Label-free Analysis Research Articles

Integrative multi-omics analyses reveal vesicle transport as a potential target for thoracic aortic aneurysm

BackgroundThoracic aortic aneurysm (TAA) refers to dilation and enlargement of the thoracic aorta caused by various reasons. Most patients have no apparent symptoms in the early stage and are subject to a poor prognosis once the aneurysm ruptures. It is crucial to identify individuals who are predisposed to TAA and to discover effective therapeutic targets for early intervention. MethodsWe conducted a label-free quantitative proteomic analysis among aorta tissue samples from TAA patients to screen differentially expressed proteins (DEPs) and key co-expression modules. Two datasets from Gene Expression Omnibus (GEO) database were included for integrative analysis, and the identified genes were subjected to immunohistochemistry (IHC) validation. Detailed vesicle transport related enrichment analysis was conducted and two FDA-approved drugs, chlorpromazine (CPZ) and chloroquine (CQ), were selected for in vivo inhibition of vesicle transport in mice TAA model. The diameter of thoracic aorta, mortality and histological differences after interventions were evaluated. ResultsWe found significant enrichments in functions involved with vesicle transport, extracellular matrix organizing, and infection diseases in TAA. Endocytosis was the most essential vesicle transport process in TAA formation. Interventions with CPZ and CQ significantly reduced the aneurysm diameter and elastin degradation in vivo and enhanced the survival rates of TAA mice. ConclusionsWe systematically screened the aberrantly regulated bioprocesses in TAA based on integrative multi-omics analyses, identified and demonstrated the importance of vesicle transport in the TAA formation. Our study provided pilot evidence that vesicular transport was a potential and promising target for the treatment of TAA.

Fibre-based fluorescence-lifetime imaging microscopy: a real-time biopsy guidance tool for suspected lung cancer.

Lung cancer is the most common cause of cancer-related deaths worldwide. Early detection improves outcomes, however, existing sampling techniques are associated with suboptimal diagnostic yield and procedure-related complications. Autofluorescence-based fluorescence-lifetime imaging microscopy (FLIM), a technique which measures endogenous fluorophore decay rates, may aid identification of optimal biopsy sites in suspected lung cancer. Our fibre-based fluorescence-lifetime imaging system, utilising 488 nm excitation, which is deliverable via existing diagnostic platforms, enables real-time visualisation and lifetime analysis of distal alveolar lung structure. We evaluated the diagnostic accuracy of the fibre-based fluorescence-lifetime imaging system to detect changes in fluorescence lifetime in freshly resected ex vivo lung cancer and adjacent healthy tissue as a first step towards future translation. The study compares paired non-small cell lung cancer (NSCLC) and non-cancerous tissues with gold standard diagnostic pathology to assess the performance of the technique. Paired NSCLC and non-cancerous lung tissues were obtained from thoracic resection patients (N=21). A clinically compatible 488 nm fluorescence-lifetime endomicroscopy platform was used to acquire simultaneous fluorescence intensity and lifetime images. Fluorescence lifetimes were calculated using a computationally-lightweight, rapid lifetime determination method. Fluorescence lifetime was significantly reduced in ex vivo lung cancer, compared with non-cancerous lung tissue [mean ± standard deviation (SD), 1.79±0.40 vs. 2.15±0.26 ns, P<0.0001], and fluorescence intensity images demonstrated distortion of alveolar elastin autofluorescence structure. Fibre-based fluorescence-lifetime imaging demonstrated good performance characteristics for distinguishing lung cancer, from adjacent non-cancerous tissue, with 81.0% sensitivity and 71.4% specificity. Our novel fibre-based fluorescence-lifetime imaging system, which enables label-free imaging and quantitative lifetime analysis, discriminates ex vivo lung cancer from adjacent healthy tissue. This minimally invasive technique has potential to be translated as a real-time biopsy guidance tool, capable of optimising diagnostic accuracy in lung cancer.

Assessing the precision of a detergent-assisted cartridge precipitation workflow for non-targeted quantitative proteomics.

