Protein Mass Research Articles

Downregulation of GPX8 in hepatocellular carcinoma: impact on tumor stemness and migration

PurposeGPX8, which is found in the endoplasmic reticulum lumen, is a member of the Glutathione Peroxidases (GPXs) family. Its role in hepatocellular carcinoma (HCC) is unknown.MethodsImmunohistochemical staining was used to detect the protein levels of GPX8 in HCC tissue microarrays. A short hairpin RNA lentivirus was used to knock down GPX8, and the main signaling pathways were investigated using transcriptome sequencing and a phosphorylated kinase array. The sphere formation assays, cloning-formation assays and cell migration assays were used to evaluate the stemness and migration ability of HCC cells. Identifying the GPX8-interacting proteins was accomplished through immunoprecipitation and protein mass spectrometry.ResultsThe GPX8 protein levels were downregulated in HCC patients. Low expression of GPX8 protein was related to early recurrence and poor prognosis in HCC patients. GPX8 knockdown could enhance the stemness and migration ability of HCC cells. Consistently, Based on transcriptome analysis, multiple signaling pathways that include the PI3K-AKT and signaling pathways that regulate the pluripotency of stem cells, were activated after GPX8 knockdown. The downregulation of GPX8 could increase the expression of the tumor stemness markers KLF4, OCT4, and CD133. The in vivo downregulation of GPX8 could also promote the subcutaneous tumor-forming and migration ability of HCC cells. MK-2206, which is a small-molecule inhibitor of AKT, could reverse the tumor-promoting effects both in vivo and in vitro. We discovered that GPX8 and the 71-kDa heat shock cognate protein (Hsc70) have a direct interaction. The phosphorylation of AKT encouraged the translocation of Hsc70 into the nucleus and the expression of the PI3K p110 subunit, thereby increasing the downregulation of GPX8.ConclusionThe findings from this study demonstrate the anticancer activity of GPX8 in HCC by inactivating the Hsc70/AKT pathway. The results suggest a possible therapeutic target for HCC.

Proteomic profiling of tumor microenvironment and prognosis risk prediction in stage I lung adenocarcinoma

ObjectivesWith the increasing popularity of CT screening, more cases of early-stage lung cancer are being diagnosed. However, 24.5% of stage I non-small-cell lung cancer (NSCLC) patients still experience treatment failure post-surgery. Biomarkers to predict lung cancer patients at high risk of recurrence are needed. Materials and methodsWe collected protein mass spectrometry data from the Taiwan Lung Cancer Moonshot Project and performed bioinformatics analysis on proteins with differential expressions between tumor and adjacent normal tissues in 74 stage I lung adenocarcinoma (LUAD) cases, aiming to explore the tumor microenvironment related prognostic biomarkers. Findings were further validated in 6 external cohorts. ResultsThe analysis of differentially expressed proteins revealed that the most enriched categories of diseases and biological functions were cellular movement, immune cell trafficking, and cancer. Utilizing proteomic profiling of the tumor microenvironment, we identified five prognostic biomarkers (ADAM10, MIF, TEK, THBS2, MAOA). We then developed a risk score model, which independently predicted recurrence-free survival and overall survival in stage I LUAD. Patients with high risk scores experienced worse recurrence-free survival (adjusted hazard ratio = 8.28, p < 0.001) and overall survival (adjusted hazard ratio = 6.88, p = 0.013). Findings had been also validated in the external cohorts. ConclusionThe risk score model derived from proteomic profiling of tumor microenvironment can be used to predict recurrence risk and prognosis of stage I LUAD.

Modelling of on-farm greenhouse gas emissions from dual-purpose meat and wool sheep production in different geographical regions of Norway

