Downregulation Of Genes Research Articles

FDA-approved disulfiram induces ferroptosis via alteration of redox balance and lipid peroxidation and overcomes carfilzomib resistance in multiple myeloma

Multiple myeloma (MM), a malignant plasma cell disorder, remains incurable due to inevitable chemo-resistance development, including carfilzomib (CFZ) leading to relapse and poor patient outcomes. Therefore, new treatment strategies, including using FDA-approved alcohol deterrent disulfiram (DSF) are under investigation. The present study investigated the effect of DSF on ferroptosis and CFZ resistance in MM cells. Our findings indicate that DSF increases the production of cytosolic and mitochondrial reactive oxygen species, and causes a loss of mitochondrial Δψ and an elevation in lipid peroxidation in MM cells. DSF treatment in MM cell lines led to the significant downregulation of ferroptotic genes, including glutathione peroxidase 4. Moreover, ferroptosis inhibitor liproxstatin-1rescued DSF-induced ferroptosis by promoting glutathione peroxidase 4 upregulation. DSF alone overcomes CFZ resistance through lipid peroxidation elevation and acts synergistically with CFZ in CFZ-resistant MM cell lines. Our results suggest that DSF is a promising anti-myeloma agent for overcoming CFZ resistance in MM through ferroptosisinduction.

CNSC-17. UPREGULATION OF CCL4 AFTER TREATMENT WITH A NOVEL MRNA VACCINE SHIFTS THE PHENOTYPE OF CCR5-EXPRESSING MICROGLIA WITHIN TUMOR MICROENVIRONMENT

Abstract INTRODUCTION Intra-tumoral immunosuppression is a major cause of treatment failure for patients with glioblastoma (GBM). Our group has developed a proprietary vaccine that combines mRNA encapsulated in lipid nanoparticles with PEG hydrogel and a chemokine (CXCL9). This hydrogel-chemokine-mRNA (HCM) vaccine is delivered subcutaneously (SQ) and results in significant anti-tumor efficacy in syngeneic murine glioma models. The objective of this study was to evaluate the effects of the vaccine on the tumor microenvironment (TME). METHODS C57BL/6 mice underwent intracranial implantation of KR158-luc tumor cells and underwent SQ injection of the HCM vaccine using total tumor mRNA. Tumors were collected for analysis at different time points. Q-PCR and flow cytometry were done to evaluate the differential gene expression and CCR5 expression (receptor for CCL4) in the TME following HCM vaccination. RESULTS CCL4 was found to be upregulated in tumor-bearing animals treated with HCM vaccine using PCR. In a separate experiment, CCL4 blockade was given prior to HCM vaccination and this resulted in abrogation of the survival benefit of the vaccine. Next, flow cytometry results revealed that microglia in the TME had the highest expression of the CCR5 receptor. Further, QPCR on the isolated microglia from TME revealed significant up-regulation of inflammatory gene CCL5 (25 fold) and down-regulation of anti-inflammatory genes as compared to untreated control and empty hydrogel control. CONCLUSION The HCM vaccine results in significant anti-tumor efficacy in murine syngeneic murine glioma which is dependent on upregulation of CCL4 in the TME. The CCL4 exerts its effect through microglia expressing CCR5 in the TME. HCM vaccination shifted the microglia phenotype from immune-suppression to inflammatory state. Further analysis of the effect of CCR5 knock out on the HCM vaccine survival efficacy are under current investigation.

Dual effects of DLG5 (disks large homolog 5 gene) modulation on chemotherapy-induced thrombocytopenia and nausea/vomiting via the hippo signalling pathway.

The CAPEOX (combination of oxaliplatin and capecitabine) chemotherapy protocol is widely used for colorectal cancer treatment, but it can lead to chemotherapy-induced adverse effects (CRAEs). To uncover the mechanisms and potential biomarkers for CRAE susceptibility, we performed whole-genome sequencing on normal colorectal tissue (CRT) before adjuvant chemotherapy. This is followed by in vivo and in vitro verifications for selected gene and CRAE pair. Our analysis revealed specific germline mutations linked to Grade 2 (or higher) chemotherapy-induced thrombocytopenia (CIT) and nausea/vomiting (CINV). Notably, both CRAEs were associated with mutations in the DLG5 gene. We found that DLG5 mutations related to CIT were associated with increased gene expression, while those associated with CINV were linked to suppressed gene expression, as indicated by the Genotype-Tissue Expression (GTEX) database. In megakaryocytes, overexpression of human DLG5 suppressed the hippo signalling pathway and induced YAP expression. In zebrafish, overexpression of human DLG5 not only reduced platelet production but also inhibited thrombus formation. Subsequent qPCR analysis revealed that DLG5 overexpression affected genes involved in cytoskeleton formation and alpha-granule formation, which could impact the normal generation of proplatelets. We identified a series of germline mutations associated with susceptibility to CIT and CINV. Of particular interest, we demonstrated that induced and suppressed DLG5 expression is respectively related to CIT and CINV. These findings shed light on the involvement of the hippo signalling pathway and DLG5 in the development of CRAEs, providing valuable insights into potential targets for therapeutic interventions.

