Single-cell intracellular epitope and transcript detection revealing signal transduction dynamics

BackgroundSingle cell RNA sequencing (scRNA-seq) is a powerful technique for studying the molecular heterogeneity of cells.1–3 However, scRNA-seq does not provide information about protein expression, which is essential for understanding...

Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRbeta has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRbeta leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene.

Gamma-Globin Gene Expression in Adult Human Erythroblasts Is Associated with Concurrent Changes in the Nuclear Protein Levels of at Least Seven Transcription Factors.

Methods Twenty-five newborn piglets were selected and then randomly assigned to the control group (n = 5) and the model group (n = 20) subjected to HI. HI was induced by blocking bilateral carotid blood flow under simultaneous inhalation of a 6% oxygen mixture. 1H-MRS data were acquired from the basal ganglia at the following time points after HI: 6, 12, 24, and 72 h. Changes in protein levels of EAAT2 and GluR2 were determined by immunohistochemical analysis. Correlations among metabolite concentrations, metabolite ratios, and the protein levels of EAAT2 and GluR2 were investigated. Results The Glu level sharply increased after HI, reached a transient low level of depletion that approached the normal level in the control group, and subsequently increased again. Negative correlations were found between concentrations of Glu and EAAT2 protein levels (Rs = −0.662, P < 0.001) and between the Glu/creatine (Cr) ratio and EAAT2 protein level (Rs = −0.664, P < 0.001). Moreover, changes in GluR2 protein level were significantly and negatively correlated with those in Glu level (the absolute Glu concentration, Rs = −0.797, P < 0.001; Glu/Cr, Rs = −0.567, P = 0.003). Conclusions Changes in Glu level measured by 1H-MRS were inversely correlated with those in EAAT2 and GluR2 protein levels following HI, and the results demonstrated that 1H-MRS can reflect the early changes of glutamatergic activity in vivo.

Dexamethasone treatment alters function of adipocytes from a mesenchymal stromal cell line

Lycopene treatment inhibits activation of Jak1/Stat3 and Wnt/β-catenin signaling and attenuates hyperproliferation in gastric epithelial cells

Specific induction of glutathione S-transferase GSTM2 subunit expression by epigallocatechin gallate in rat liver

The aim of the current study was to investigate whether doublecortin (DCX), insulin-like growth factor receptor 1 (IGF-1R) and metabotropic glutamate receptor 5 (mGluR5) levels are indeed modified in the aging rat hippocampal individual subareas (rather than total hippocampal tissue as in previous reports) at the protein and mRNA level and whether the methylation status contributes to these changes. Since the aging population is not homogeneous in terms of spatial memory performance, we examined whether changes in DCX, IGF-1R and mGluR5 are linked to cognitive aging. Aged (22months) male Sprague Dawley rats were trained in the hole-board, a spatial memory task, and were subdivided according to performance to aged impaired and aged unimpaired groups. Age- and memory performance-dependent changes in mRNA steady-state levels, protein levels and DNA methylation status of DCX, IGF-1R and mGluR5 were evaluated by RT-PCR, immunoblotting and bisulfite pyrosequencing. Extending previous findings, we detected decreased DCX protein and mRNA levels in dentate gyrus (DG) of aged animals. IGF-1 signaling is a key event and herein we show that mRNA levels for IGF-1R were unchanged although reduced at the protein level. This finding may simply reflect that these protein levels are regulated at the level of protein synthesis as well as protein degradation. We provide evidence that promoter methylation is not involved in regulation of mRNA and protein levels of DCX, IGF-1R and mGluR5 during aging. Moreover, there was no significant difference between aged rats with impaired and aged rats with unimpaired memory at the protein and mRNA level. Findings propose that changes in the abovementioned protein levels may not be relevant for performance in the spatial memory task used in aged rats.