The whole-farm model, HolosNorSheep, was developed to estimate net greenhouse gas emissions from dual purpose sheep production in Norway. The model adopted a cradle-to-farm gate system boundary and is based on the International Panel on Climate Change methodology and includes direct emissions from enteric and manure methane (CH4; GWP100=27), direct and indirect nitrous oxide (N2O; GWP100=273) from manure and soils, direct carbon dioxide (CO2) emissions from energy use and indirect CO2 emissions from the production of input factors. Soil carbon balance is calculated using the ICBM-model. Emission intensities (CO2-eq/kg product) for sheep carcass and greasy wool, was estimated for five geographical regions of Norway (East, West, South, Mid and North). The geographical regions varied considerably in climate, feed resources, days on pasture and herd/farm management. Estimated emission intensities per kg carcass and greasy wool varied from 23.5 to 19.9 (Mid region) to 26.8 and 22.8 kg CO2-eq (North region) respectively. The difference between the Mid and North region was mainly due to higher CO2 and N2O emissions from use of fuel and N-fertilizer in the North, where a short growth season, and thus reduced grass yields, are compensated by using greater land area. Based on the distribution of sheep in the regions, and the emission intensities in each region, weighted average emission intensities per kg carcass and greasy wool were estimated as 25.1 and 23.3 kg CO2-eq, respectively. Five alternative methods for the allocation of the co-products were compared with the amounts allocated to greasy wool ranging from 0 % when using no allocation (i.e., all emissions allocated to the sheep carcass), 11 % for economic allocation, 12 % for mass allocation, 19 % for biophysical allocation based on energy requirement up to 31 % for allocation based on protein mass. The emission intensity for wool was highly sensitive to the allocation method used (range from 0 to 61 kg CO2-eq per kg greasy wool), while the effect on sheep carcass was lower (range 19 to 28 kg CO2-eq per kg carcass). For production systems where meat is the main product, as in Norway, allocation should be based on biophysical allocation based on energy requirement.

Association of indoor solid fuel pollution with risk factors for cardiovascular disease among Chinese adults: A nationally multi-center study

Few studies have comprehensively assessed the associations of household air pollution (HAP) from solid fuel burning with cardiovascular risk factors (eg, hypertension, diabetes, dyslipidemia, and obesity). Moreover, whether these associations with cardiovascular disease (CVD) risk factors varied by residential environment characteristics remains unclear. Using two waves data (2011 and 2015) of 13,953 participants aged at least 45 years from 125 cities in the China Health and Retirement Longitudinal Study, we explored how solid fuel exposure is associated with CVD risk factors among Chinese adults. Blood pressure (BP), fasting blood glucose (FBG), lipid profiles, C-reactive protein, and body mass index (BMI) were measured using standard methods. Multivariate logistic regression and generalized linear models explored the associations of solid fuel use for heating and cooking with CVD risk factors. Stratified analyses examined whether the associations varied by sociodemographics and living environmental characteristics. Compared with clean fuels, solid fuel used for heating and cooking was associated with a higher risk of hypertension, diabetes, and dyslipidemia, respectively. Solid fuel was related to elevated systolic BP (adjusted β 1.369 mmHg, 0.481–2.258), diastolic BP (0.704 mmHg, 0.183–1.225), FBG (0.309 mmol/l, 0.218–0.399), total cholesterol (0.055 mmol/l, 0.012–0.099), and low-density lipoprotein (0.100 mmol/l, 0.061–0.139), while inversely with BMI (−0.437 kg/m2, -0.599-–0.275). Women, who live in a house with poor cleanliness (according to the overall tidiness and whether have tap water in the house) had a stronger association for hypertension or dyslipidemia. Our findings provide implications for policymakers to implement interventions to improve cardiovascular health by reducing HAP.

Allosteric activation unveils protein-mass modulation of ATP phosphoribosyltransferase product release

Heavy-isotope substitution into enzymes slows down bond vibrations and may alter transition-state barrier crossing probability if this is coupled to fast protein motions. ATP phosphoribosyltransferase from Acinetobacter baumannii is a multi-protein complex where the regulatory protein HisZ allosterically enhances catalysis by the catalytic protein HisGS. This is accompanied by a shift in rate-limiting step from chemistry to product release. Here we report that isotope-labelling of HisGS has no effect on the nonactivated reaction, which involves negative activation heat capacity, while HisZ-activated HisGS catalytic rate decreases in a strictly mass-dependent fashion across five different HisGS masses, at low temperatures. Surprisingly, the effect is not linked to the chemical step, but to fast motions governing product release in the activated enzyme. Disruption of a specific enzyme-product interaction abolishes the isotope effects. Results highlight how altered protein mass perturbs allosterically modulated thermal motions relevant to the catalytic cycle beyond the chemical step.

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein 1 (FABP1) and FABP1 Binding Alters Drug Metabolism.

