Relative Protein Research Articles

Tissue-based absolute quantification using large-scale TMT and LFQ experiments.

Relative and absolute intensity-based protein quantification across cell lines, tissue atlases and tumour datasets is increasingly available in public datasets. These atlases enable researchers to explore fundamental biological questions, such as protein existence, expression location, quantity and correlation with RNA expression. Most studies provide MS1 feature-based label-free quantitative (LFQ) datasets; however, growing numbers of isobaric tandem mass tags (TMT) datasets remain unexplored. Here, we compare traditional intensity-based absolute quantification (iBAQ) proteome abundance ranking to an analogous method using reporter ion proteome abundance ranking with data from an experiment where LFQ and TMT were measured on the same samples. This new TMT method substitutes reporter ion intensities for MS1 feature intensities in the iBAQ framework. Additionally, we compared LFQ-iBAQ values to TMT-iBAQ values from two independent large-scale tissue atlas datasets (one LFQ and one TMT) using robust bottom-up proteomic identification, normalisation and quantitation workflows.

Combination of Zearalenone and Deoxynivalenol Induces Apoptosis by Mitochondrial Pathway in Piglet Sertoli Cells: Role of Endoplasmic Reticulum Stress.

Zearalenone (ZEA) and deoxynivalenol (DON) are widely found in various feeds, which harms livestock's reproductive health. Both mitochondria and endoplasmic reticulum (ER) can regulate cell apoptosis. This study aimed to explore the regulatory mechanism of endoplasmic reticulum stress (ERS) on ZEA- combined with DON-induced mitochondrial pathway apoptosis in piglet Sertoli cells (SCs). The results showed that ZEA + DON damaged the ultrastructure of the cells, induced apoptosis, decreased mitochondrial membrane potential, promoted the expression of cytochrome c (CytC), and decreased the cell survival rate. Furthermore, ZEA + DON increased the relative mRNA and protein expression of Bid, Caspase-3, Drp1, and P53, while that of Bcl-2 and Mfn2 declined. ZEA + DON was added after pretreatment with 4-phenylbutyric acid (4-PBA). The results showed that 4-PBA could alleviate the toxicity of ZEA + DON toward SCs. Compared with the ZEA + DON group, 4-PBA improved the cell survival rate, decreased the apoptosis rate, inhibited CytC expression, and increased mitochondrial membrane potential, and the damage to the cell ultrastructure was alleviated. Moreover, after pretreatment with 4-PBA, the relative mRNA and protein expression of Bid, Caspase-3, Drp1, and P53 were downregulated, while the relative mRNA and protein expression of Bcl-2 and Mfn2 were upregulated. It can be concluded that ERS plays an important part in the apoptosis of SCs co-infected with ZEA-DON through the mitochondrial apoptosis pathway, and intervention in this process can provide a new way to alleviate the reproductive toxicity of mycotoxins.

Protein Corona of Anionic Fluid-Phase Liposomes Compromises Their Integrity Rather than Uptake by Cells.

Despite the undisputable role of the protein corona in the biointeractions of liposome drug carriers, the field suffers from a lack of knowledge regarding the patterns of protein deposition on lipid surfaces with different compositions. Here, we investigated the protein coronas formed on liposomes of basic compositions containing combinations of egg phosphatidylcholine (PC), palmitoyloleoyl phosphatidylglycerol (POPG), and cholesterol. Liposome-protein complexes isolated by size-exclusion chromatography were delipidated and analyzed using label-free LC-MS/MS. The addition of the anionic lipid and cholesterol both affected the relative protein abundances (and not the total bound proteins) in the coronas. Highly anionic liposomes, namely those containing 40% POPG, carried corona enriched with cationic proteins (apolipoprotein C1, beta-2-glycoprotein 1, and cathelicidins) and were the least stable in the calcein release assay. Cholesterol improved the liposome stability in the plasma. However, the differences in the corona compositions had little effect on the liposome uptake by endothelial (EA.hy926) and phagocytic cells in the culture (U937) or ex vivo (blood-derived monocytes and neutrophils). The findings emphasize that the effect of protein corona on the performance of the liposomes as drug carriers occurs through compromising particle stability rather than interfering with cellular uptake.

