Protein Abundance Research Articles

Evaluation of the Composite Skin Patch Loaded with Bioactive Functional Factors Derived from Multicellular Spheres of EMSCs for Regeneration of Full-thickness Skin Defects in Rats.

Transplantation of stem cells/scaffold is an efficient approach for treating tissue injury including full-thickness skin defects. However, the application of stem cells is limited by preservation issues, ethical restriction, low viability, and immune rejection in vivo. The mesenchymal stem cell conditioned medium is abundant in bioactive functional factors, making it a viable alternative to living cells in regeneration medicine. Nasal mucosa-derived ecto-mesenchymal stem cells (EMSCs) of rats were identified and grown in suspension sphere-forming 3D culture. The EMSCs-conditioned medium (EMSCs-CM) was collected, lyophilized, and analyzed for its bioactive components. Next, fibrinogen and chitosan were further mixed and cross-linked with the lyophilized powder to obtain functional skin patches. Their capacity to gradually release bioactive substances and biocompatibility with epidermal cells were assessed in vitro. Finally, a full-thickness skin defect model was established to evaluate the therapeutic efficacy of the skin patch. The EMSCs-CM contains abundant bioactive proteins including VEGF, KGF, EGF, bFGF, SHH, IL-10, and fibronectin. The bioactive functional composite skin patch containing EMSCs-CM lyophilized powder showed the network-like microstructure could continuously release the bioactive proteins, and possessed ideal biocompatibility with rat epidermal cells in vitro. Transplantation of the composite skin patch could expedite the healing of the full-thickness skin defect by promoting endogenous epidermal stem cell proliferation and skin appendage regeneration in rats. In summary, the bioactive functional composite skin patch containing EMSCs-CM lyophilized powder can effectively accelerate skin repair, which has promising application prospects in the treatment of skin defects.

Proteomic changes in Alzheimer disease associated with progressive Aβ plaque and tau tangle pathologies.

Proteomics can shed light on the dynamic and multifaceted alterations in neurodegenerative disorders like Alzheimer disease (AD). Combining radioligands measuring β-amyloid (Aβ) plaques and tau tangles with cerebrospinal fluid proteomics, we uncover molecular events mirroring different stages of AD pathology in living humans. We found 127 differentially abundant proteins (DAPs) across the AD spectrum. The strongest Aβ-related proteins were mainly expressed in glial cells and included SMOC1 and ITGAM. A dozen proteins linked to ATP metabolism and preferentially expressed in neurons were independently associated with tau tangle load and tau accumulation. Only 20% of the DAPs were also altered in other neurodegenerative diseases, underscoring AD's distinct proteome. Two co-expression modules related, respectively, to protein metabolism and microglial immune response encompassed most DAPs, with opposing, staggered trajectories along the AD continuum. We unveil protein signatures associated with Aβ and tau proteinopathy in vivo, offering insights into complex neural responses and potential biomarkers and therapeutics targeting different disease stages.

Structural variants linked to Alzheimer's Disease and other common age-related clinical and neuropathologic traits.

Advances have led to a greater understanding of the genetics of Alzheimer's Disease (AD). However, the gap between the predicted and observed genetic heritability estimates when using single nucleotide polymorphisms (SNPs) and small indel data remains. Large genomic rearrangements, known as structural variants (SVs), have the potential to account for this missing genetic heritability. By leveraging data from two ongoing cohort studies of aging and dementia, the Religious Orders Study and Rush Memory and Aging Project (ROS/MAP), we performed genome-wide association analysis testing around 20,000 common SVs from 1,088 participants with whole genome sequencing (WGS) data. A range of Alzheimer's Disease and Related Disorders (AD/ADRD) clinical and pathologic traits were examined. Given the limited sample size, no genome-wide significant association was found, but we mapped SVs across 81 AD risk loci and discovered 22 SVs in linkage disequilibrium (LD) with GWAS lead variants and directly associated with AD/ADRD phenotypes (nominal P < 0.05). The strongest association was a deletion of an Alu element in the 3'UTR of the TMEM106B gene. This SV was in high LD with the respective AD GWAS locus and was associated with multiple AD/ADRD phenotypes, including tangle density, TDP-43, and cognitive resilience. The deletion of this element was also linked to lower TMEM106B protein abundance. We also found a 22 kb deletion associated with depression in ROSMAP and bearing similar association patterns as AD GWAS SNPs at the IQCK locus. In addition, genome-wide scans allowed the identification of 7 SVs, with no LD with SNPs and nominally associated with AD/ADRD traits. This result suggests potentially new ADRD risk loci not discoverable using SNP data. Among these findings, we highlight a 5.6 kb duplication of coding regions of the gene C1orf186 at chromosome 1 associated with indices of cognitive impairment, decline, and resilience. While further replication in independent datasets is needed to validate these findings, our results support the potential roles of common structural variations in the pathogenesis of AD/ADRD.