Liver fatty acid binding protein 1 (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data are available for human FABP1 (hFABP1). FABP1 has a large binding pocket, and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1 = 0.2 μM) and low (Kd,2 > 10 μM) affinity binding sites. Nine drugs bound to hFABP1 with equilibrium dissociation constant (Kd) values ranging from 1 to 20 μM. None of the tested drugs completely displaced DAUDA from hFABP1, and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The catalytic rate constant of diclofenac hydroxylation by CYP2C9 was decreased by ∼50% (P < 0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs bind fatty acid binding protein 1 (FABP1), forming ternary complexes with FABP1 and the fluorescent fatty acid 11-(dansylamino)undecanoic acid. These findings suggest that drugs will bind to apo-FABP1 and fatty acid-bound FABP1 in the human liver. The high expression of FABP1 in the liver, together with drug binding to FABP1, may alter drug disposition processes in vivo.

Inhibition of Verticillium Wilt in Cotton through the Application of Pseudomonas aeruginosa ZL6 Derived from Fermentation Residue of Kitchen Waste.

To isolate and analyze bacteria with Verticillium wilt-resistant properties from the fermentation residue of kitchen wastes, as well as explore their potential for new applications of the residue. A total of six bacterial strains exhibiting Verticillium wilt-resistant capabilities were isolated from the biogas residue of kitchen waste fermentation. Using a polyphasic approach, strain ZL6, which displayed the highest antagonistic activity against cotton Verticillium wilt, was identified as belonging to the Pseudomonas aeruginosa. Bioassay results demonstrated that this strain possessed robust antagonistic abilities, effectively inhibiting V. dahliae spore germination and mycelial growth. Furthermore, P. aeruginosa ZL6 exhibited high temperature resistance (42°C), nitrogen fixation, and phosphorus removal activities. Pot experiments revealed that P. aeruginosa ZL6 fermentation broth treatment achieved a 47.72% biological control effect compared to the control group. Through activity tracking and protein mass spectrometry identification, a neutral metalloproteinase (Nml) was hypothesized as the main virulence factor. The mutant strain ZL6ΔNml exhibited a significant reduction in its ability to inhibit cotton Verticillium wilt compared to the strain P. aeruginosa ZL6. While the inhibitory activities could be partially restored by a complementation of nml gene in the mutant strain ZL6CMΔNml. This research provides a theoretical foundation for the future development and application of biogas residue as biocontrol agents against Verticillium wilt and as biological preservatives for agricultural products. Additionally, this study presents a novel approach for mitigating the substantial amount of biogas residue generated from kitchen waste fermentation.

The GFPT2-O-GlcNAcylation-YBX1 axis promotes IL-18 secretion to regulate the tumor immune microenvironment in pancreatic cancer

The immunosuppressive microenvironment caused by several intrinsic and extrinsic mechanism has brought great challenges to the immunotherapy of pancreatic cancer. We identified GFPT2, the key enzyme in hexosamine biosynthesis pathway (HBP), as an immune-related prognostic gene in pancreatic cancer using transcriptome sequencing and further confirmed that GFPT2 promoted macrophage M2 polarization and malignant phenotype of pancreatic cancer. HBP is a glucose metabolism pathway leading to the generation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which is further utilized for protein O-GlcNAcylation. We confirmed GFPT2-mediated O-GlcNAcylation played an important role in regulating immune microenvironment. Through cellular proteomics, we identified IL-18 as a key downstream of GFPT2 in regulating the immune microenvironment. Through CO-IP and protein mass spectrum, we confirmed that YBX1 was O-GlcNAcylated and nuclear translocated by GFPT2-mediated O-GlcNAcylation. Then, YBX1 functioned as a transcription factor to promote IL-18 transcription. Our study elucidated the relationship between the metabolic pathway of HBP in cancer cells and the immune microenvironment, which might provide some insights into the combination therapy of HBP vulnerability and immunotherapy in pancreatic cancer.