Statistical Detection of Differentially Abundant Proteins in Experiments with Repeated Measures Designs and Isobaric Labeling.

Repeated measures experimental designs, which quantify proteins in biological subjects repeatedly over multiple experimental conditions or times, are commonly used in mass spectrometry-based proteomics. Such designs distinguish the biological variation within and between the subjects and increase the statistical power of detecting within-subject changes in protein abundance. Meanwhile, proteomics experiments increasingly incorporate tandem mass tag (TMT) labeling, a multiplexing strategy that gains both relative protein quantification accuracy and sample throughput. However, combining repeated measures and TMT multiplexing in a large-scale investigation presents statistical challenges due to unique interplays of between-mixture, within-mixture, between-subject, and within-subject variation. This manuscript proposes a family of linear mixed-effects models for differential analysis of proteomics experiments with repeated measures and TMT multiplexing. These models decompose the variation in the data into the contributions from its sources as appropriate for the specifics of each experiment, enable statistical inference of differential protein abundance, and recognize a difference in the uncertainty of between-subject versus within-subject comparisons. The proposed family of models is implemented in the R/Bioconductor package MSstatsTMT v2.2.0. Evaluations of four simulated datasets and four investigations answering diverse biological questions demonstrated the value of this approach as compared to the existing general-purpose approaches and implementations.

Body Weight Support Treadmill Training Combined With Sciatic Nerve Electrical Stimulation Ameliorating Motor Function by Enhancing PI3K/Akt Proteins Expression via BDNF/TrkB Signaling Pathway in Rats with Spinal Cord Injury

To investigate the effects of body weight support treadmill training (BWSTT) and sciatic nerve electrical stimulation (SNES) on motor function recovery in spinal cord injury (SCI) rats and its possible mechanism. Modified Allen's method was utilized for T10 incomplete SCI. The Basso-Beattie-Bresnahan (BBB) score and modified Tarlov score were applied to assess motor function. Pathologic alterations of the spinal cord and muscles were observed by hematoxylin and eosin (HE) staining. The positive staining region of collagen fibers was assessed with Masson staining. Immunofluorescence was applied to count the positive cells of brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB). BDNF, TrkB, phosphatidylinositol-3-kinase (PI3K), and protein kinase B (Akt) relative mRNA and protein expressions were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. On the 21st day of the intervention, the motor scores in SNES and BWSTT+ SNES groups were higher than that in SCI group (P < 0.05). Compared with SCI group, mRNA and protein expressions of BDNF/TrkB and PI3K/Akt were more significant on the 21st day of the intervention in SNES and BWSTT+ SNES groups (P < 0.05), but there was no difference in BWSTT group (P > 0.05). This experiment demonstrated that BWSTT combined with SNES contributed to alleviating spinal cord tissue injury, delaying muscle atrophy and improving locomotion. One of the possible mechanisms may be related to the regulation of the BDNF/TrkB signaling pathway, which changes the expression of PI3K/Akt protein. Furthermore, it was discovered that the ultra-early BWSTT may not be conducive to recovery.

Fenbufen Alleviates Severe Acute Pancreatitis by Suppressing Caspase-1/Caspase-11-mediated Pyroptosis in Mice.

