protein (BiP). BiP bound directly to FKBP9 with high affinity. BiP prolonged the half-life of the FKBP9 protein and stabilized the FKBP9 protein. BiP and FKBP9 protein levels were positively correlated in patients with glioma, and patients with high expression of BiP and FKBP9 showed a worse prognosis. Further studies showed that FKBP9 knockout in genetically engineered mice inhibited intracranial glioblastoma formation and prolonged survival by decreasing cellular proliferation and ER stress-induced CHOP-related apoptosis. Moreover, normal cells may depend less on FKBP9, as shown by the absence of apoptosis upon FKBP9 knockdown in a non-transformed human cell line and overall normal development in homozygous knockout mice. These findings suggest an important role of BiP-regulated FKBP9-associated signaling in glioma progression and the BiP–FKBP9 axis may be a potential therapeutic target for glioma.

The interaction network of the proteasome assembly chaperone PSMD9 regulates proteostasis.

Functional networks in cells are created by physical, genetic, and regulatory interactions. Mapping them and annotating their functions by available methods remains a challenge. We use affinity purification mass spectrometry (AP-MS) coupled with SLiMFinder to discern such a network involving 26S proteasome non-ATPase regulatory subunit 9 (PSMD9), a chaperone of proteasome assembly. Approximately 20% of proteins within the PSMD9 interactome carry a short linear motif (SLiM) of the type 'EXKK'. The binding of purified PSMD9 with the peptide sequence ERKK, proteins heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2B1; containing ERKK), and peroxiredoxin-6 (PRDX6; containing EAKK) provided proof of principle for this motif-driven network. The EXKK motif in the peptide primarily interacts with the coiled-coil N domain of PSMD9, a unique interaction not reported for any coiled-coil domain. PSMD9 knockout (KO) HEK293 cells experience endoplasmic reticulum (ER) stress and respond by increasing the unfolded protein response (UPR) and reducing the formation of aggresomes and lipid droplets. Trans-expression of PSMD9 in the KO cells rescues lipid droplet formation. Overexpression of PSMD9 in HEK293 cells results in reduced UPR, and increased lipid droplet and aggresome formation. The outcome argues for the prominent role of PSMD9 in maintaining proteostasis. Probable mechanisms involve the binding of PSMD9 to binding immunoglobulin protein (BIP/GRP78; containing EDKK), an endoplasmic reticulum chaperone and key regulator of the UPR, and fatty acid synthase (FASN; containing ELKK), involved in fatty acid synthesis/lipid biogenesis. We propose that PSMD9 acts as a buffer in the cellular milieu by moderating the UPR and enhancing aggresome formation to reduce stress-induced proteotoxicity. Akin to waves created in ponds that perpetuate to a distance, perturbing the levels of PSMD9 would cause ripples down the networks, affecting final reactions in the pathway, one of which is altered proteostasis.

The Endoplasmic Reticulum Is a Key Battleground between Phytoplasma Aggression and Host Plant Defense.

Phytoplasmas are intracellular plant pathogens that heavily rely on host cell nutrients for survival and propagation due to their limited ability to synthesize essential substrates. The endoplasmic reticulum (ER), which plays a vital role in various cellular processes, including lipid and protein biosynthesis, is an attractive target for numerous intracellular pathogens to exploit. This study investigated the impact of potato purple top (PPT) phytoplasma infection on the ER in tomato plants. Abnormal accumulation of ER-resident proteins, disrupted ER network structures, and formation of protein aggregates in the phloem were observed using confocal microscopy and transmission electron microscopy, indicating a phytoplasma-infection-induced disturbance in ER homeostasis. The colocalization of phytoplasmas with the accumulated ER-resident proteins suggests an association between ER stress, unfolded protein response (UPR) induction, and phytoplasma infection and colonization, with the ER stress response likely contributing to the host plant's defense mechanisms. Quantitative real-time PCR revealed a negative correlation between ER stress/UPR activation and PPT phytoplasma titer, implying the involvement of UPR in curbing phytoplasma proliferation. Inducing ER stress and activating the UPR pathway effectively decreased phytoplasma titer, while suppressing the ER-resident protein, binding immunoglobulin protein (BiP) increased phytoplasma titer. These results highlight the ER as an intracellular battleground where phytoplasmas exploit host components for survival and multiplication, while host plants deploy defense mechanisms to counteract the invasion. Understanding the intricate interactions between phytoplasmas and plant hosts at the subcellular level, particularly within the ER, provides valuable insights for developing new strategies to control phytoplasma diseases.