Bcl-xL regulates radiation-induced ferroptosis through chaperone-mediated autophagy of GPX4 in tumor cells

ObjectiveTo investigate the role and the molecular mechanisms of apoptotic signaling in ferroptosis to regulate tumor radiosensitivity. MethodsReactive oxygen species (ROS) and lipid peroxide levels were detected in Mouse embryonic fibroblasts(MEFs) with Bcl-xL or Mcl-1 deficiency induced by erastin. Colony formation, ROS, lipid peroxidation and the transcription/translation levels of PTGS2 were measured in Bcl-xL knockdown tumor cells induced by 5 ​Gy γ-rays or co-treated with ferrostatin-1 (Ferr-1). The protein levels of LPCAT3, ACSL4 and PEBP1 in Bcl-xL knockout MEF cells were evaluated in Bcl-xL knockout MEF cells post-radiation. Moreover, the interaction of heat shock protein 90 (HSP90) with Bcl-xL, GPX4, or LAMP2A was detected by protein mass spectrometry and immunoprecipitation assays. ResultsManipulating Bcl-xL levels facilitated radiation-induced ferroptosis by augmenting the enzymatic oxidation of polyunsaturated fatty acids (PUFAs) and enhancing chaperone-mediated autophagy (CMA) of glutathione peroxidase 4 (GPX4) (MEF cell line: t=4.540, P＜0.01; A549 ​cell line: t=56.16, P＜0.0001; t=4.885, P＜0.01; HCT116 ​cell line: t=14.75, P＜0.01; t=7.363, P＜0.05). Downregulating Bcl-xL expression promoted the activity of acyl-CoA synthetase long-chain family member 4 (ACSL4), thus increasing the enzymatic oxidation of PUFAs (t=4.258, P＜0.01). Moreover, depletion of Bcl-xL expedited the CMA process targeting GPX4 by facilitating the association of GPX4 with heat shock protein 90 (HSP90) and LAMP2A following radiation exposure. Subsequent degradation of GPX4 led to the accumulation of lipid peroxides, ultimately triggering ferroptosis. ConclusionsOur study provides initial insights into the regulatory role of Bcl-xL in ferroptosis and underscores the potential of targeting Bcl-xL as a promising therapeutic strategy for cancer by modulating both apoptotic and ferroptotic pathways.

208 Preliminary Results of Novel Method for Identifying Ischemic Stroke Vesicular Protein Signature in Cardiac Surgery Patients

INTRODUCTION: Acute ischemic stroke (AIS) treatment depends on timely diagnosis. Identifying an efficient, blood-based protein signature for AIS has been limited by the lack of a pre-ischemia control group. Here we report the preliminary results of a model for AIS protein signature discovery using cardiac surgery patients and novel mass spectrometry techniques. METHODS: Twenty patients undergoing aortic arch repair with deep hypothermic circulatory arrest (n = 17) or coronary artery bypass graft (n = 3) were enrolled. Blood samples were obtained immediately pre- and postoperatively. Magnetic resonance imaging (MRI) of the brain was obtained within 72 hours of surgery. Presence of infarct and total ischemic volume was determined. A novel protein enrichment and mass spectrometry analysis technique (MAG-Net) was used to characterize the pre- and post-surgical proteomes present in extracellular vesicles. Analysis of variance (ANOVA) (false discovery rate &lt; 0.05) was used to analyze protein signatures associated with infarction. RESULTS: Post-surgical infarct occurred in 15 patients (75%), with a median volume of 108mm3. A total of 5,376 proteins were identified, 1,125 of which showed a significant difference between paired pre- and post-operative concentrations. Of these 1,125 proteins, 524 proteins were unique to patients without infarction, 340 proteins had a significant change in both populations, and 261 proteins were unique to patients with infarcts. There were no significant proteins for the interaction term comparing pre- and post-operative differences between the infarct/no infarct groups. CONCLUSIONS: This clinical model for AIS protein signature development relies on the high rate of ischemia during cardiac surgery to allow for pre-stroke sample collection. The MAG-Net technique enables efficient processing of biologically relevant extracellular vesicle proteins and analysis of an unbiased protein signature. The preliminary results demonstrate the feasibility of combining these innovations for AIS protein signature discovery.

QUALITY OF MARE'S MILK AND KOUMISS

Nowadays mare's milk and koumiss are in great demand in Kazakhstan. Therefore, the study of its qualitative indicators is relevant.In the article the physical and chemical parameters and vitamin content (B and C groups) of mare's milk and koumiss samples taken from peasant farms of Almaty region in the fall-winter period are studied in a comparative form.The study showed that mare's milk samples had no significant difference in total protein mass fraction in the fall and winter. In mass fraction terms of fat in the sample obtained in the fall the fat content was 2.18% higher than in the sample obtained in winter. And the mass fraction of lactose in the sample obtained in the fall was 0.14% higher than in the sample obtained in winter.According to the study results of obtained koumiss samples’ physical and chemical parameters in the fall and winter periods, the mass fraction of protein was 1.86-1.89%. Fat content of koumiss proportion prepared in the fall period was 1.84%, in the winter period-2.19%, and the average mass fraction of lactose was from 3.64 to 3.98%.The content of water-soluble vitamins B and C in samples of mare's milk and koumiss was investigated. According to the study results the content of vitamins B1, B2, B3, B5 B6 and C in the composition of mare's milk and koumiss obtained in the fall and winter periods was established.According to the general research results samples of mare's milk and koumiss products obtained from peasant farms of Almaty region have proven high quality, nutritious in food value and easily assimilated by human body.