IMMU-15. CD74/MIF SIGNALING AXIS DISRUPTION IN BONE MARROW-DERIVED MYELOID CELLS DELAYS MALIGNANT PROGRESSION IN A PRECLINICAL MODEL OF GLIOMA

Abstract INTRODUCTION CD74, a chaperone associated with the major histocompatibility complex class II (MHC-II), plays a crucial role in assembling and transporting MHC-II molecules on the cellular surface. Beyond its physiological function in antigen presentation, CD74 is a negative regulator of inflammation, and its dysregulation is associated with inflammatory disorders and cancer. Notably, tumor stem cells release macrophage migration inhibitory factor (MIF) to trigger CD74 immunosuppressive signaling in tumor-infiltrating immune cells, thereby abrogating CD8+ T cell activation and promoting angiogenesis. While CD74 expression is known in dendritic cells, macrophages and B cells, our recent findings reveal its upregulation in immunosuppressive macrophages within the low-grade glioma (LGG) tumor microenvironment, and its negative correlation with the survival of adolescent and young adult patients with LGG. HYPOTHESIS. We hypothesize that inhibiting CD74/MIF in immunosuppressive myeloid cells may affect tumor progression and prevent malignant transformation (MT) to high-grade glioma (HGG). METHODS To test our hypothesis, we isolated bone marrow cells from immunocompetent RCAS n/tv-a LGG bearing animals, differentiated them into bone marrow-derived myeloid cells (BMDMs), silenced CD74 expression, and co-cultured them with primary CD8+T cells. Moreover, we inhibited CD74/MIF signaling in vivo. RESULTS. Deconvolution analysis of tumor-infiltrating immune cells revealed upregulation of CD74 and its network in myeloid cells during LGG compared to HGG. Silencing CD74 in LGG BMDMs in vitro resulted in the downregulation of genes associated with migration, proliferation, and immunosuppression. The co-culture of LGG CD74KD-BMDMs with CD8+ T cells increased the expression of T cell activation genes. In vivo treatment of LGG mice with MIF inhibitors significantly decreased myeloid cell infiltration in the tumor microenvironment (TME) and prolonged survival. CONCLUSIONS These preclinical studies highlight a role for the CD74/MIF signaling axis in glioma and present a novel immunotherapeutic target to modulate the activity of immunosuppressive myeloid cells in order to delay MT.

CSIG-21. THE ROLE OF GUT MICROBIOTA IN GLIOBLASTOMA: INSIGHTS FROM MICROBIOME-GENE EXPRESSION INTERACTIONS

Abstract Glioblastoma, the most aggressive form of primary brain tumor, remains a formidable clinical challenge due to its invasiveness and resistance to therapy. Emerging evidence suggests that the gut microbiome plays a crucial role in modulating cancer progression through complex interactions with host physiology. In this study, we investigated the influence of gut microbiota and tumor gene expression by analyzing stool and tissue samples from glioblastoma patients. Utilizing 16S rRNA sequencing of stool samples and NanoString gene expression analysis of tumor tissue samples, we explored the relationship between gut microbiome taxa and gene expression levels in tissue. Pathway analysis using KEGG and Gene Ontology databases was employed to elucidate the functional implications of these interactions. Our findings reveal contrasting effects of specific gut microbiota taxa and gene expression, with Fusobacterium demonstrating a potential oncogenic role, while Bifidobacteriaceae and Akkermansiaceae display potential tumor-suppressive effects. Fusobacterium is associated with the upregulation of key oncogenes and pathways promoting cancer cell survival, growth, and metabolism, whereas Bifidobacteriaceae and Akkermansiaceae are linked to the downregulation of genes involved in pathways promoting cancer cell survival and proliferation, such as MAPK signaling and stress response pathways, potentially inhibiting cancer cell growth. These results highlight the intricate interplay between gut microbiota composition and cancer biology and underscore the potential of gut microbiota modulation as a therapeutic strategy in glioblastoma management.