Regulation of cytokinesis and necroptosis pathways by diosgenin inhibits the proliferation of NCI-H460 lung cancer cells

AimOvercoming resistance to apoptosis and antimitotic chemotherapy is crucial for effective treatment of lung cancer. Diosgenin (DG), a promising phytochemical, can regulate various molecular pathways implicated in tumor formation and progression. However, the precise biological activity of DG in lung cancer remains unclear. This study aimed to investigate the antiproliferative activity of DG in NCI-H460 lung carcinoma cells to explore the underlying antimitotic mechanisms and alternative cell death pathways. Materials and methodsIn a 2D culture system, we analyzed cell viability, multinucleated cell frequency, cell concentration, cell cycle changes, cell death induction, intracellular reactive oxygen species (ROS) production, and nuclear DNA damage, particularly in relation to target gene expression. We also evaluated the antiproliferative activity of DG in a 3D culture system of spheroids, assessing volume changes, cell death induction, and inhibition of proliferation recovery and clonogenic growth. Key findingsDG reduced cell viability and concentration while increasing the frequency of cells with multiple nuclei, particularly binucleated cells resulting from daughter cell fusion. This effect was associated with genes involved in cytokinesis regulation (RAB35, OCRL, BIRC5, and AURKB). Additionally, DG-induced cell death was linked to necroptosis, as evidenced by increased intracellular ROS production and RIPK3, MLKL, TRAF2, and HSPA5 gene expression. In tumor spheroids, DG increased spheroid volume, induced cell death, and inhibited proliferation recovery and clonogenic growth. SignificanceOur study provides new insights into the biological activities of DG in lung cancer cells, contributing to the development of novel oncological therapies.

Ruta graveolens: Boost Melanogenic Effects and Protection against Oxidative Damage in Melanocytes.

Vitiligo, an acquired depigmentation disorder, is characterized by the loss of functional melanocytes and epidermal melanin. In recent years, research has focused on promoting melanin biosynthesis and protecting melanocytes to reduce stress-related damage for the purpose of applying it to vitiligo treatment. Ruta graveolens L. has been utilized as a medicinal herb in diverse traditional medicine systems to address conditions like vitiligo. In this investigation, we isolated and purified 16 unique alkaloid compounds from the chloroform extracts of R. graveolens, encompassing a new quinoline alkaloid and several recognized compounds. Bioactivity analysis showed that compound 13, an alkaloid derived from R. graveolens, promotes melanin production while protecting PIG3V melanocytes against 4-tert-butylphenol (4-TBP)-induced oxidative damage by downregulating endoplasmic reticulum (ER) stress and pro-inflammatory cytokines through interleukin-6 (IL-6) regulation. Additionally, the compound suppressed the expression of Bip, IRE1, p-IRE1, and XBP-1 proteins, suggesting a potential antioxidant function. These findings suggest that compound 13 isolated from R. graveolens can augment melanogenesis in melanocytes, reduce endoplasmic reticulum (ER) stress, and ameliorate vitiligo exacerbation. The melanogenic activity observed in the chloroform fraction emphasizes R. graveolens's potential as a novel therapeutic target for vitiligo treatment, warranting further exploration in future studies.

AMPK Mediates Early Activation of the Unfolded Protein Response through a Positive Feedback Loop in Palmitate-Treated Muscle Cells.

Activation of the unfolded protein response (UPR) is closely related to the pathogenesis of many metabolic disorders. Accumulating evidence also shows that UPR and metabolic signaling pathways are interdependent. The AMP-activated protein kinase (AMPK) signal pathway controls the energy balance of eukaryotes. The aim of this study was therefore to investigate the possible interaction between AMPK signaling and UPR in muscle cells exposed to saturated fatty acids, as well as the potential mechanism. The saturated fatty acid palmitate was used to induce UPR in C2C12 myotubes. Compound C or knockdown of AMPKα with short hairpin RNA (shRNA) were used to inhibit the AMPK signaling pathway in palmitate-treated muscle cells. AMPK signaling in myotubes was activated using 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICAR) or ex229. C2C12 myotubes were pre-treated with taurourdodeoxycholic acid (TUDCA) to inhibit UPR before adding palmitate. Real-time PCR and Western blotting were performed to evaluate the expression of UPR markers and activation of AMPK. Palmitate treatment induced UPR in C2C12 myotubes while activating AMPK signaling. Inhibition of the AMPK pathway with compound C or AMPK shRNA reduced palmitate-induced activation of UPR, while inhibition of UPR with TUDCA reduced palmitate-induced AMPK activation. This indicates a positive feedback loop between UPR and AMPK. Furthermore, activation of the AMPK pathway with AICAR or ex229 caused a dose-dependent upregulation of UPR markers, including activating transcription factor 4 (ATF4), binding immunoglobulin protein (BIP), and growth arrest and DNA damage-inducible 34 (GADD34) protein. These results provide the first evidence that AMPK signaling is involved in the early activation of UPR caused by saturated fatty acids in skeletal muscle. Furthermore, they indicate that physiological or pharmacological activation of the AMPK pathway (e.g., by exercise or phenformin, respectively) can promote muscle health and function, thereby improving the quality of life in individuals with metabolic disorders due to a high-fat diet or obesity.