Detergent-based workflows incorporating sodium dodecyl sulfate (SDS) necessitate additional steps for detergent removal ahead of mass spectrometry (MS). These steps may lead to variable protein recovery, inconsistent enzyme digestion efficiency, and unreliable MS signals. To validate a detergent-based workflow for quantitative proteomics, we herein evaluate the precision of a bottom-up sample preparation strategy incorporating cartridge-based protein precipitation with organic solvent to deplete SDS. The variance of data-independent acquisition (SWATH-MS) data was isolated from sample preparation error by modelling the variance as a function of peptide signal intensity. Our SDS-assisted cartridge workflow yield a coefficient of variance (CV) of 13%-14%. By comparison, conventional (detergent-free) in-solution digestion increased the CV to 50%; in-gel digestion provided lower CVs between 14% and 20%. By filtering peptides predicting to display lower precision, we further enhance the validity of data in global comparative proteomics. These results demonstrate the detergent-based precipitation workflow is a reliable approach for in depth, label-free quantitative proteome analysis.

Tumor-derived Prevotella intermedia aggravates gastric cancer by enhancing Perilipin 3 expression.

The indigenous microbial milieu within tumorous tissues exerts a pivotal influence on the genesis and advancement of gastric cancer (GC). This investigation scrutinizes the functions and molecular mechanisms attributed to Prevotella intermedia in the malignant evolution of GC. Isolation of P. intermedia from paired GC tissues was undertaken. Quantification of P. intermedia abundance in 102 tissues was accomplished using quantitative real-time PCR (qRT-PCR). Assessment of the biological effects of P. intermedia on GC cells was observed using culture medium supernatant. Furthermore, the protein profile of GC cells treated with tumor-derived P. intermedia was examined through label-free protein analysis. The functionality of perilipin 3 (PLIN3) was subsequently confirmed using shRNA. Our investigation revealed that the relative abundance of P. intermedia in tumor tissues significantly surpassed that of corresponding healthy tissues. The abundance of P. intermedia exhibited correlations with tumor differentiation (p = 0.006), perineural invasion (p = 0.004), omentum majus invasion (p = 0.040), and the survival duration of GC patients (p = 0.042). The supernatant derived from tumor-associated P. intermedia bolstered the proliferation, clone formation, migration, and invasion of GC cells. After indirect co-cultivation with tumor-derived P. intermedia, dysregulation of 34 proteins, including PLIN3, was discerned in GC cells. Knockdown of PLIN3 mitigated the malignancy instigated by P. intermedia in GC cells. Our findings posit that P. intermedia from the tumor microenvironment plays a substantial role in the malignant progression of GC via the modulation of PLIN3 expression. Moreover, the relative abundance of P. intermedia might serve as a potential biomarker for the diagnosis and prognosis of GC.

MAGOH promotes gastric cancer progression via hnRNPA1 expression inhibition-mediated RONΔ160/PI3K/AKT signaling pathway activation

BackgroundGastric cancer (GC) is associated with high mortality and heterogeneity and poses a great threat to humans. Gene therapies for the receptor tyrosine kinase RON and its spliceosomes are attracting increasing amounts of attention due to their unique characteristics. However, little is known about the mechanism involved in the formation of the RON mRNA alternative spliceosome RONΔ160.MethodsFourteen human GC tissue samples and six normal gastric tissue samples were subjected to label-free relative quantitative proteomics analysis, and MAGOH was identified as a candidate protein for subsequent studies. The expression of MAGOH in clinical specimens was verified by quantitative real-time PCR and western blotting. We then determined the biological function of MAGOH in GC through in vitro and in vivo experiments. RNA pulldown, RNA sequencing and RNA immunoprecipitation (RIP) were subsequently conducted to uncover the underlying mechanism by which MAGOH regulated the formation of RONΔ160.ResultsProteomic analysis revealed that MAGOH, which is located at key nodes and participates in RNA processing and mRNA splicing, was upregulated in GC tissue and GC cell lines and was associated with poor prognosis. Functional analysis showed that MAGOH promoted the proliferation, migration and invasion of GC cells in vitro and in vivo. Mechanistically, MAGOH inhibited the expression of hnRNPA1 and reduced the binding of hnRNPA1 to RON mRNA, thereby promoting the formation of RONΔ160 to activate the PI3K/AKT signaling pathway and consequently facilitating GC progression.ConclusionsOur study revealed that MAGOH could promote the formation of RONΔ160 and activate the PI3K/AKT signaling pathway through the inhibition of hnRNPA1 expression. We elucidate a novel mechanism and potential therapeutic targets for the growth and metastasis of GC based on the MAGOH-RONΔ160 axis, and these findings have important guiding significance and clinical value for the future development of effective therapeutic strategies for GC.