In the present study, we aimed to investigate the effects of Fenbufen treatment on the SAP model induced by caerulein and lipopolysaccharide. Severe acute pancreatitis (SAP) is an extremely dangerous disease with high mortality, which is associated with inflammatory response and acinar cell death. The caspase family plays an important role in cell death, such as caspase-1 and caspase-11 in pyroptosis. In recent years, caspases have been shown to be a novel pharmacological target of Fenbufen. Effects of Fenbufen on pancreatic tissue damage and serum levels of lipase and amylase in SAP in mice; Effect of Fenbufen on caspase-1 pathway in SAP in mice; Effect of Fenbufen on caspase-1/caspase-11-mediated pyroptosis of PACs in SAP in mice; Effect of Fenbufen on isolated PACs and caspase-1/caspase-11-mediated pyroptosis in vitro. In vivo, eighteen female C57BL/6 mice were randomly divided into 3 groups: the NC group, the SAP group, and the Fenbufen +SAP group with 6 mice in each group. The SAP model was induced by intraperitoneal injection of caerulein and lipopolysaccharide. The pathological changes in pancreatic and the serum levels of lipase and amylase and the relative gene and protein expressions in each group were compared. In vitro, pancreatic acinar cells were assigned to 5 groups: medium group, SAP group, Fenbufen 100μM group, Fenbufen 200μM group, and Fenbufen 400μM group. The cell damage and the relative gene and protein expressions in each group were evaluated. Our results showed that Fenbufen ameliorated the severity of SAP and decreased the serum levels of lipase and amylase. Meanwhile, the in vivo and in vitro data demonstrated that Fenbufen inhibited the activation of caspase-1 and caspase-11, decreasing the levels of IL-1β, IL-18, and GSDMD. In in vitro experiments, we found that by inhibiting the activation of caspase-1 and caspase-11, Fenbufen significantly reduced lactate dehydrogenase (LDH) excretion by acinar cells. In general, our data showed that Fenbufen could protect the pancreatic acinar cell from injury by inhibiting pyroptosis.

Acetylation at the O-6 position of t-Glc improved immunoactivity of α-1,6-glucan from longan by additionally activating Dectin-1 and CD14 receptors

Acetylation is an important approach to improve the bioactivity of polysaccharides; however, the mechanisms have not been fully understood. As a key component of longan for exerting health promoting function, longan polysaccharide was hypothesized may achieve elevated immunoregulatory activity after acetylation. A bioactive longan polysaccharide (LP) composed of (1 → 6)-α-d-glucan (84.1 %) and with an average Mw of 9.68 × 104 kDa was acetylated to different degree of substitutions (DS) in this study. Key structural changes responsible for improvement in immunoregulatory activity were identified, and underlying mechanisms were investigated. Acetylated LP (Ac-LP) with DS 0.37, 0.78 and 0.92 were obtained. Structural characterization identified the substitution of acetyl groups occurs at O-6 positions of t-Glc non-selectively, while the backbone structure was not apparently changed. This resulted in increased expression of cytokines (IL-10, IL-6 and TNF-α) and ROS production in RAW264.7 macrophages, indicating improved immune activity which is positively related to the DS of Ac-LP. This is attribute to additional cellular receptors for Ac-LP (CD14 and Dectin-1) apart from receptors for LP (TLR4 and Ca2+ receptors), as well as the relative higher protein expression of TLR4-MyD88 signaling pathways. These results would provide guidance for the utilization of acetylated polysaccharides with improved immunoactivity.

New proapoptotic chemotherapeutic agents based on the quinolone-3-carboxamide scaffold acting by VEGFR-2 inhibition

In the current study, we designed and synthesized a series of new quinoline derivatives 10a-p as antiproliferative agents targeting cancer through inhibition of VEGFR-2. Preliminary molecular docking to assess the interactions of the designed derivatives with the binding site of VEGFR-2 (PDB code: 4ASD) displayed binding poses and interactions comparable to sorafenib. The synthesized compounds exhibited VEGFR-2 inhibitory activity with IC50 ranging from 36 nM to 2.23 μM compared to sorafenib (IC50 = 45 nM), where derivative 10i was the most potent. Additionally, the synthesized derivatives were evaluated in vitro for their cytotoxic activity against HepG2 cancer cell line. Seven compounds 10a, 10c, 10d, 10e, 10i, 10n and 10o (IC50 = 4.60, 4.14, 1.07, 0.88, 1.60, 2.88 and 2.76 μM respectively) displayed better antiproliferative activity than sorafenib (IC50 = 8.38 μM). Compound 10i was tested against Transformed Human Liver Epithelial-2 normal cell line (THLE-2) to evaluate its selective cytotoxicity. Furthermore, 10i, as a potent representative of the series, was assayed for its apoptotic activity and cell cycle kinetics’ influence on HepG2, its effects on the gene expression of VEGFR-2, and protein expression of the apoptotic markers Caspase-7 and Bax. Compound 10i proved to have a potential role in apoptosis by causing significant increase in the early and late apoptotic quartiles, a remarkable activity in elevating the relative protein expression of Bax and Caspase-7 and a significant reduction of VEGFR-2 gene expression. Collectively, the obtained results indicate that compound 10i has a promising potential as a lead compound for the development of new anticancer agents.