Human Serum Albumin Protein Corona in Prussian Blue Nanoparticles

Prussian Blue nanoparticles (PBnps) are now popular in nanomedicine thanks to the FDA approval of PB. Despite the numerous papers suggesting or describing the in vivo use of PBnps, no studies have been carried out on the formation of a protein corona on the PBnp surface and its stabilizing role. In this paper, we studied qualitatively and quantitatively the corona formed by the most abundant protein of blood, human serum albumin (HSA). Cubic PBnps (41 nm side), prepared in citric acid solution at PB concentration 5 × 10−4 M, readily form a protein corona by redissolving ultracentrifuged PBnp pellets in HSA solutions, with CHSA ranging from 0.025 to 7.0 mg/mL. The basic decomposition of PBnp@HSA was studied in phosphate buffer at the physiological pH value of 7.4. Increased stability with respect to uncoated PBnps was observed at all concentrations, but a minimum CHSA value of 3.0 mg/mL was determined to obtain stability identical to that observed at serum-like HSA concentrations (35–50 mg/mL). Using a modified Lowry protocol, the quantity of firmly bound HSA in the protein corona (hard corona) was determined for all the CHSA used in the PBnp@HSA synthesis, finding increasing quantities with increasing CHSA. In particular, an HSA/PBnp number in the 1500–2300 range was found for CHSA 3.0–7.0 mg/mL, largely exceeding the 180 HSA/PBnp value calculated for an HSA monolayer on a PBnp. Finally, the stabilization brought by the HSA corona allowed us to carry out pH-spectrophotometric titrations on PBnp@HSA in the 3.5-9-0 pH range, revealing a pKa value of 6.68 for the water molecules bound to the Fe3+ centers on the PBnp surface, whose deprotonation is responsible for the blue-shift of the PBnp band from 706 nm (acidic solution) to 685 nm (basic solution).

Cross-species transcriptomics reveals differential regulation of essential photosynthesis genes in Hirschfeldia incana.

Photosynthesis is the only yield-related trait not yet substantially improved by plant breeding. Previously, we have established Hirschfeldia incana as the model plant for high photosynthetic light-use efficiency (LUE). Now we aim to unravel the genetic basis of this trait in H. incana, potentially contributing to the improvement of photosynthetic LUE in other species. Here, we compare its transcriptomic response to high light with that of Arabidopsis thaliana, Brassica rapa, and Brassica nigra, three fellow Brassicaceae members with lower photosynthetic LUE. We built a high-light, high-uniformity growing environment, in which the plants developed normally without signs of stress. We compared gene expression in contrasting light conditions across species, utilizing a panproteome to identify orthologous proteins. In-depth analysis of three key photosynthetic pathways showed a general trend of lower gene expression under high-light conditions for all four species. However, several photosynthesis-related genes in H. incana break this trend. We observed cases of constitutive higher expression (like antenna protein LHCB8), treatment-dependent differential expression (as for PSBE), and cumulative higher expression through simultaneous expression of multiple gene copies (like LHCA6). Thus, Hirschfeldia incana shows differential regulation of essential photosynthesis genes, with the light-harvesting complex as the first point of deviation. The effect of these expression differences on protein abundance and turnover, and ultimately the high photosynthetic LUE phenotype is relevant for further investigation. Furthermore, this transcriptomic resource of plants fully grown under, rather than briefly exposed to, a very high irradiance, will support the development of highly efficient photosynthesis in crops.

LacdiNAc to LacNAc: remodelling of bovine α-lactalbumin N-glycosylation during the transition from colostrum to mature milk.