ANGI-08. SPATIAL TRANSCRIPTOMIC COMPARISONS OF GLIOBLASTOMA TISSUE REVEAL UPREGULATION OF NEURODEVELOPMENTAL PATHWAYS AND SYNAPTIC GENES IN INFILTRATING TUMOR CELLS

Abstract Glioblastoma remains one of the deadliest brain malignancies. First-line therapy consists of maximal surgical tumor resection, chemotherapy, and radiotherapy. Malignant cells escape surgical resection by migrating into the surrounding healthy brain tissue, where they give rise to the recurrent tumor. Gene expression profiling allows glioblastoma tumor cores to be classified into mesenchymal, proneural, and classical subtypes, each with distinct genetic alterations and cellular compositions. In contrast, the adjacent brain parenchyma where infiltrating malignant cells escape surgical resection is less characterized in patients. Using single-cell and multicellular resolution spatial transcriptomics on tissues from both the tumor core and infiltrated brain tissue (n = 11), we compared the transcriptional profiles of malignant cells in these regions. Malignant cells near regions with microvascular proliferation showed increased expression of genes related to vascular homeostasis. Within tumor cores, proneural and mesenchymal subtypes exhibited distinct spatial gene expression patterns, although these differences were reduced in the infiltrated brain tissue, apart from gene expression due to chromosomal alterations specific to the proneural subtype. We identified two transcriptional patterns of brain infiltration, with the dominant pattern showing a transition from mesenchymal to proneural states. This transition was accompanied by increased expression of genes linked to neurodevelopmental pathways and glial cell differentiation. Additionally, one gene module upregulated in infiltrating malignant cells was predictive of poor patient survival and enriched for genes associated with differentiated neuronal cells. Together, our findings provide an updated view of the spatial landscape of glioblastomas and infiltrated brain tissue, furthering our understanding of the malignant cells that invade healthy brain. This insight offers new avenues for targeted therapy after surgical resection of the primary tumor.

The perspective of ceRNA regulation of circadian rhythm on choroidal neovascularization

Abnormal growth of blood vessels (choroidal neovascularization) can lead to age-related macular degeneration (AMD) and eventually cause vision loss due to detachment of the retinal pigmented epithelium. This indicates that choroidal neovascularization is important for the treatment of AMD. The circadian clock in the mammalian retina is responsible for controlling various functions of the retina, enabling it to adjust to changes in light and darkness. Recent studies have revealed a potential connection between the circadian clock and eye diseases, although a cause-and-effect relationship has not been definitively established. C57BL/6J male mice (aged 6 weeks) were randomly divided into two groups (Control group: 9:00–21:00 light period (300 lx); Jet lag group: 8-hour phase advance once every 4 days). A laser-induced CNV model was created after 2 weeks of feeding in a controlled or jet-lagged environment. Then, full transcriptome sequencing was performed. The pathways regulated by differentially expressed mRNAs were identified by GO analysis and GSEA. Further protein networks were constructed with the STRING database and Cytoscape software. WGCNA was used to further explore the co-expression modules of these differential genes and the correlation between these differential genes and phenotypes. ceRNA networks were constructed with miRanda and TargetScan. The pathways associated with the overlapping differentially expressed mRNAs in the ceRNA network were identified, and the hub genes were validated by qPCR. A total of 661 important DEGs, 31 differentially expressed miRNAs, 106 differentially expressed lncRNAs and 87 differentially expressed circRNAs were identified. GO and GSEA showed that the upregulated DEGs were mainly involved in reproductive structure development and reproductive system development. The STRING database and Cytoscape were used to determine the protein interaction relationships of these DEGs. WGCNA divided the expression of these genes into several modules and screened the hub genes of each module separately. Furthermore, a ceRNA network was constructed. GO analysis and GSEA showed that these target DEmRNAs mainly function in wound healing, cell spreading, epiboly involved in wound healing, epiboly, and morphogenesis of an epithelial sheet. Finally, ten key genes were identified, and their expression patterns were confirmed by real-time qPCR. In this study, we investigated the regulatory mechanism of ceRNAs in choroidal neovascularization according to different light-dark cycles in the eyeball.