Differential Protein Expression in Response to Varlitinib Treatment in Oral Cancer Cell Line: an In Vitro Therapeutic Approach.

Epidermal growth factor receptor (EGFR) is the most frequently overexpressed receptor histologically exhibited by oral squamous cell carcinoma (OSCC) patients. Aberrated EGFR signaling may lead to recurrence and metastasis, thus laying the foundation of targeted therapy. Deactivating EGFR is likely to prevent downstream signaling thus resulting in apoptosis. Tyrosine kinase inhibitors (TKIs) have come into play to revert aggressiveness of OSCC. We exploited comparative proteomic analyses based on anti-EGFR potential of varlitinib, using cellular proteomes from treated and untreated groups of oral cancer cells to identify protein players functional during oral carcinogenesis. Following separation by two-dimensional electrophoresis, differentially expressed cellular proteins (varlitinib-treated and untreated cells) were analyzed and later identified using QTOF mass spectrometer. In silico analysis for protein-protein interaction was carried out using STRING. Six differentially expressed proteins were identified as binding immunoglobulin protein (BiP), heat shock protein 7 C (HSP7C), protein disulfide isomerase 1 A (PDIA1), vimentin (VIME), keratin type I cytoskeletal 14 (K1C14), and β-Actin (ACTB). Relative expression of five proteins was found to be downregulated upon varlitinib treatment, whereas only K1C14 was upregulated in treated cells compared to control. Protein network analysis depicts the interaction between BiP, PDIA1, VIME, etc. indicating their role in oral carcinogenesis. Oral cancer cells show proteome shift based on varlitinib treatment compared to corresponding controls. Our data suggest candidature of varlitinib as a potent therapeutic agent and BiP, PDIA1, HSP7C, VIME, and β-Actin as complementary/prognostic markers of OSCC.

Comparison of Three Lateral Flow Immunoassay Formats for the Detection of Antibodies against the SARS-CoV-2 Antigen.

Reliable detection of specific antibodies against pathogens by lateral flow immunoassay (LFIA) greatly depends on the composition of the detectable complex and the order of its assembly. We compared three LFIA formats for revealing anti-SARS-CoV-2 antibodies in sera with the following detected complexes in the analytical zone of the strip: antigen-antibodies-labeled immunoglobulin-binding protein (Scheme A); antigen-antibodies-labeled antigen (Scheme B); and immunoglobulin-binding protein-antibodies-labeled antigen (Scheme C). The lowest detection limit was observed for Scheme C, and was equal to 10 ng/mL of specific humanized monoclonal antibodies. When working with pooled positive sera, Scheme C had a detection limit 15 times lower than Scheme B and 255 times lower than Scheme A. Due to the high sensitivity of Scheme C, its application for the panel of human sera (n = 22) demonstrated 100% diagnostic specificity and sensitivity. These consistent results be useful for designing the format of LFIA serodiagnosis for other diseases.

High fat diet induces brain injury and neuronal apoptosis via down-regulating 3-β hydroxycholesterol 24 reductase (DHCR24).

Hyperlipidemia (HLP) is one of the risk factors for memory impairment and cognitive impairment. However, its pathological molecular mechanism remained unclear. 3β-hydroxysterol Δ24- reductase (DHCR24) is a key enzyme in cholesterol synthesis and has been reported to decrease in the affected areas in the brain of neurodegenerativedisorders. In this study, hyperlipidemic mouse model was established to study the effect of high blood lipid on brain. The data obtained from HPLC analysis demonstrated that the cholesterol level in the brain of mice with hyperlipidemia was significantly elevated compared to the control group. While the pathological damages were observed in both cerebral cortex and hippocampus in the brain of hyperlipidemic mice. Furthermore, the protein level of DHCR24 was downregulated accompanied by elevated ubiquitination level in the hyperlipidemic mice brain. The mouse neuroblastoma cells N2a were exposed to the excess cholesterol loading, the cells underwent apoptosis and the mRNA and protein of DHCR24 in cholesterol-loaded N2a cells were significantly reduced. In addition, the expression level of endoplasmic reticulum stress marker protein (Bip and Chop) was markedly increased in response to the cholesterol loading. More importantly, overexpression of DHCR24 in N2a reversed neuronal apoptosis induced by the cholesterol loading. Conclusively, these findings suggested that hyperlipidemia could cause brain tissue injuries via down-regulating DHCR24, and overexpression of DHCR24 may alleviate hyperlipidemia-induced neuronal cells damage by reversing the endoplasmic reticulum stress-mediated apoptosis.