Potential strategic allocation of nuptial gift proteins of the neotropical katydid Conocephalus ictus (Orthoptera Tettigoniidae)

In many katydids, the male feeds his mate with a large gelatinous spermatophore. While providing large spermatophores can increase female fecundity and lifespan, it may also decrease their sexual receptivity, benefiting male fitness. Allocating resources to these edible gifts may entail a lower apportionment of them to other functions, generating a trade-off between somatic and reproductive functions. Despite their effect on male and female fitness, little is known of the compounds associated with katydid spermatophores. Our study found 177 different putative proteins in the spermatophore of Conocephalus ictus, with no correlation between male body size with spermatophore mass, number, concentration and mass of proteins. However, we did observe a negative relationship between male forewing length and protein concentration, and a negative relationship between the mass of the spermatophore transferred to the females and their body size, suggesting a resource allocation trade-off in males, but also strategic transference of resources based on female quality.

Preparation of a superior soy protein adhesive with high solid content by enzymatic hydrolysis combined with cross-linking modification

The production of traditional wood adhesives relies on petrochemical resources and puts enormous pressure on the ecosystem. Therefore, it is important to use bio-based soy protein adhesives to replace traditional adhesives. However, soy protein adhesives have the problems of low solid content, high viscosity and poor water resistance. In this study, we combined enzymatic hydrolysis with cross-linking modification to reduce the molecular mass of soy protein and increase the solid content of adhesive, constructed a uniform and tight covalent bond cross-linking network in the adhesive. Through this modification strategy, we prepared a soy protein adhesive with high solid content, suitable viscosity and excellent bonding strength. The results showed that compared with 0-SPI adhesive, the viscosity of 4-SPI-18/PA/TA adhesive decreased by 61.6%, reached 21899 mPa·s. Meanwhile, the residual rate of the adhesive increased by 18% (59%), the moisture absorption rate reduced by 7.8% to reach 3.8%. The dry/wet shear strength of modified adhesive increased by 56.1% (1.28 MPa) and 44.3% (0.88 MPa), respectively, and the tensile strain at break increased by 58.1% (14.84%). This is because enzymolysis improved its coating performance, and formed mechanical interlocking with wood. In addition, the amount of SPI increased, and more protein molecules participated in the cross-linking reaction, forming a more uniform and compact cross-linking network structure in the adhesive, effectively improving the bonding strength and toughness. This study provides a novel approach for the preparation of cleaning and tenacious adhesives, aiming to explore a green development model.

Using High-Throughput Experiments To Screen N-Glycosyltransferases with Altered Specificities.

The important roles that protein glycosylation plays in modulating the activities and efficacies of protein therapeutics have motivated the development of synthetic glycosylation systems in living bacteria and in vitro. A key challenge is the lack of glycosyltransferases that can efficiently and site-specifically glycosylate desired target proteins without the need to alter primary amino acid sequences at the acceptor site. Here, we report an efficient and systematic method to screen a library of glycosyltransferases capable of modifying comprehensive sets of acceptor peptide sequences in parallel. This approach is enabled by cell-free protein synthesis and mass spectrometry of self-assembled monolayers and is used to engineer a recently discovered prokaryotic N-glycosyltransferase (NGT). We screened 26 pools of site-saturated NGT libraries to identify relevant residues that determine polypeptide specificity and then characterized 122 NGT mutants, using 1052 unique peptides and 52,894 unique reaction conditions. We define a panel of 14 NGTs that can modify 93% of all sequences within the canonical X-1-N-X+1-S/T eukaryotic glycosylation sequences as well as another panel for many noncanonical sequences (with 10 of 17 non-S/T amino acids at the X+2 position). We then successfully applied our panel of NGTs to increase the efficiency of glycosylation for three protein therapeutics. Our work promises to significantly expand the substrates amenable to in vitro and bacterial glycoengineering.