Single-cell RNA-sequencing of BK polyomavirus replication in primary human renal proximal tubular epithelial cells identifies specific transcriptome signatures and a novel mitochondrial stress pattern.

BK polyomavirus (BKPyV) contributes to premature renal failure in 10%-20% of kidney transplant recipients. Current treatment relies on reducing immunosuppression to regain BKPyV-specific immune control. Subsequently, declining allograft function may result from persisting viral cytopathology, BKPyV-specific immune reconstitution, or alloimmunity/rejection, all being poorly distinguishable by current histological or molecular approaches. To reduce the complexity encountered in BKPyV-replicating kidneys, we analyzed differentially expressed genes (DEGs) in primary human renal proximal tubular epithelial cells at 24 and 48 h post-infection (hpi) using single-cell RNA-sequencing (10x-Genomics-3´ kit). At 24 hpi, viral transcript reads predominantly mapped to the early viral gene region (EVGR) and shifted to >100-fold higher late viral gene region (LVGR) levels at 48 hpi, matching the sequential bi-directional viral protein expression from the circular double-stranded BKPyV-DNA genome. Besides expected coverage "hills" at viral 3´-poly-A sites, unexpected "spike" and "pulse" reads resulted from off-target TSO priming. "Spike" and "pulse" patterns were rare for the mostly unidirectional reads mapping to the circular mitochondrial genome. Bioinformatic curation removed "spikes" and "pulses" and reclassified 10% of DEGs in renal proximal tubular epithelial cells (RPTECs). Up-regulated gene ontologies included S and G2/M phase, double-stranded DNA repair, proximal tubulopathy, and renal tubular dysfunction, whereas allograft rejection, antigen presentation, innate immunity, translation, and autophagy were down-regulated. BKPyV-LVGR expression induced a novel mitochondrial cell stress pattern consisting of discordant up-regulation and down-regulation of mitochondria-encoded and nucleus-encoded mitochondrial genes, respectively. We explored which top-scoring gene sets of late-phase BKPyV-replicating RPTECs can identify BKPyV-associated nephropathy in kidney transplant biopsies. The results should facilitate distinguishing BKPyV-associated pathology from other entities in kidney transplant biopsies.IMPORTANCEBK polyomavirus (BKPyV) infects more than 90% of the general population and then persists in the reno-urinary tract. Subsequently, low-level urinary shedding is seen in 10% of healthy BKPyV-seropositive persons, indicating that BKPyV replication occurs despite the presence of virus-specific cellular and humoral immunity. Notably, transplantation of donor kidneys with low-level BKPyV replication is a risk factor for progression to high-level BKPyV viruria, new-onset BKPyV-DNAemia and biopsy-proven BKPyV nephropathy. Here, we identify a short list of robust up- and down-regulated nucleus-encoded differentially expressed genes potentially allowing to discriminate viral from allograft immune damage. By carefully curating viral and mitochondrial transcriptomes, we identify a novel virus-associated mitochondrial stress pattern of up-regulated mitochondria-encoded and down-regulated nucleus-encoded mitochondrial transcripts which heralds the BKPyV-agnoprotein-mediated immune escape by breakdown of the mitochondrial membrane potential and network and mitophagy. The results may prove useful when assessing the role of BKPyV replication in kidney transplant patients with suspected acute rejection and/or BKPyV nephropathy.

Klrb1 Loss Promotes Chronic Hepatic Inflammation and Metabolic Dysregulation

Background/Objectives: CD161, encoded by the KLRB1 gene, is an inhibitory receptor expresses on various immune cell and has gained attention in immune checkpoint research. In recent studies, KLRB1 has been found to be one of the potential markers of liver diseases such as cirrhosis. Therefore, it will be important to understand what process KLRB1 involved in the liver for the prevention of liver diseases. Methods: We compared KO mice with wild-type controls by routine blood analysis and RNA-seq, and additionally performed H&E staining and qPCR to validate the differentially expressed genes (DEGs). Results:KO mice had fewer lymphocytes compared to the wild-type mice. A transcriptomic analysis showed that Klrb1 loss causes the upregulation of immune-related genes and pathways like NOD-like receptor and p53 signaling, while causing the downregulation of lipid metabolism-related genes. A protein interaction analysis indicated a potential cancer risk under chronic inflammation. Histological examination with H&E staining reveals an inflammatory response around the central venous vessels in the liver tissue of the KO mice. Conclusions: We conclude that Klrb1 knockout disrupts the immune and metabolic functions in the liver, which may possibly lead to chronic inflammation and malignancy risks. These findings highlight the role of Klrb1 in hepatic health.