Proteomic profiling of formalin-fixed paraffine-embedded tissue reveals key proteins related to lung dysfunction in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) severely affects the lung leading to aberrant deposition of extracellular matrix and parenchymal stiffness with progressive functional derangement. The limited availability of fresh tissues represents one of the major limitations to study the molecular profiling of IPF lung tissue. The primary aim of this study was to explore the proteomic profiling yield of archived formalin-fixed paraffin-embedded (FFPE) specimens of IPF lung tissues. We further determined the protein expression according to respiratory functional decline at the time of biopsy. The total proteins isolated from 11 FFPE samples of IPF patients compared to 3 FFPE samples from a non-fibrotic lung defined as controls, were subjected to label-free quantitative proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS) and resulted in the detection of about 400 proteins. After the pairwise comparison between controls and IPF, functional enrichment analysis identified differentially expressed proteins that were involved in extracellular matrix signaling pathways, focal adhesion and transforming growth factor β (TGF-β) signaling pathways strongly associated with IPF onset and progression. Five proteins were significantly over- expressed in the lung of IPF patients with either advanced disease stage (Stage II) or impaired pulmonary function (FVC<75, DLCO<55) compared to controls; these were lymphocyte cytosolic protein 1 (LCP1), peroxiredoxin-2 (PRDX2), transgelin 2 (TAGLN2), lumican (LUM) and mimecan (OGN) that might play a key role in the fibrogenic processes. Our work showed that the analysis of FFPE samples was able to identify key proteins that might be crucial for the IPF pathogenesis. These proteins are correlated with lung carcinogenesis or involved in the immune landscape of lung cancer, thus making possible common mechanisms between lung carcinogenesis and fibrosis progression, two pathological conditions at risk for each other in the real life.

Label-Free Surface-Enhanced Raman Scattering Detection of Fire Blight Pathogen Using a Pathogen-Specific Bacteriophage.

Fire blight is one of the most devastating plant diseases, causing severe social and economic problems. Herein, we report a novel method based on label-free surface-enhanced Raman scattering (SERS) combined with an Erwinia amylovora-specific bacteriophage that allows detecting efficiently fire blight bacteria E. amylovora for the first time. To achieve the highest SERS signals for E. amylovora, we synthesized and compared plasmonic nanoparticles (PNPs) with different sizes, i.e., bimetallic gold core-silver shell nanoparticles (Au@AgNPs) and monometallic gold nanoparticles (AuNPs) and utilized the coffee-ring effect for the self-assembly of PNPs and enrichment of fire blight bacteria. Furthermore, we investigated the changes in the SERS spectra of E. amylovora after incubation with an E. amylovora-specific bacteriophage, and we found considerable differences in the SERS signals as a function of the bacteriophage incubation time. The results indicate that our bacteriophage-based label-free SERS analysis can specifically detect E. amylovora without the need for peak assignment on the SERS spectra but simply by monitoring the changes in the SERS signals over time. Therefore, our facile method holds great potential for the label-free detection of pathogenic bacteria and the investigation of viral-bacterial interactions.

Mucous cell histopathology and label-free quantitative proteomic analysis of skin mucus in fat greenling (Hexagrammos otakii) infected with Vibrio harveyi

Hexagrammos otakii is favored by consumers and aquaculture practitioners because of its strong adaptability and fast growth. However, recently, frequent outbreaks of diseases in the breeding of H. otakii have led to significant economic losses, especially due to bacterial diseases, which limit the healthy breeding of H. otakii. As a luminescent Gram-negative bacterium, Vibrio harveyi is the main pathogenic bacteria of H. otakii. In this study, the histopathology and label-free quantitative proteomics analysis were performed to reveal the changes of skin mucus proteins in H. otakii after infection with V. harveyi. The histopathological changes in the skin of H. otakii showed that when the bacteria were injected into the epithelial cells, it caused an increase in the number of mucous cells and a certain degree of damage and deformation in skin. Moreover, the quantitative proteomics analysis revealed a total of 364 differentially expressed proteins (DEPs), and these DEPs were found to be involved in environmental information processing, metabolism, infectious diseases: bacteria, replication and repair. More importantly, the enrichment analysis of the DEPs revealed that these different proteins were mainly targeted immune-related pathways. After infection of bacteria, the host's immune ability will be weakened, causing V. harveyi to enter the organism more easily, resulting in increased mucus in H. otakii, which will eventually lead to a decline in its physical function. These results provided an insight into a series of physiological changes after the bacterial infection of fish at the proteomic level and basic data for further exploration of the potential mechanism of skin mucus. Taken together, the results indicated more opportunities for the future designs and discoveries of effective antibacterial vaccines and antibacterial drugs for H. otakii.