Immunomodulatory drugs lenalidomide and pomalidomide inhibit multiple myeloma-induced osteoclast formation and the RANKL/OPG ratio in the myeloma microenvironment targeting the expression of adhesion molecules

A recent advance that has revitalized toxicology studies in the past couple of decades is Frontiers in Genetics “toxicogenomics,” the study of the effects of environmental toxins on global changes within a cell or tissue as related to genomic variables. “Transcriptional toxicogenomics” is the study of global gene expression patterns after administering a toxin. Similarly, “toxicoproteomics” is the study of global changes in protein levels or post-translational modifications; while “toxicometabolomics” is the study of global changes in metabolite levels after administering a toxin. A new field called “toxicoepigenomics” or more simply, the “epigenetics of toxicology,” is the study of global epigenetic changes in a cell that are caused by exposure of a cell, tissue, or organism to a toxin or drug. Frontiers in Toxicogenomics provides a format for presenting cutting-edge reviews and studies in the frontiers of all branches of toxicogenomics. One type of study within the area of transcriptional toxicogenomics called “genetical genomics” or “genetics of gene expression,” is the combination of quantitative trait-locus (QTL) and whole-genome expression analyses (Brem et al., 2002; Broman, 2005). In genetical genomics, the investigator treats the expression of each of the ∼20,000 genes in an organism as a quantitative trait and identifies cis-expression QTL (eQTL) in cis-regulatory sequences and trans-eQTL in trans-acting factors that regulate expression of co-regulated genes in specific pathways. Genetical toxicogenomics adds a new dimension to genetical genomics by studying the effects of an environmental toxin on global gene expression patterns (Ruden et al., 2009). Such studies are theoretically very powerful because they not only tell which genes are induced in a particular tissue (such as a brain region) by the toxin, but they also allow the identification of trans-acting factors and cis-acting sequences that are regulated by the toxin. Toxicogenetical genomic studies in mice have the advantage that thousands of recombinant inbred lines have recently been generated in a collaborative cross amongst eight different wild type strains of mice (Churchill et al., 2004). Rats are sometimes preferred models for toxicological screens because they have much larger organs than mice, allowing for easier gene and protein expression analyses because much more material can be obtained compared with mice. Recombinant inbred lines have also been developed in rats, and some laboratories have conducted genetical genomics and genetical toxicogenomics analyses in rats, especially in the rat brain. The transcriptional toxicogenomic studies have mostly been done with recombinant inbred lines, such as in mice, C. elegans, and Drosophila, and utilized gene expression microarrays. However, next-generation sequencing technologies allow new types of transcriptional toxicogenomic studies to be conducted. For example, “speed QTL” analyses can be done by selecting a large population of model organisms for a particular trait, such as lifespan after feeding a toxin, for several generations and then sequencing the genome of the long-living descendents (Lai et al., 2007). The single nucleotide polymorphisms (SNPs) that are enriched in the long-living descendents are located in candidate toxin-survival genes which can be studied in follow up genetic and biochemical studies. Third generation sequencing technologies will soon make the “$1000 genome” a reality and will not only allow “personalized medicine,” which is using a person's genome sequence to optimize a drug regimen to treat a disease (Ruden, 2007), but also “personalized toxicogenomics” which uses this genome information to determine a person's susceptibility to an environmental toxin. Toxicoproteomics and toxicometabolimics are new areas that utilize state-of-the-art mass spectrometry and other sensitive assay systems that can quantify protein, peptide, and metabolic levels in a population of model organisms. Instead of gene expression being the quantitative trait, as in transcriptional toxicogenomics, protein, and metabolite levels can be used as quantitative traits. In this manner, protein QTL (pQTL) and metabolite-QTL (mQTL) can be identified in both cis- and trans-genes that regulate the levels of molecules. Toxicoepigenomics is the study of global epigenomic changes, such as DNA methylation changes, histone modifications, or microRNA changes, that are caused by exposure to toxins. In this manner, epigenetic-QTL (epiQTL) can be identified that identify cis- and trans-regulatory elements and factors that alter the epigenome in response to a toxin. It is known for instance that prenatal lead exposure causes hypomethylation of repeats in humans. It would be interesting to study global methylation patterns after exposure to lead and other toxins in large populations. In conclusion, toxicogenomics is an exciting new field that has revitalized toxicology research. No longer need toxicologists study only LD50 values (lethal dose that kills 50% of the population) and use death alone as a toxicology endpoint. Now toxicologists can perform studies with much more subtle endpoints, such as gene expression, protein or metabolite levels, and epigenomic changes. Frontiers in Toxicogenomics provides a format for present research and reviews in this exciting new field.