Sugar beet root susceptibility to storage rots and downregulation of plant defense genes increases with time in storage

Storage rots are a significant cause of postharvest losses for the sugar beet crop, however, intrinsic physiological and genetic factors that determine the susceptibility of roots to pathogen infection and disease development are unknown. Research, therefore, was carried out to evaluate the disease development in sugar beet roots caused by two common storage pathogens as a function of storage duration and storage temperature, and to identify changes in the expression of defense genes that may be influencing the root susceptibility to disease. To evaluate root susceptibility to disease, freshly harvested roots were inoculated with Botrytis cinerea or Penicillium vulpinum on the day of harvest or after 12, 40, or 120 d storage at 5 or 12 °C and the weight of rotted tissue present in the roots after incubation for 35 d after inoculation were determined. Disease susceptibility and progression to B. cinerea and P. vulpinum increased with storage duration with elevations in susceptibility occurring more rapidly to B. cinerea than P. vulpinum. Also, B. cinerea was more aggressive than P. vulpinum and caused greater rotting and tissue damage in postharvest sugar beet roots. Storage temperature had minimal effect on root susceptibility to these rot-causing pathogens. Changes in defense gene expression were determined by sequencing mRNA isolated from uninoculated roots that were similarly stored for 12, 40 or 120 d at 5 or 12 °C. As susceptibility to rot increased during storage, concurrent changes in defense-related gene expression were identified, including the differential expression of 425 pathogen receptor and 275 phytohormone signal transduction pathway-related genes. Furthermore, plant resistance and hormonal signaling genes that were significantly altered in expression coincident with the change in root susceptibility to storage rots were identified. Further investigation into the function of these genes may ultimately elucidate methods by which storage rot resistance in sugar beet roots may be improved in the future.

Inhibition of Bruton's tyrosine kinase with PD-1 blockade modulates T cell activation in solid tumors.

BACKGROUNDInhibition of Bruton's tyrosine kinase with ibrutinib blocks the function of myeloid-derived suppressor cells (MDSC). The combination of ibrutinib and nivolumab was tested in patients with metastatic solid tumors.METHODSSixteen patients received ibrutinib 420 mg p.o. daily with nivolumab 240 mg i.v. on days 1 and 15 of a 28-day cycle. The effect of ibrutinib and nivolumab on MDSC, the immune profile, and cytokine levels were measured. Single-cell RNA-Seq and T cell receptor sequencing of immune cells was performed.RESULTSCommon adverse events were fatigue and anorexia. Four patients had partial responses and 4 had stable disease at 3 months (average 6.5 months, range 3.5-14.6). Median overall survival (OS) was 10.8 months. Seven days of Bruton's tyrosine kinase (BTK) inhibition significantly increased the proportion of monocytic-MDSC (M-MDSC) and significantly decreased chemokines associated with MDSC recruitment and accumulation (CCL2, CCL3, CCL4, CCL13). Single-cell RNA-Seq revealed ibrutinib-induced downregulation of genes associated with MDSC-suppressive function (TIMP1, CXCL8, VEGFA, HIF1A), reduced MDSC interactions with exhausted CD8+ T cells, and decreased TCR repertoire diversity. The addition of nivolumab significantly increased circulating NK and CD8+ T cells and increased CD8+ T cell proliferation. Exploratory analyses suggest that MDSC and T cell gene expression and TCR repertoire diversity were differentially affected by BTK inhibition according to patient response.CONCLUSIONIbrutinib and nivolumab were well tolerated and affected MDSC and T cell function in patients with solid metastatic tumors.TRIAL REGISTRATIONClinicalTrials.gov NCT03525925.FUNDINGNIH; National Cancer Institute Cancer; National Center for Advancing Translational Sciences; Pelotonia.

Molecular basis for the disease-modifying effects of belimumab in systemic lupus erythematosus and molecular predictors of early response: blood transcriptome analysis implicates the innate immunity and DNA damage response pathways

ObjectivesBelimumab is a putative disease-modifying agent in systemic lupus erythematosus (SLE), yet the molecular underpinnings of its effects and the ability to predict early clinical response remain unexplored. To address...

A probiotic multi-strain mixture combined with hydroxyectoine improves intestinal barrier function by alleviating inflammation in lipopolysaccharide stimulated differentiated Caco-2 cells.