Cancer screening through surface-enhanced Raman spectroscopy fingerprinting analysis of urinary metabolites using surface-carbonized silver nanowires on a filter membrane

BackgroundLabel-free surface-enhanced Raman spectroscopy (SERS)-based metabolic profiling has great potential for early cancer diagnosis, but further advancements in analytical methods and clinical evidence studies are required for clinical applications. To improve the cancer diagnostic accuracy of label-free SERS spectral analysis of complex biological fluids, it is necessary to obtain specifically enhanced SERS signals of cancer-related metabolites present at low concentrations. ResultsThis study presents a novel 3D SERS sensor, comprising a surface-carbonized silver nanowire (AgNW)-stacked filter membrane, alongside an optimized urine/methanol/chloroform extraction technique, which specifically changes the molecular adsorption and orientation of aromatic metabolites onto SERS substrates. By analyzing the pretreated urine samples on the surface-carbonized AgNW 3D SERS sensor, distinct and highly enhanced SERS peaks derived from semi-polar aromatic metabolites were observed for pancreatic cancer and prostate cancer samples compared with normal controls. Urine metabolite analysis using SERS fingerprinting successfully differentiated pancreatic cancer and prostate cancer groups from normal control group: normal control (n = 56), pancreatic cancer (n = 40), and prostate cancer (n = 39). Significance and noveltyWe confirmed the clinical feasibility of performing fingerprint analysis of urinary metabolites based on the surface-carbonized AgNW 3D SERS sensor and methanol/chloroform extraction for noninvasive cancer screening. This technology holds potential for large-scale screening owing to its high accuracy, and cost effective, simple and rapid detection method.

Dehydration-responsive cytoskeleton proteome of rice reveals reprograming of key molecular pathways to mediate metabolic adaptation and cell survival

The plant cytoskeletal proteins play a key role that control cytoskeleton dynamics, contributing to crucial biological processes such as cell wall morphogenesis, stomatal conductance and abscisic acid accumulation in repercussion to water-deficit stress or dehydration. Yet, it is still completely unknown which specific biochemical processes and regulatory mechanisms the cytoskeleton uses to drive dehydration tolerance. To better understand the role of cytoskeleton, we developed the dehydration-responsive cytoskeletal proteome map of a resilient rice cultivar. Initially, four-week-old rice plants were exposed to progressive dehydration, and the magnitude of dehydration-induced compensatory physiological responses was monitored in terms of physicochemical indices. The organelle fractionation in conjunction with label-free quantitative proteome analysis led to the identification of 955 dehydration-responsive cytoskeletal proteins (DRCPs). To our knowledge, this is the first report of a stress-responsive plant cytoskeletal proteome, representing the largest inventory of cytoskeleton and cytoskeleton-associated proteins. The DRCPs were apparently involved in a wide array of intra-cellular molecules transportation, organelles positioning, cytoskeleton organization followed by different metabolic processes including amino acid metabolism. These findings presented open a unique view on global regulation of plant cytoskeletal proteome is intimately linked to cellular metabolic rewiring of adaptive responses, and potentially confer dehydration tolerance, especially in rice, and other crop species, in general.