Mechanism of oxalate decarboxylase Oxd_S12 from Bacillus velezensis BvZ45-1 in defence against cotton verticillium wilt.

Verticillium wilt, a soilborne vascular disease caused by Verticillium dahliae, strongly affects cotton yield and quality. In this study, an isolated rhizosphere bacterium, designated Bacillus velezensis BvZ45-1, exhibited >46% biocontrol efficacy against cotton verticillium wilt under greenhouse and field conditions. Moreover, through crude protein extraction and mass spectrometry analyses, we found many antifungal compounds present in the crude protein extract of BvZ45-1. The purified oxalate decarboxylase Odx_S12 from BvZ45-1 inhibited the growth of V. dahliae Vd080 by reducing the spore yield, causing mycelia to rupture, spore morphology changes, cell membrane rupture, and cell death. Subsequently, overexpression of Odx_S12 in Arabidopsis significantly improved plant resistance to V. dahliae. Through studies of the resistance mechanism of Odx_S12, V. dahliae was shown to produce oxalic acid (OA), which has a toxic effect on Arabidopsis leaves. Odx_S12 overexpression reduced Arabidopsis OA content, enhanced tolerance to OA, and improved resistance to verticillium wilt. Transcriptomics and quantitative real-time PCR analysis revealed that Odx_S12 promoted a reactive oxygen species burst and a salicylic acid- and abscisic acid-mediated defence response in Arabidopsis. In summary, this study not only identified B. velezensis BvZ45-1 as an efficient biological control agent, but also identified the resistance gene Odx_S12 as a candidate for cotton breeding against verticillium wilt.

Anti-cancer drug-mediated increase in mitochondrial mass limits the application of metabolic viability-based MTT assay in cytotoxicity screening.

The high-throughput metabolic viability-based colorimetric MTT test is commonly employed to screen the cytotoxicity of different chemotherapeutic drugs. The assay assumes a cell density-dependent linear correlation with the MTT spectral absorbance. Therefore, the present study aimed to compare the cytotoxicity assessment between the MTT assay and gold standard cell number enumeration. The cytotoxicity was induced by Cisplatin, Etoposide, and Doxorubicin in human lung epithelial adenocarcinoma cells (A549) and cervix carcinoma (HeLa) cell lines. The mitochondrial mass was estimated, and immunoblotting of succinate dehydrogenase (SDH-A) was performed following drug treatment in both cell lines. Student's t-test paired analysis was employed to calculate the significance of the results, where the value p < 0.05 was considered statistically significant. The drug-induced cytotoxic response estimated by MTT absorbance did not show any significant difference with respect to control, and no correlation was observed with the enumerated cell number in both A549 and HeLa cells. Interestingly, per-cell metabolic viability was found to be increased by 1.18 to 3.26-fold (p < 0.05) following drug treatment. Further, mechanistic investigation revealed a drug concentration-dependent significant increase in mitochondrial mass (1.21 to 4.2-fold) and upregulation of SDH protein (50-70%) as well as enzymatic activity with respect to control in both A549 and Hela cells. The limitation of the MTT assay for drug-induced cytotoxicity assessment is due to increased mitochondrial mass and SDH upregulation in surviving cells, leading to enhanced formazan formation. This leads to a lack of correlation between cell number and MTT spectral absorbance, suggesting that the MTT assay may provide an erroneous conclusion for cytotoxicity assessment.

Solid-Phase Microextraction-Aided Capillary Zone Electrophoresis-Mass Spectrometry: Toward Bottom-Up Proteomics of Single Human Cells.

Capillary zone electrophoresis-mass spectrometry (CZE-MS) has been recognized as a valuable technique for the proteomics of mass-limited biological samples (i.e., single cells). However, its broad adoption for single cell proteomics (SCP) of human cells has been impeded by the low sample loading capacity of CZE, only allowing us to use less than 5% of the available peptide material for each measurement. Here we present a reversed-phase-based solid-phase microextraction (RP-SPME)-CZE-MS platform to solve the issue, paving the way for SCP of human cells using CZE-MS. The RP-SPME-CZE system was constructed in one fused silica capillary with zero dead volume for connection via in situ synthesis of a frit, followed by packing C8 beads into the capillary to form a roughly 2 mm long SPME section. Peptides captured by SPME were eluted with a buffer containing 30% (v/v) acetonitrile and 50 mM ammonium acetate (pH 6.5), followed by dynamic pH junction-based CZE-MS. The SPME-CZE-MS enabled the injection of nearly 40% of the available peptide sample for each measurement. The system identified 257 ± 24 proteins and 523 ± 69 peptides (N = 2) using a Q-Exactive HF mass spectrometer when only 0.25 ng of a commercial HeLa cell digest was available in the sample vial and 0.1 ng of the sample was injected. The amount of available peptide is equivalent to the protein mass of one HeLa cell. The data indicate that SPME-CZE-MS is ready for SCP of human cells.