&lt;div&gt;AbstractPurpose:&lt;p&gt;The management of chronic lymphocytic leukemia (CLL) has significantly improved with targeted therapies. However, many patients experience a suboptimal response. To optimally select the best therapy, predictive biomarkers are necessary. In this study, we used the phosphoinositide 3-kinase (PI3K) inhibitor umbralisib as a model to (i) understand the impact of targeted treatment on cell signaling and immunophenotypes in responders and nonresponders, (ii) identify molecular features that predict individual treatment responses, and (iii) suggest alternative treatment options for the nonresponders.&lt;/p&gt;Experimental Design:&lt;p&gt;We performed functional phenotyping of CLL cells from patients enrolled in two clinical trials with umbralisib, administered either as a monotherapy (NCT02742090, &lt;i&gt;n&lt;/i&gt; = 55) or in combination with the Bruton tyrosine kinase (BTK) inhibitor acalabrutinib (NCT04624633, &lt;i&gt;n&lt;/i&gt; = 12).&lt;/p&gt;Results:&lt;p&gt;We found that umbralisib monotherapy led to significant changes in (phospho)protein levels, including AKT (pS473), in responders but not in nonresponders. Furthermore, the proportion of cytotoxic natural killer (NK) cells increased at the end of the study but only in responders, suggesting a role in the antitumor response. To identify molecular predictors of response, we used the baseline levels of 30 (phospho)proteins in the monotherapy cohort as input features for a machine learning model, which achieved significant prediction accuracy in cross-validation and maintained its predictive power in the combination cohort. Drug sensitivity profiling of the CLL cells at baseline suggested that PI3K + Bcl-2 inhibitors are effective in umbralisib nonresponders.&lt;/p&gt;Conclusions:&lt;p&gt;Functional phenotyping reveals differential cellular responses to umbralisib treatment in responders and nonresponders; predicts treatment response of individual patients with CLL; and suggests alternative treatment options for the nonresponders.&lt;/p&gt;&lt;/div&gt;

Simple SummaryThe health of farmed marine fish, including the Japanese flounder (Paralichthys olivaceus), is severely compromised by temperature fluctuations and infections such as Edwardsiella tarda, resulting in significant economic losses. A previous study revealed that elevated temperature enhanced E. tarda dissemination in flounder tissues. However, the underlying mechanism has not been fully explained, especially the changes in protein level. This study utilized label-free proteomics to investigate the immune response of flounder to E. tarda infection under low (15 °C) and high (23 °C) temperature conditions. Multiple differentially abundant proteins (DAPs) were identified in each group. GO and KEGG analyses highlighted immune-related pathways and proteins, with key DAPs forming extensive interaction networks. Venn analysis revealed distinct responses: high temperature promoted endocytosis and complement activation, while low temperature increased histone levels and impaired RNA transport. These results enhance our understanding of how water temperature changes affect antibacterial immunity in fish.Fluctuating water temperatures and bacterial pathogens such as Edwardsiella tarda pose a serious threat to mariculture, resulting in significant economic losses within the flounder industry. A previous study revealed that elevated temperature enhanced E. tarda dissemination in flounder tissues. However, the underlying mechanism has not been fully explained, especially the changes in protein level. In this study, label-free proteomics was utilized to investigate the impact of high temperature (23 °C) and low temperature (15 °C) on flounder immune response to E. tarda infection. Our results identified 317 differentially abundant proteins (DAPs) in the low-temperature group (LI-LC) and 302 DAPs in the high-temperature group (HI-HC). GO and KEGG analyses of DAPs revealed numerous immune-related proteins and pathways. Twenty-six key DAPs in the LI-LC group and twenty-seven key DAPs in the HI-HC group were further identified and formed extensive interaction networks, respectively. Through the analysis of key immune-related DAPs that were specifically identified in both groups via Venn diagram analysis, we demonstrated that the endocytosis capacity and complement activity were enhanced in the HI-HC group, while histone abundance and RNA transport function were, respectively, increased and severely interfered with in the LI-LC group. These findings highlight a clear divergence in the immune response of flounder to E. tarda infection between 15 °C and 23 °C, providing valuable insights into how temperature variation influences antibacterial immunity in fish.