Microcavity well-plate for automated parallel bioelectronic analysis of 3D cell cultures

Three-dimensional (3D) in vitro cell culture models serve as valuable tools for accurately replicating cellular microenvironments found in vivo. While cell culture technologies are rapidly advancing, the availability of non-invasive, real-time, and label-free analysis methods for 3D cultures remains limited. To meet the demand for higher-throughput drug screening, there is a demanding need for analytical methods that can operate in parallel. Microelectrode systems in combination with microcavity arrays (MCAs), offer the capability of spatially resolved electrochemical impedance analysis and field potential monitoring of 3D cultures. However, the fabrication and handling of small-scale MCAs have been labour-intensive, limiting their broader application. To overcome this challenge, we have established a process for creating MCAs in a standard 96-well plate format using high-precision selective laser etching. In addition, to automate and ensure the accurate placement of 3D cultures on the MCA, we have designed and characterized a plug-in tool using SLA-3D-printing. To characterize our new 96-well plate MCA-based platform, we conducted parallel analyses of human melanoma 3D cultures and monitored the effect of cisplatin in real-time by impedance spectroscopy. In the following we demonstrate the capabilities of the MCA approach by analysing contraction rates of human pluripotent stem cell-derived cardiomyocyte aggregates in response to cardioactive compounds. In summary, our MCA system significantly expands the possibilities for label-free analysis of 3D cell and tissue cultures, offering an order of magnitude higher parallelization capacity than previous devices. This advancement greatly enhances its applicability in real-world settings, such as drug development or clinical diagnostics.

Effects of nano freezing-thawing on myofibrillar protein of Atlantic salmon fillets: Protein structure and label-free proteomics

This study investigated the impact of magnetic nanoparticles (MNPs) -assisted cryogenic freezing integrated with MNPs combined microwave thawing (NNMT) on the structural integrity of myofibrillar proteins and alterations in protein profiles in salmon fillets. The NNMT showed the lowest myofibrillar fragmentation index (MFI) value (2.73 ± 0.31) among the four freezing-thawing groups. The myofibrillar structure exhibited the highest level of integrity, while the myofibrillar proteins demonstrated minimal aggregation and displayed the most stable secondary and tertiary structures in response to NNMT treatment. Compared with the other three treatments, NNMT exhibited a high abundance of ionic and hydrogen bonds, resulting in stronger interactions between the proteins and water molecules. The label-free proteomics analysis revealed that different freezing-thawing methods primarily affected the cytoskeletal proteins, with collagen and myosin being down-regulated due to degradation caused by cold stress and recrystallization. Additionally, NNMT demonstrated a superior capability in stabilizing salmon cytoskeletal proteins.

Optimized protocol for shotgun label-free proteomic analysis of pancreatic islets.

Pancreatic islets are crucial in diabetes research. Consequently, this protocol aims at optimizing both the protein-extraction process and the proteomic analysis via shotgun methods for pancreatic islets. Six protocols were tested, combining three types of chemical extraction with two mechanical extraction methods. Furthermore, two protocols incorporated a surfactant to enhance enzymatic cleavage. The steps involved extraction and concentration of protein, protein quantification, reduction, alkylation, digestion, purification and desalination, sample concentration to ∼1 µl, and proteomic analysis using the mass spectrometer. The most effective protocol involves either a milder chemical extraction paired with a more intensive mechanical process, or a more robust chemical extraction paired with a gentle mechanical process, tailored to the sample's characteristics. Additionally, it was observed that the use of a surfactant proved ineffective for these types of samples. Protocol 5 was recently used with success to examine metabolic changes in pancreatic islets of non-obese diabetic mice exposed to low doses of fluoride ions (F-) and the primary pathways altered by the treatment.

Plasma-derived exosomal protein SHP2 deficiency induces neutrophil hyperactivation in Behcet's uveitis

To investigate the effect of plasma-derived exosomal proteins on neutrophil hyperactivation in Behcet's uveitis (BU), we treated neutrophils from healthy controls with plasma-derived exosomes from active BU patients, and determined the level of neutrophil activation by real-time quantitative PCR (RT-qPCR) and cytokine detection assay. The results revealed that exosomes from active BU patients could activate neutrophils as shown by increasing the expression levels of pro-inflammatory cytokines (IL-17 and IL-6), chemokines (IL-8 and MCP-1), and NETs (MPO and ELANE). Label-free quantitative proteomic analysis of plasma-derived exosomes from patients and healthy controls found a remarkably distinct protein profile and identified differentially expressed proteins (DEPs) between the two groups. The results of GO, KEGG, and GSEA enrichment analysis showed that DEPs were enriched in innate immune-mediated and neutrophil hyperactivation-related signaling pathways. The protein-protein interaction (PPI) analysis determined that SHP2 was a downregulated key hub protein in the exosomes of active BU patients. Knockdown of SHP2 in human neutrophil cell lines (NB4 cells) was shown to promote the secretion of pro-inflammatory cytokines, chemokines, and NETs. The converse effects were observed following SHP2 overexpression. In conclusion, we highlighted a pathogenic role of plasma-derived exosomal SHP2 deficiency in facilitating neutrophil activation and suggested that SHP2 might be an immunoprotective factor in BU pathologic process.