Coal fly ash application as an eco-friendly approach for modulating the growth, yield, and biochemical constituents of Withania somnifera L. plants.

The solid waste known as fly ash, which is produced when coal is burned in thermal power plants, is sustainably used in agriculture. It is an excellent soil supplement for plant growth and development since it contains some desired nutrients (macro and micro), as well as being porous. The present study was done to evaluate the effect of different fly ash levels on Withania somnifera. The present study aimed to assess the impact of various fly ash (FA) concentrations on growth, yield, photosynthetic pigments, biochemical parameters, and cell viability of W. somnifera. The results showed that FA enhanced physical and chemical properties of soil like pH, electric conductivity, porosity, water-holding capacity, and nutrients. The low doses of FA-amended soil (15%) significantly increased the shoot length (36%), root length (24.5%), fresh weight of shoots and roots (107.8 and 50.6%), dry weight of shoots and roots (61.9 and 47.1%), number of fruits (70.4%), carotenoid (43%), total chlorophyll (44.3%), relative water content (109.3%), protein content (20.4%), proline content (110.3%), total phenols (116.1%), nitrogen (20.3%), phosphorus (16.9%), and potassium (26.4%). On the other hand, the higher doses, i.e., 25% of fly ash showed a negative effect on all the above parameters and induced oxidative stress by increasing lipid peroxidation (33.1%) and hydrogen peroxide (102.0%) and improving the activities of antioxidant enzymes and osmolytes. Compared to the control plants, the plants growing in soil enriched with 15 and 25% fly ash had larger stomata pores when examined using a scanning electron microscope. In addition, according to a confocal microscopic analysis of the roots of W. somnifera, higher fly ash concentrations caused membrane damage, as evidenced by an increase in the number of stained nuclei. Moreover, several functional groups and peaks of the biomolecules represented in the control and 15% of fly ash were alcohols, phenols, allenes, ketenes, isocynates, and hydrocarbons. Gas chromatography-mass spectrometryanalysis of the methanol extract of W. somnifera leaves cultivated in soil amended with 15% fly ash shows the presence of 47 bioactive compounds. The most abundant compounds in the methanol extract were cis-9-hexadecenal (22.33%), n-hexadecanoic acid (9.68%), cinnamic acid (6.37%), glycidyl oleate (3.88%), nonanoic acid (3.48%), and pyranone (3.57%). The lower concentrations of FA (15%) can be used to enhance plant growth and lower the accumulation of FA that results in environmental pollution.

Ethanol-induced AMPA alterations are mediated by mGLU5 receptors through miRNA upregulation in hippocampal slices

Prenatal alcohol exposure (PAE) affects neuronal networks and brain development causing a range of physical, cognitive and behavioural disorders in newborns that persist into adulthood. The array of consequences associated with PAE can be grouped under the umbrella-term ‘fetal alcohol spectrum disorders’ (FASD). Unfortunately, there is no cure for FASD as the molecular mechanisms underlying this pathology are still unknown. We have recently demonstrated that chronic EtOH exposure, followed by withdrawal, induces a significant decrease in AMPA receptor (AMPAR) expression and function in developing hippocampus in vitro. Here, we explored the EtOH-dependent pathways leading to hippocampal AMPAR suppression. Organotypic hippocampal slices (2 days in cultures) were exposed to EtOH (150 mM) for 7 days followed by 24 h EtOH withdrawal. Then, the slices were analysed by means of RT-PCR for miRNA content, western blotting for AMPA and NMDA related-synaptic proteins expression in postsynaptic compartment and electrophysiology to record electrical properties from CA1 pyramidal neurons. We observed that EtOH induces a significant downregulation of postsynaptic AMPA and NMDA subunits and relative scaffolding protein expression and, accordingly, a decrease of AMPA-mediated neurotransmission. Simultaneously, we found that chronic EtOH induced-upregulation of miRNA 137 and 501-3p and decreased AMPA-mediated neurotransmission are prevented by application of the selective mGlu5 antagonist MPEP during EtOH withdrawal. Our data indicate mGlu5 via miRNA137 and 501-3p expression as key factors in the regulation of AMPAergic neurotransmission that may contribute, at least in part, to the pathogenesis of FASD.