Many studies have highlighted the role of probiotics in re-establishing the gut microbiota balance and preventing intestinal barrier dysfunction. In fact, they can also contribute to the upregulation of anti-inflammatory genes and the downregulation of pro-inflammatory genes, which are known to contribute to the development of the inflammatory bowel disease (IBD) syndrome. The present study aims to investigate the effect of the compatible solute hydroxyectoine (HOE), to be used as a cryopreservant but also for its intrinsic biological properties, to obtain a new formula containing three probiotic strains (Limosilactobacillus fermentum (L. fermentum), Levilactobacillus brevis SP-48 (L. brevis), and Bifidobacterium lactis HN019 (B. lactis)), and evaluate the latter for its ability to prevent lipopolysaccharide (LPS)-induced inflammation in an in vitro bi-dimensional model of the intestinal barrier using a Caco-2 cell monolayer. The mRNA expression levels of the inflammatory cytokines (IL-6, IL-1β, and TNF-α) were analyzed by real-time PCR. Changes in the modulation of (TLR-4 and NF-κB) proteins were assessed by western blotting, and the effect of the HOE/PRO formula on the intestinal epithelial barrier function was also assessed using an immunofluorescence microscope for the tight junction protein zonula occludens-1 (ZO-1). This study found that this novel probiotic formulation containing HOE is capable of decreasing LPS-induced cytokines, as confirmed by the results of RT-PCR and ELISA and preserving the integrity of tight junctions as demonstrated by the relevant expression of ZO-1. HOE/PRO was shown to be effective in reducing the expression of TLR-4 and NF-κB. The latter plays a key role as an inflammation modulator as shown through experiments run with the THP-1/NF-κB reporter gene. Collectively, our data indicate that the HOE/PRO formula is a good candidate for potential preventive and/or therapeutic implementation in IBD.

GDF15 is required for maintaining subcutaneous adipose tissue lipid metabolic signature

Recent research has identified growth differentiation factor 15 (GDF15) as a crucial factor in various physiological and pathological processes, particularly in energy balance regulation. While the role of GDF15 in modulating energy metabolism through hindbrain GDNF family receptor alpha-like (GFRAL) signaling has been extensively studied, emerging evidence suggests direct peripheral metabolic actions of GDF15. Using knockout mouse models, we investigated GDF15 and GFRAL’s roles in adipose tissue metabolism. Our findings indicate that C57BL/6/129/SvJ Gdf15-KO mice exhibit impaired expression of de novo lipogenesis enzymes in subcutaneous adipose tissue (sWAT). In contrast, C57BL/6J Gfral-KO mice showed no impairments compared to wild-type (WT) littermates. RNA-Seq analysis of sWAT in Gdf15-KO mice revealed a broad downregulation of genes involved in lipid metabolism. Importantly, our study uncovered sex-specific effects, with females being more affected by GDF15 loss than males. Additionally, we observed a fasting-induced upregulation of GDF15 gene expression in sWAT of both mice and humans, reinforcing this factor’s role in adipose tissue lipid metabolism. In conclusion, our research highlights an essential role for GDF15 in sWAT lipid metabolic homeostasis. These insights enhance our understanding of GDF15’s functions in adipose tissue physiology and underscore its potential as a therapeutic target for metabolic disorders.

Hypoxia adaptation mechanism in rats’ peripheral auditory system in high altitude migration: a time series transcriptome analysis

High altitude is characterized by low oxygen, low pressure, and high radiation. When migrates from low to high altitudes, the body’s tissues and organs experience hypoxic stress and will present acoustic adaptation as the protective response. However, the mechanisms of acoustic adaptation at high altitudes remain unclear. In this study, cochlear tissues from Wistar rats were collected at 15, 30, 60, 120, and 180 days after high-altitude migration. Transcriptome sequencing was conducted and DESeq algorithm revealed expression patterns of Differentially Expressed Genes(DEGs) after high altitude migration. Day 60 is a critical stage for cochlear tissue “damage” and “repair” in high-altitude conditions. Transmission Electron Microscopy (TEM) observations of structures also support the findings. A time-series gene co-expression network algorithm was used to investigate gene regulatory patterns and key genes after migration. Immunofluorescence, immunohistochemistry, and qPCR were per-formed for key gene validation and localization. At Day 60, the peak DEG count occurs in rats migrating to high altitude, aligning with the critical phase for cochlear tissue damage and repair at high altitudes. Repair hinges on synaptic plasticity and myelination-linked processes, influencing modules M4 to M6. Module M4’s activation gradually diminishes from its peak. However, the ‘damage’ effect is orchestrated by inflammation-related processes in modules M3 to M5, with module M3’s activation also waning. Key gene module M4, pivotal for repair during this pivotal phase, encompasses Sptbn5, Cldn1, Gfra2, and Lims2 as its core genes. Immunohistochemistry reveals Sptbn5’s presence in cochlear neurons, hair cells, Schwann cells and stria vascularis tissue. Cldn1 and Gfra2 predominantly localize within the cochlear neuron region. These results may suggest new directions for future research on acoustic acclimatization to high altitude.