Label-Free and Real-Time Optical Detection of Affinity Binding of the Antibody on Adherent Live Cells.

Oblique-incidence reflectivity difference (OIRD) is a novel real-time, label-free, and nondestructive optical detection method and exhibits encouraging application in the detection of antibody/DNA microarrays. In this study, for the first time, an OIRD label-free immunoassay was achieved by using adherent live cells as the probe. The cells were cultured on glass cells, and the affinity binding of antibodies targeted on the HLA class I antigen of the cell surface was detected with an OIRD. The results show that an OIRD is able to detect the binding process of anti-human HLA-A, B, and C antibodies on MDA-MB-231 cells and HUVEC cells. Control experiments and complementary fluorescence analysis confirmed the high detection specificity and good quantitative virtue of the OIRD label-free immunoassay. Label-free OIRD imaging analysis of cell microarrays was further demonstrated successfully, and the underlying optical mechanism was revealed by combining the theoretical modeling. This work explores the use of live cells as probes for an OIRD immunoassay, thus expanding the potential applications of the OIRD in the field of pathological analysis, disease diagnosis, and drug screening, among others.

Motion-resolved, reference-free holographic imaging via spatiotemporally regularized inversion

Holography is a powerful technique that records the amplitude and phase of an optical field simultaneously, enabling a variety of applications such as label-free biomedical analysis and coherent diffraction imaging. Holographic recording without a reference wave has been long pursued because it obviates the high experimental requirements of conventional interferometric methods. However, due to the ill-posed nature of the underlying phase retrieval problem, reference-free holographic imaging is faced with an inherent tradeoff between imaging fidelity and temporal resolution. Here, we propose a general computational framework, termed spatiotemporally regularized inversion (STRIVER), to achieve motion-resolved, reference-free holographic imaging with high fidelity. Specifically, STRIVER leverages signal priors in the spatiotemporal domain to jointly eliminate phase ambiguities and motion artifacts, and, when combined with diversity measurement schemes, produces a physically reliable, time-resolved holographic video from a series of intensity-only measurements. We experimentally demonstrate STRIVER in near-field ptychography, where dynamic holographic imaging of freely swimming paramecia is performed at a framerate-limited speed of 112 fps. The proposed method can be potentially extended to other measurement schemes, spectral regimes, and computational imaging modalities, pushing the temporal resolution toward higher limits.

Integrated 4D Analysis of Intramuscular Fat Deposition: Quantitative Proteomic and Transcriptomic Studies in Wannanhua Pig Longissimus Dorsi Muscle.

Wannanhua (WH) is a pig breed indigenous to Anhui Province, China. This breed has a high intramuscular fat (IMF) content, making it an ideal model for investigating lipid deposition mechanisms in pigs. IMF content is one of the main indicators of meat quality in pigs and is regulated by multiple genes and metabolic pathways. Building upon our prior transcriptomic investigation, the present study focused on the longissimus dorsi muscle tissue of Wannanhua (WH) pigs in the rapid fat-deposition stages (120 and 240 days of age). Employing 4D label-free quantitative proteomic analysis, we identified 106 differentially expressed proteins (DEPs). Parallel reaction monitoring (PRM) technology was used to verify the DEPs, and the results showed that the 4D label-free results were reliable and valid. Functional enrichment and protein-protein interaction analyses showed that the DEPs were mainly involved in the skeletal-muscle-associated structural proteins, mitochondria, energy metabolism, and fatty acid metabolism. By integrating transcriptomic data, we identified seven candidate genes including ACADL, ACADM, ANKRD2, MYOZ2, TNNI1, UCHL1, and ART3 that play a regulatory role in fat deposition and muscle development. These findings establish a theoretical foundation for future analyses of lipid deposition traits, contributing to potential enhancements in pig meat quality during breeding and advancing the selection process for Chinese indigenous breeds.