α-Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.

Plasminogen activator inhibitor-1 mediates cerebral ischemia-induced astrocytic reactivity.

Although ischemia increases the abundance of plasminogen activator inhibitor-1 (PAI-1), its source and role in the ischemic brain remain unclear. We detected PAI-1-immunoreactive cells with morphological features of reactive astrocytes in the peri-ischemic cortex of mice after an experimentally-induced ischemic lesion, and of a chimpanzee that suffered a naturally-occurring stroke. We found that although the abundance of PAI-1 increases 24 hours after the onset of the ischemic injury in a non-reperfusion murine model of ischemic stroke, at that time-point there is no difference in astrocytic reactivity and the volume of the ischemic lesion between wild-type (Wt) animals and in mice either genetically deficient (PAI-1-/-) or overexpressing PAI-1 (PAI-1Tg). In contrast, 72 hours later astrocytic reactivity and the volume of the ischemic lesion were decreased in PAI-1-/- mice and increased in PAI-1Tg animals. Our immunoblottings and fractal analysis studies show that the abundance of astrocytic PAI-1 rises during the recovery phase from a hypoxic injury, which in turn increases the abundance of glial fibrillary acidic protein (GFAP) and triggers morphological features of reactive astrocytes. These studies indicate that cerebral ischemia-induced release of astrocytic PAI-1 triggers astrocytic reactivity associated with enlargement of the necrotic core.

Comparison of Relative Activity versus Relative Expression Factors (RAF versus REF) in Predicting Glucuronidation Mediated Drug Clearance Using Recombinant UGTs.

Predicting the quantitative fraction of glucuronidation (fgluc) by individual UDP-glucuronosyltransferase enzymes (UGTs) is challenging due to the lack of selective inhibitors and inconsistent activity of recombinant UGT systems (rUGTs). Our study compares the relative expression versus activity factors (REF versus RAF) to predict fgluc based on rUGT data to human liver and intestinal microsomes (HLM and HIM). REF scalars were derived from a previous in-house proteomics study for eleven UGT enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17), whereas RAF was calculated by measuring activities in rUGTs to microsomes of selective UGT probe substrates. Protein-normalized activity factor (pnAF) values weregenerated after correcting activity of individual UGTs to their corresponding protein abundance. The utility of REF and RAF in predicting fgluc was assessed for three UGT substrates-diclofenac, vorinostat, and raltegravir. The REF values ranged from 0.02 to 1.75, RAF based on activity obtained in rUGTs to HLM/HIM were from 0.1 to 274. pnAF values were ~ 5 to 80-fold, except for UGT2B4 and UGT2B15, where pnAF was ~ 180 and > 1000, respectively. The results revealed confounding effect of differential specific activities (per pmol) of rUGTs in fgluc prediction. The data suggest that the activity of UGT enzymes was significantly lower when compared to their activity in microsomes at the same absolute protein amount (pmol). Collectively, results of this study demonstrate poor and variable specific activity of different rUGTs (per pmol protein), as determined by pnAF values, which should be considered in fgluc scaling.

Circular RNA ACVR2A promotes the progression of hepatocellular carcinoma through mir-511-5p targeting PI3K-Akt signaling pathway

Circular RNA (circRNA) is thought to mediate the occurrence and development of human cancer and usually acts as a tiny RNA (miRNA) sponge to regulate downstream gene expression. However, it is not clear whether and how circACVR2A (hsa_circ_0001073) is involved in the progression of HCC. The purpose of this study is to clarify the potential role and molecular mechanism of circACVR2A in regulating the progression of hepatocellular carcinoma cells (HCC). The abundance of related proteins in circACVR2A, microRNA (miR511-5p) and PI3K-Akt signaling pathway was determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) or Western blotting. Cell viability, invasion and apoptosis were analyzed by CCK-8, Transwell analysis and Tunel staining, respectively. The interaction between circACVR2A and microRNA was evaluated by double luciferase reporter gene assay. The results showed that circACVR2A was highly expressed in hepatocellular carcinoma cell lines. Our in vivo and in vitro data showed that circACVR2A promoted the proliferation, migration and invasion of HCC. In terms of mechanism, we found that circACVR2A can directly interact with miR511-5p and act as a miRNA sponge to regulate the expression of related proteins in PI3K-Akt signaling pathway.In HCC, circACVR2A can mediate miR-511-5p/mRNA network to activate PI3K signal pathway. This shows that the molecular regulatory network with circACVR2A as the core is a new potential target for diagnosis and treatment of hepatocellular carcinoma.