The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used high-throughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems.

Background/Aims: Efficient uptake at the basolateral plasma membrane of hepatocytes is required for maintenance of the enterohepatic circulation of bile salts. Uptake occurs mainly via a Na +-dependent process mediated by ntcp, a recently cloned and characterized 51 kDa glycoprotein. The aim of this study was to evaluate the role of variations in hepatic bile salt flux through the liver in the regulation of ntcp activity and expression under non-cholestatic conditions. Methods: We determnied the kinetics of Na +-dependent taurocholate transport in isolated basolateral plasma membrane vesicles as well as hepatic ntcp protein and ntcp mRNA levels in long-term (8 days) bile-diverted rats, with a transhepatic bile salt flux of 0, and in streptozotocin-induced diabetic rats with a 2.5-fold increased bile salt flux. Results: We found no changes in the kinetics of taurocholate transport in the absence of transhepatic bile salt flux due to bile diversion. Ntcp protein and ntcp mRNA levels were also unaffected in bile-diverted rats. Likewise, no changes in taurocholate transport kinetics, ntcp protein or ntcp mRNA levels were detected in streptozotocin-diabetic rats when compared to non-diabetic controls. Thus, variation in hepatic bile salt flux from 0 to 250% of normal values had no effect on hepatic ntcp expression or taurocholate transport activity in basolateral plasma membrane vesicles in rats. In contrast, 4 days of bile duct ligation resulted in a strong decrease in ntcp mRNA and protein levels, as recently also reported by others. Conclusions: Our data indicate that ntcp is not regulated by the transhepatic flux of bile acids under non-cholestatic conditions.

Recent studies have highlighted the role of ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, in cancer biology. This study aims to investigate the effect of lactotransferrin (LTF) upregulation on the pathological changes related to non-small cell lung cancer (NSCLC) via the inhibition of ferroptosis. LTF's involvement in NSCLC was investigated through cell experiments and clinical samples. Cell models with stable LTF knockdown or overexpression were established by lentiviral transduction. Cell viability and cytotoxicity were evaluated through cell counting kit 8 (CCK8) and lactate dehydrogenase (LDH) experiments. Scratch and Transwell experiments were conducted to verify the effect of LTF expression on the migration and invasion abilities of lung cancer cells. Protein and mRNA expression were analyzed using Western blotting and qPCR. Malondialdehyde (MDA), glutathione (GSH), free iron ions (Fe2+), and reactive oxygen species (ROS) levels were measured with appropriate kits. The intracellular localization and expression of the protein was detected through immunofluorescence (IF). Peripheral blood of healthy controls and patients with preliminarily diagnosed non-small cell lung cancer was collected, and the expression levels of LTF protein and mRNA were detected by Western blotting and quantitative polymerase chain reaction (qPCR) experiments. The results demonstrate that LTF was upregulated in NSCLC and it's overexpression could significantly enhance the migration, invasion, and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer cells. The overexpression of LTF significantly inhibited ferroptosis in non-small cell lung cancer cells. LTF modulates the expression of critical regulators of ferroptosis including glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4), leading to altered cellular redox status. The protein and mRNA expression levels of LTF were both increased in the peripheral blood of patients with NSCLC, with changes in protein level being more significant. Additionally, the overexpression of LTF was significantly correlated with the stage of NSCLC. In conclusion, these findings suggest that LTF upregulation plays a crucial role in inhibiting ferroptosis, thereby influencing the pathological progression of NSCLC. This study provides a potential therapeutic avenue for targeting ferroptosis in NSCLC treatment strategies.