Paradoxical imbalance between activated lymphocyte protein synthesis capacity and rapid division rate.

Rapid lymphocyte cell division places enormous demands on the protein synthesis machinery. Flow cytometric measurement of puromycylated ribosome-associated nascent chains after treating cells or mice with translation initiation inhibitors reveals that ribosomes in resting lymphocytes in vitro and in vivo elongate at typical rates for mammalian cells. Intriguingly, elongation rates can be increased up to 30% by activation in vivo or fever temperature in vitro. Resting and activated lymphocytes possess abundant monosome populations, most of which actively translate in vivo, while in vitro, nearly all can be stalled prior to activation. Quantitating lymphocyte protein mass and ribosome count reveals a paradoxically high ratio of cellular protein to ribosomes insufficient to support their rapid in vivo division, suggesting that the activated lymphocyte proteome in vivo may be generated in an unusual manner. Our findings demonstrate the importance of a global understanding of protein synthesis in lymphocytes and other rapidly dividing immune cells.

Paradoxical imbalance between activated lymphocyte protein synthesis capacity and rapid division rate

Rapid lymphocyte cell division places enormous demands on the protein synthesis machinery. Flow cytometric measurement of puromycylated ribosome-associated nascent chains after treating cells or mice with translation initiation inhibitors reveals that ribosomes in resting lymphocytes in vitro and in vivo elongate at typical rates for mammalian cells. Intriguingly, elongation rates can be increased up to 30% by activation in vivo or fever temperature in vitro. Resting and activated lymphocytes possess abundant monosome populations, most of which actively translate in vivo, while in vitro, nearly all can be stalled prior to activation. Quantitating lymphocyte protein mass and ribosome count reveals a paradoxically high ratio of cellular protein to ribosomes insufficient to support their rapid in vivo division, suggesting that the activated lymphocyte proteome in vivo may be generated in an unusual manner. Our findings demonstrate the importance of a global understanding of protein synthesis in lymphocytes and other rapidly dividing immune cells.

WNT2B activates macrophages via NF-κB signaling pathway in inflammatory bowel disease.

Inflammation is a significant pathological manifestation of inflammatory bowel disease (IBD), yet its mechanism has remained unclear. Although WNT2B is enriched in the intestinal inflammatory tissue of IBD patients, the specific mechanism of WNT2B in the formation of intestinal inflammation remains unclear. This study was aimed to investigate whether macrophages expressing WNT2B can aggravate intestinal tissue inflammation. Samples were collected from both normal individuals and patients with IBD at multiple colon sites. Macrophages were identified using tissue immunofluorescence. IκB kinase (IKK)-interacting protein (IKIP), which interacts with WNT2B, was found by protein cross-linking and protein mass spectrometry. The expression of WNT2B, IKIP, the NF-κB pathway, and downstream molecules were analyzed. An acute colitis model of C57BL/6J mice was established using an adeno-associated virus (AAV)-mediated WNT2B knockdown system and 3% dextran sulfate sodium (DSS). The degree of intestinal inflammation in mice was assessed upon WNT2B knockdown in macrophages. Macrophages expressing WNT2B were found to be enriched in the colitis tissues of IBD patients. WNT2B in macrophages activated the NF-κB pathway and enhanced the expression of downstream inflammatory cytokines. By competitively binding IKIP, WNT2B reduced the binding of IKIP to IKKβ and promoted the activation of the NF-κB pathway. Using an AAV-mediated WNT2B knockdown system, WNT2B expression in intestinal macrophages was suppressed, leading to a reduction in intestinal inflammation. WNT2B activated the NF-κB pathway and enhanced the expression of downstream inflammatory cytokines by competitively binding to IKIP, potentially contributing to colon inflammatory injury in IBD.