Chemerin affects the cytokine production and the expression of their receptors in the porcine endometrium during early pregnancy and the estrous cycle: an in vitro study.

Interactions between female metabolic status, immune response, and reproductive system functioning are complex and not fully understood. We hypothesized that chemerin, considered a hormonal link between the above-mentioned processes, influences endometrial functions, particularly cytokine secretion and signalling. Using porcine endometrial explants collected during early pregnancy and the estrous cycle, we investigated chemerin effects on the secretion of interleukins (IL-1β, IL-6, IL-8), leukaemia inhibitory factor (LIF), tumour necrosis factor α (TNFα), transforming growth factor α (TGFα), and protein abundances of their respective receptors. Our results demonstrate chemerin modulation of cytokine secretion and receptor expression, with effects dependent on the stage of pregnancy and dose of chemerin. Furthermore, chemerin influences the phosphorylation of stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ) in the endometrium. Chemerin multifaceted actions, such as involvement in immune response, cell proliferation, and tissue remodelling seem to be mediated by cytokines, at least in the endometrium. These findings underscore the potential crosstalk between chemerin and hormonal signalling pathways, providing insights into the complex mechanisms underlying early pregnancy establishment and maintenance.

Bacterial infection-biased abundance of proteins in the skin mucus of obscure puffer (Takifugu Obscurus)

The skin mucus of fish is equipped with immunological and antimicrobial peptides that confer protection against invading pathogens. The skin mucus has been studied in fish however information regarding its immunological roles in bacterial infection is rare. This study highlighted the proteins and peptides in the skin mucus of Obscure puffer Takifugu obscurus that quantitatively altered against Aeromonas hydrophila infection. We infected the fish through bath immersion, intraperitonially, and treated with PBS (control) then compared the level of proteins in the skin mucus among the groups using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The Tandem Mass Tag (TMT) based quantification showed that 4896 proteins were Deferentially Quantified Proteins (DQPs), based on 19,751 unique peptides. Of which 170 were depleted (decreased in abundance) and 69 were abundant in comparison of Bath Treated (BT) vs Control (C) groups. Similarly, 76 DQPs were depleted and 70 were abundant in comparison of Treated (T) vs BT groups. Further, 126 DQPs were depleted, and 34 were abundant in comparison to T vs C groups. The DQPs we report were mostly immunological and were involved in unique biological functions and pathways. The interesting protein we report, where some of the proteins are for the first time in fish, shows the protein-rich structure of the mucus of fish, which may act as a biomarker to be targeted for bacterial disease therapy in fish and ultimately hint to the way of making resistance in fish against bacterial pathogens.

Identifying novel risk genes in intracranial aneurysm by integrating human proteomes and genetics.

Genome-wide association studies (GWAS) have become increasingly popular for detecting numerous loci associated with intracranial aneurysm (IA), but how these loci function remains unclear. In this study, we employed an integrative analytical pipeline to efficiently transform genetic associations and identify novel genes for IA. Using multidimensional high-throughput data, we integrated proteome-wide association studies (PWAS), transcriptome-wide association studies (TWAS), Mendelian randomization (MR) and Bayesian co-localization analyses to prioritize genes that can increase IA risk by altering their expression and protein abundances in the brain and blood. Moreover, single-cell RNA sequencing (scRNA-seq) of the circle of Willis was performed to enrich filtered genes in cells, and gene set enrichment analysis (GSEA) was conducted for each gene using bulk RNA-seq data for IA. No significant genes with cis-regulated plasma protein levels were proven to be associated with IA. The protein abundances of five genes in the brain were found to be associated with IA. According to cellular enrichment analysis, these five genes were expressed mainly in the endothelium, fibroblasts and vascular smooth muscle cells. Only three genes, CNNM2, GPRIN3 and UFL1, passed MR and Bayesian co-localization analyses. While UFL1 was not validated in confirmation PWAS as it was not profiled, it was validated in TWAS. GSEA suggested these three genes are associated with the cell cycle. In addition, the protein abundance of CNNM2 was found to be associated with IA rupture (based on PWAS, MR and co-localization analyses). Our findings indicated that CNNM2, GPRIN3 and UFL1 (CNNM2 correlated with IA rupture) are potential IA risk genes that may provide a broad hint for future research on possible mechanisms and therapeutic targets for IA.