Zolmitriptan niosomal transdermal patches: combating migraine via epigenetic and endocannabinoid pathways and reversal of migraine hypercoagulability.

Conventional zolmitriptan (ZOL) has limited oral bioavailability, many adverse effects, and poor membrane penetrability that negatively influences its accessibility to its 5-HT1B/1D receptor binding pocket, located transmemberanous. This work aimed at preparing transdermal ZOL-nanoformulation (niosomes) to surpass these limitations and to explore novel antimigraine mechanisms for ZOL via modulation of the epigenetically-altered chronification genes (RAMP-1, NPTX-2) or microRNAs and affecting the endocannabinoid CB-1/MAPK pathway. The prepared ZOL niosomes (Fsp60/6-1:1) exhibited %EE of 57.28%, PS of 472.3nm, PDI of 0.366, and ZP of -26 mV were cast into patch with content uniformity of 93.12%, maintained endurance after 200-times folding, no interaction between its components (FT-IR), a biphasic release pattern and good stability after storage at 4°C for 6 months. In-vivo ZOL-patch application in rats with nitroglycerin-induced migraine showed significant management of migraine pain symptoms and photophobia assessed behaviorally, decreased brain levels of the trigeminal neuronal activation marker (c-fos), the migraine pain neurotransmitter (CGRP) and the serum levels of different migraine pain markers (substance P, nitric-oxide, and TNF-α). It also significantly decreased RAMP-1, NPTX-2, miR-382-5p, and CB-1/MAPK gene expression reflecting improved efficacy and brain receptors delivery to a much greater extent than conventional ZOL has done. Additionally, this nanoformulation significantly opposed migraine-induced platelet activation and hypercoagulable status in both central and peripheral circulations as evidenced by the significant decrease in adenosine diphosphate, thrombin, factor X, CD41, and Von-Willebrand factor levels assessed peripherally and centrally. TPFsp60/6-1:1 significantly improved ZOL efficacy and accessibility to brain-receptors to a much greater extent than conventional ZOL-solution.KeywordsEndocannabinoid receptors; Epigenetically-altered genes; Hemostatic pathways; Niosomal patch; Transdermal; Zolmitriptan.

New Horizons in Micro/Nanoplastic-Induced Oxidative Stress: Overlooked Free Radical Contributions and Microbial Metabolic Dysregulations in Anaerobic Digestion.

Excessive production of reactive oxygen species (ROS) induced by micro/nanoplastics (MPs/NPs) is highly toxic to microbes. However, the mechanisms underlying ROS generation and metabolic regulation within anaerobic guilds remain poorly understood. In this study, we investigated the effects of environmentally relevant levels of polypropylene (PP)-MPs/NPs on oxidative stress and microbial ecology during anaerobic digestion (AD). Electron paramagnetic resonance spectroscopy revealed that PP-MPs/NPs elevated the concentrations of environmentally persistent free radicals (EPFRs) and derived hydroxyl radicals (•OH). EPFRs were identified as the primary contributors to •OH generation, as evidenced by a high Spearman correlation coefficient (r = 0.884, p < 0.001) and free radical-quenching studies. The formation of •OH enhanced ROS production by 86.2-100.9%, resulting in decreased cellular viability and methane production (by 37.5-50.5%) at 100 mg/g TS PP-MPs/NPs. Genome-centric metagenomic and metatranscriptomic analyses suggested that PP-MPs/NPs induced the reassembly of community structures, re-evolution of functional traits, and remodeling of interspecies interactions. Specifically, PP-MPs/NPs induced a shift in methanogen consortia from hydrogenotrophic Methanofollis sp. to acetoclastic and hydrogenotrophic Methanothrix soehngenii, primarily because of the latter's diverse ingestion patterns, electron bifurcation complexes, and ROS-scavenging abilities. Downregulation of genes associated with antioxidative defense systems (i.e., sodN, katA, and osmC) and ROS-driven redox signal transduction pathways (c-di-AMP and phosphorylation signaling pathways) provided insights into the mechanisms underlying ROS-induced microbial metabolic dysregulation. Our findings enhance the understanding of microbial ecological and metabolic traits under MPs/NPs stressors, facilitating the control of MPs/NPs toxicity and the stabilization of AD processes.