Metalloenzymes play major roles to achieve high-rate nitrogen removal in N-damo communities: Lessons from metaproteomics

Nitrite-driven anaerobic methane oxidation (N-damo) is a promising biological process to achieve carbon–neutral wastewater treatment solutions, aligned with the sustainable development goals. Here, the enzymatic activities in a membrane bioreactor highly enriched in N-damo bacteria operated at high nitrogen removal rates were investigated. Metaproteomic analyses, with a special focus on metalloenzymes, revealed the complete enzymatic route of N-damo including their unique nitric oxide dismutases. The relative protein abundance evidenced that “Ca. Methylomirabilis lanthanidiphila” was the predominant N-damo species, attributed to the induction of its lanthanide-binding methanol dehydrogenase in the presence of cerium. Metaproteomics also disclosed the activity of the accompanying taxa in denitrification, methylotrophy and methanotrophy. The most abundant functional metalloenzymes from this community require copper, iron, and cerium as cofactors which was correlated with the metal consumptions in the bioreactor. This study highlights the usefulness of metaproteomics for evaluating the enzymatic activities in engineering systems to optimize microbial management.

Capsaicin improves hypertension and cardiac hypertrophy via SIRT1/NF-κB/MAPKs pathway in the hypothalamic paraventricular nucleus

BackgroundHypertension has seriously affected a large part of the adult and elderly population. The complications caused by hypertension are important risk factors for cardiovascular disease accidents. Capsaicin, a pungent component of chili pepper has been revealed to improve hypertension. However, its potential mechanism in improving hypertension remains to be explored. PurposeIn the present study, we aimed to investigate whether capsaicin could attenuate the SIRT1/NF-κB/MAPKs pathway in the paraventricular nucleus of hypothalamus (PVN). MethodsWe used spontaneous hypertensive rats (SHRs) as animal model rats. Micro osmotic pump was used to give capsaicin through PVN for 28 days, starting from age12-week-old. ResultsThe results showed that capsaicin significantly reduced blood pressure from the 16th day of infusion onward. At the end of the experimental period, we measured cardiac hypertrophy index and the heart rate (HR), and the results showed that the cardiac hypertrophy and heart rate of rats was significantly improved upon capsaicin chronic infusion. Norepinephrine (NE) and epinephrine (EPI) in plasma of SHRs treated with capsaicin were also decreased. Additionally, capsaicin increased the protein expression and number of positive cells of SIRT1 and the 67-kDa isoform of glutamate decarboxylase (GAD67), decreased the production of reactive oxygen species (ROS), number of positive cells of NOX2, those of Angiotensin Converting Enzyme (ACE) and p-IKKβ, tyrosine hydroxylase (TH), the gene expression levels of NOX4 and pro-inflammatory cytokines. Capsaicin also decreased the relative protein expressions of protein in MAPKs pathway. ConclusionCurrent data indicated that capsaicin within the PVN improves hypertension and cardiac hypertrophy via SIRT1/NF-κB/MAPKs pathway in the PVN of SHRs, supporting its potential as candidate drug for preventing and improving hypertension.

FoxO4 mediates macrophage M2 polarization by promoting LXA4R expression in an ovalbumin-induced allergic asthma model in mice