Targeted proteomic approach for quantification of collagen type I and type III in formalin-fixed paraffin-embedded tissue

Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC–MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer sources, demonstrating its generalizability. In addition, the targeted LC–MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straightforward, accurate, precise, and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.

Disordered proteins interact with the chemical environment to tune their protective function during drying.

The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins combined with the exposure of their residues accounts for this sensitivity. One context in which IDPs play important roles that is concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family, synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results suggest that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.

Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment.

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (microdroplet processing in one pot for trace samples), multiplexed isobaric labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (∼60,000 μm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.

Reconstituted HDL ameliorated renal injury of diabetic kidney disease in mice.

Diabetic kidney disease (DKD) is a devastating kidney disease and lacks effective therapeutic interventions. The present study was aimed to determine whether reconstituted high-density lipoprotein (rHDL) ameliorated renal injury in eNOS-/- dbdb mice, a mouse model of DKD. Three groups of mice, wild type C57BLKS/J (non-diabetes), eNOS-/- dbdb (diabetes), and eNOS-/- dbdb treated with rHDL (diabetes+rHDL) with both males and females were used. The rHDL nanoparticles were administered to eNOS-/- dbdb mice at Week 16 at 5 μg/g body weight in ~100 μL of saline solution twice per week for 4 weeks via retroorbital injection. We found that rHDL treatment significantly blunted progression of albuminuria and GFR decline observed in DKD mice. Histological examinations showed that the rHDLs significantly alleviated glomerular injury and renal fibrosis, and inhibited podocyte loss. Western blots and immunohistochemical examinations showed that increased protein abundances of fibronectin and collagen IV in the renal cortex of eNOS-/- dbdb mice were significantly reduced by the rHDLs. Taken together, the present study suggests a renoprotective effect of rHDLs on DKD.

Affinity purification mass spectrometry characterisation of the interactome of receptor tyrosine kinase proline-rich motifs in cancer

Receptor tyrosine kinase (RTK) overexpression is linked to the development and progression of multiple cancers. RTKs are classically considered to initiate cytoplasmic signalling pathways via ligand-induced tyrosine phosphorylation, however recent evidence points to a second tier of signalling contingent on interactions mediated by the proline-rich motif (PRM) regions of non-activated RTKs. The presence of PRMs on the C-termini of >40 % of all RTKs and the abundance of PRM-binding proteins encoded by the human genome suggests that there is likely to be a large number of previously unexplored interactions which add to the RTK intracellular interactome. Here, we explore the RTK PRM interactome and its potential significance using affinity purification mass spectrometry and in silico enrichment analyses. Peptides comprising PRM-containing C-terminal tail regions of EGFR, FGFR2 and HER2 were used as bait to affinity purify bound proteins from different cancer cell line lysates. 490 unique interactors were identified, amongst which proteins with metabolic, homeostatic and migratory functions were overrepresented. This suggests that PRMs from RTKs may sustain a diverse interactome in cancer cells. Since RTK overexpression is common in cancer, RTK PRM-derived signalling may be an important, but as yet underexplored, contributor to negative cancer outcomes including resistance to kinase inhibitors.

Unraveling the assembly mechanism of SADS-CoV virus nucleocapsid protein: insights from RNA binding, dimerization, and epitope diversity profiling.

SADS-CoV is a porcine coronavirus that originated from a bat HKU2-related coronavirus. It causes devastating swine diseases and poses a high risk of spillover to humans. The coronavirus N protein, as the most abundant viral protein in infected cells, likely plays a key role in viral assembly and replication. However, the structure and function of this protein remain unclear. Therefore, this study employed a combination of biochemistry and X-ray crystallography to uncover distinct structural domains in the N protein, including RNA-binding domains, two disordered domains, and dimerization domains. Additionally, we made the novel discovery that the disordered domain elicited a significant antibody response. These findings provide new insights into the structure and functions of the SADS-CoV N protein, which have important implications for future studies on SADS-CoV diagnosis, as well as the development of vaccines and anti-viral drugs.