Altered immune response is associated with sex difference in vulnerability to Alzheimer's disease in human prefrontal cortex.

Alzheimer's disease (AD) is a neurodegenerative disorder with a higher risk incidence in females than in males, and there are also differences in AD pathophysiology between sexes. The role of sex in the pathogenesis of AD may be crucial, yet the cellular and molecular basis remains unclear. Here, we performed a comprehensive analysis using four public transcriptome datasets of AD patients and age-matched control individuals in prefrontal cortex, including bulk transcriptome (295 females and 402 males) and single-nucleus RNA sequencing (snRNA-seq) data (224 females and 219 males). We found that the transcriptomic profile in female control was similar to those in AD. To characterize the key features associated with both the pathogenesis of AD and sex difference, we identified a co-expressed gene module that positively correlated with AD, sex, and aging, and was also enriched with immune-associated pathways. Using snRNA-seq datasets, we found that microglia (MG), a resident immune cell in the brain, demonstrated substantial differences in several aspects between sexes, such as an elevated proportion of activated MG, altered transcriptomic profile and cell-cell interaction between MG and other brain cell types in female control. Additionally, genes upregulated in female MG, such as TLR2, MERTK, SPP1, SLA, ACSL1, and FKBP5, had high confidence to be identified as biomarkers to distinguish AD status, and these genes also interacted with some approved drugs for treatment of AD. These findings underscore the altered immune response in female is associated with sex difference in susceptibility to AD, and the necessity of considering sex factors when developing AD biomarkers and therapeutic strategies, providing a scientific basis for further in-depth studies on sex differences in AD.

Identification and validation of interferon-stimulated gene 15 as a biomarker for dermatomyositis by integrated bioinformatics analysis and machine learning

BackgroundDermatomyositis (DM) is an autoimmune disease that primarily affects the skin and muscles. It can lead to increased mortality, particularly when patients develop associated malignancies or experience fatal complications such as pulmonary fibrosis. Identifying reliable biomarkers is essential for the early diagnosis and treatment of DM. This study aims to identify and validate pivotal diagnostic biomarker for DM through integrated bioinformatics analysis and clinical sample validation.MethodsGene expression datasets GSE46239 and GSE142807 from the Gene Expression Omnibus (GEO) database were merged for analysis. Differentially expressed genes (DEGs) were identified and subjected to enrichment analysis. Advanced machine learning methods were utilized to further pinpoint hub genes. Weighted gene co‐expression network analysis (WGCNA) was also conducted to discover key gene modules. Subsequently, we derived intersection gene from these methods. The diagnostic performance of the candidate biomarker was evaluated using analysis with dataset GSE128314 and confirmed by immunohistochemistry (IHC) in skin lesion biopsy specimens. The CIBERSORT algorithm was used to analyze immune cell infiltration patterns in DM, then the association between the hub gene and immune cells was investigated. Gene set enrichment analysis (GSEA) was performed to understand the biomarker’s biological functions. Finally, the drug-gene interactions were predicted using the DrugRep server.ResultsInterferon-stimulated gene 15 (ISG15) was identified by intersecting DEGs, advanced machine learning-selected genes and key module genes from WGCNA. ROC analysis showed ISG15 had a high Area under the curve (AUC) of 0.950. IHC findings confirmed uniformly positive expression of ISG15, particularly in perivascular regions and lymphocytes, contrasting with universally negative expression in controls. Further analysis revealed that ISG15 is involved in abnormalities in various immune cells and inflammation-related pathways. We also predicted three drugs targeting ISG15, supported by molecular docking studies.ConclusionOur study identifies ISG15 as a highly specific diagnostic biomarker for DM, ISG15 may be closely related to the pathogenesis of DM, demonstrating promising potential for clinical application.

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

BackgroundSomatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.MethodsA landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.ResultsWe observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.ConclusionssPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.