Quantitative label-free proteomic analysis of excretory-secretory proteins in different developmental stages of Trichinella spiralis

Trichinella spiralis (T. spiralis) is a zoonotic parasitic nematode with a unique life cycle, as all developmental stages are contained within a single host. Excretory-secretory (ES) proteins are the main targets of the interactions between T. spiralis and the host at different stages of development and are essential for parasite survival. However, the ES protein profiles of T. spiralis at different developmental stages have not been characterized. The proteomes of ES proteins from different developmental stages, namely, muscle larvae (ML), intestinal infective larvae (IIL), preadult (PA) 6 h, PA 30 h, adult (Ad) 3 days post-infection (dpi) and Ad 6 dpi, were characterized via label-free mass spectrometry analysis in combination with bioinformatics. A total of 1217 proteins were identified from 9341 unique peptides in all developmental stages, 590 of which were quantified and differentially expressed. GO classification and KEGG pathway analysis revealed that these proteins were important for the growth of the larvae and involved in energy metabolism. Moreover, the heat shock cognate 71 kDa protein was the centre of protein interactions at different developmental stages. The results of this study provide comprehensive proteomic data on ES proteins and reveal that these ES proteins were differentially expressed at different developmental stages. Differential proteins are associated with parasite survival and the host immune response and may be potential early diagnostic antigen or antiparasitic vaccine candidates.

Comparative Proteomic Analysis Reveals Candidate Pathways Related to the Effect of Different Light Qualities on the Development of Mycelium and Fruiting Body of Pleurotus ostreatus.

Light affects the morphology and physiology of Pleurotus ostreatus. However, the underlying molecular mechanism of this effect remains unclear. In this study, a label-free comparative proteomic analysis was conducted to investigate the global protein expression profile of the mycelia and fruiting bodies of P. ostreatus PH11 growing under four different light quality treatments. Among all the 2234 P. ostreatus proteins, 1349 were quantifiable under all tested conditions. A total of 1100 differentially expressed proteins were identified by comparing the light group data with those of the darkness group. GO and KEGG enrichment analyses indicated that the oxidative phosphorylation, proteasome, and mRNA surveillance pathways were the most related pathways under the light condition. qRT-PCR verified that the expression of the white collar 1 protein was significantly enhanced under white light. Additionally, glutamine synthetase and aldehyde dehydrogenase played important roles during light exposure. This study provides valuable insight into the P. ostreatus light response mechanism, which will lay the foundation for improved cultivation.

The sparing effect of ultra-high dose rate irradiation on the esophagus.

Current studies have substantiated the sparing effect of ultra-high dose rate irradiation (FLASH) in various organs including the brain, lungs, and intestines. Whether this sparing effect extends to esophageal tissue remains unexplored. This study aims to compare the different responses of esophageal tissue in histological and protein expression levels following conventional dose rate irradiation (CONV) and FLASH irradiation to ascertain the presence of a sparing effect. C57 female mice were randomly divided into three groups: control, CONV, and FLASH groups. The chest region of the mice in the radiation groups was exposed to a prescribed dose of 20 Gy using a modified electron linear accelerator. The CONV group received an average dose rate of 0.1 Gy/s, while the FLASH group received an average dose rate of 125 Gy/s. On the 10th day after irradiation, the mice were euthanized and their esophagi were collected for histopathological analysis. Subsequently, label-free proteomic quantification analysis was performed on esophageal tissue. The validation process involved analyzing transmission electron microscopy images and utilizing the parallel reaction monitoring method. Histopathology results indicated a significantly lower extent of esophageal tissue damage in the FLASH group compared to the CONV group (p < 0.05). Label-free quantitative proteomic analysis revealed that the sparing effect observed in the FLASH group may be attributed to a reduction in radiation-induced protein damage associated with mitochondrial functions, including proteins involved in the tricarboxylic acid cycle and oxidative phosphorylation, as well as a decrease in acute inflammatory responses. Compared with CONV irradiation, a sparing effect on esophageal tissue can be observed after FLASH irradiation. This sparing effect is associated with alleviated mitochondria damage and acute inflammation.