Genomics and proteomics to determine novel molecular subtypes and predict the response to immunotherapy and the effect of bevacizumab in glioblastoma

Glioblastoma (GBM) is a highly aggressive, infiltrative malignancy that cannot be completely cured by current treatment modalities, and therefore requires more precise molecular subtype signatures to predict treatment response for personalized precision therapy. Expression subtypes of GBM samples from the Cancer Genome Atlas (TCGA) were identified using BayesNM and compared with existing molecular subtypes of GBM. Biological features of the subtypes were determined by single-sample gene set enrichment analysis. Genomic and proteomic data from GBM samples were combined and Genomic Identification of Significant Targets in Cancer analysis was used to screen genes with recurrent somatic copy-number alterations phenomenon. The immune environment among subtypes was compared by assessing the expression of immune molecules and the infiltration of immune cells. Molecular subtypes adapted to immunotherapy were identified based on Tumor Immune Dysfunction and Exclusion (TIDE) score. Finally, least absolute shrinkage and selection operator (LASSO) logistic regression was performed on the expression profiles of S2, S3 and S4 in TCGA-GBM and RPPA to determine the respective corresponding best predictive model. Four novel molecular subtypes were classified. Specifically, S1 exhibited a low proliferative profile; S2 exhibited the profile of high proliferation, IDH1 mutation, TP53 mutation and deletion; S3 was characterized by high immune scores, innate immunity and adaptive immune infiltration scores, with the lowest TIDE score and was most likely to benefit from immunotherapy; S4 was characterized by high proliferation, EGFR amplification, and high protein abundance, and was the most suitable subtype for bevacizumab. LASSO analysis constructed the best prediction model composed of 13 genes in S2 with an accuracy of 96.7%, and the prediction model consisting of 17 genes in S3 with an accuracy of 86.7%, and screened 14 genes as components of the best prediction model in S4 with an accuracy of 93%. To conclude, our study classified reproducible and robust molecular subtypes of GBM, and these findings might contribute to the identification of patients responding to immunotherapy, thereby improving GBM prognosis.

Astragaloside IV relieves passive heymann nephritis and podocyte injury by suppressing the TRAF6/NF-κb axis

The pathogenesis of membranous nephropathy (MN) involves podocyte injury that is attributed to inflammatory responses induced by local immune deposits. Astragaloside IV (AS-IV) is known for its robust anti-inflammatory properties. Here, we investigated the effects of AS-IV on passive Heymann nephritis (PHN) rats and TNF-α-induced podocytes to determine the underlying molecular mechanisms of MN. Serum biochemical parameters, 24-h urine protein excretion and renal histopathology were evaluated in PHN and control rats. The expression of tumor necrosis factor receptor associated factor 6 (TRAF6), the phosphorylation of nuclear factor kappa B (p-NF-κB), the expression of associated proinflammatory cytokines (TNF-α, IL-6 and IL-1β) and the ubiquitination of TRAF6 were measured in PHN rats and TNF-α-induced podocytes. We detected a marked increase in mRNA expression of TNF-α, IL-6 and IL-1β and in the protein abundance of p-NF-κB and TRAF6 within the renal tissues of PHN rats and TNF-α-induced podocytes. Conversely, there was a reduction in the K48-linked ubiquitination of TRAF6. Additionally, AS-IV was effective in ameliorating serum creatinine, proteinuria, and renal histopathology in PHN rats. This effect was concomitant with the suppression of NF-κB pathway activation and decreased expression of TNF-α, IL-6, IL-1β and TRAF6. AS-IV decreased TRAF6 levels by promoting K48-linked ubiquitin conjugation to TRAF6, which triggered ubiquitin-mediated degradation. In summary, AS-IV averted renal impairment in PHN rats and TNF-α-induced podocytes, likely by modulating the inflammatory response through the TRAF6/NF-κB axis. Targeting TRAF6 holds therapeutic promise for managing MN.