Background: Asthma imposes a heavy burden due to its high prevalence. Forkhead box O4 (FoxO4) proteins participate in the modulation of cell progression. However, the role and mechanism of FoxO4 in asthma remains uncharted. Methods: An allergic asthma model was constructed by the induction of ovalbumin and interleukin (IL)-4 in mice and monocyte/macrophage-like Raw264.7 cells, respectively. The role and mechanism of FoxO4 in asthma was determined by pathological staining, immunofluorescence assay, measurement of inflammatory cells in the blood, reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, and flow cytometry. Results: Ovalbumin treatment triggered an obvious inflammatory cell infiltration with a prominent increase in F4/80+ cell numbers. The relative messenger RNA (mRNA) and protein expressions of FoxO4 were increased in both ovalbumin-induced mice and interleukin-4 (IL-4)-induced Raw264.7 cells. Inhibition of FoxO4 via AS1842856 reduced inflammatory cell infiltration, the number of Periodic Acid Schiff+ (PAS+) goblet cells, the numbers of inflammatory cells in the blood, and the airway resistance in ovalbumin-induced mice. Besides, interference of FoxO4 decreased the number of F4/80+CD206+ cells, and the relative protein expressions of CD163 and Arg1 in vivo and in vitro. Mechanically, suppression of FoxO4 diminished the relative mRNA and protein expressions of LXA4R in both ovalbumin-induced mice and IL-4-induced Raw264.7 cells. Overexpression of LXA4R reversed the outcomes caused by repression of FoxO4, including airway resistance, the number of F4/80+ cells, the proportion of CD206+ cells in ovalbumin-induced mice, and the proportion of F4/80+CD206+ cells in IL-4-induced Raw264.7 cells. Conclusion: FoxO4/LXA4R axis mediated macrophage M2 polarization in allergic asthma.

Therapeutic effect and mechanism of Xiaoyao Kangai Jieyu Recipe on mice with breast cancer related depression through regulating COX pathway

This study aimed to investigate the intervention effect and mechanism of Xiaoyao Kangai Jieyu Recipe(XKJR) on hip-pocampal microglia and neuronal damage in mice with breast cancer related depression. The mouse model of breast cancer related depression was established by inoculation of 4T1 breast cancer cells in axilla and subcutaneous injection of corticosterone(30 mg·kg~(-1)). The successfully modeled mice were randomly divided into a model group, a positive drug group(capecitabine 60 mg·kg~(-1)+fluoxetine 19.5 mg·kg~(-1)), and XKJR group(19.5 mg·kg~(-1) crude drug), with 6 in each group. Another 6 normal mice were taken as a normal group. The administration groups were given corresponding drugs by gavage, while the normal and model groups were given an equal volume of distilled water, once a day for 21 consecutive days. The depressive behavior of mice was assessed by glucose consumption test, open field test and novelty-suppressed feeding test. Hematoxylin and eosin(HE) staining and tumor suppression rate were used to evaluate the changes of axillary tumors. The mRNA expressions and the relative protein expressions of interleukin-1β(IL-1β), interleukin-18(IL-18), cyclooxyganese-2(COX-2) and glutamyl-prolyl-tRNA synthetase(EPRs) in the hippocampus of mice were determined by quantitative real-time polymerase chain reaction(qRT-PCR) and immunohistochemistry, respectively. Immunofluorescence was performed to detect the mean fluorescence intensity of CD11b, a marker of hippocampal microglia activation. Nissler staining and transmission electron microscopy were employed to observe the morphological changes and the ultramorphological changes of hippocampal neurons, respectively. The experimental results indicated that compared with the normal group, the model group had reduced glucose consumption and lowered number of total activities in open field test(P&lt;0.05, P&lt;0.01), prolonged first feeding latency in no-velty-suppressed feeding test(P&lt;0.01), and significant depression-like behavior; the contents of IL-1β, IL-18, COX-2, and EPRs in hippocampus were increased(P&lt;0.05, P&lt;0.01), with hippocampal microglia activation and obvious neuronal damage. Compared with the model group, the positive drug group and the XKJR group presented an improvement in depressive behaviors, a decrease in the contents of IL-1β, IL-18, COX-2 and EPRs in hippocampus, and an alleviation in the activation of hippocampal microglia and neuronal damage; the tumor suppression rates of positive drug and XKJR were 40.32% and 48.83%, respectively, suggesting a lower tumor growth rate than that of the model group. In summary, XKJR may improve hippocampal microglia activation and neuronal damage in mice with breast cancer related depression through activating COX signaling pathway.

How the West(ern) was won: Solutions for immunoblotting large and small proteins

Relative protein quantification is a well-established technique in the vast majority of molecular biology laboratories. However, western blot standard protocols may not detect proteins of certain sizes. When the molecular weight of protein of interest is out of the 10–250 kDa range, its migration through the gel or transfer to the membrane is compromised, making its detection difficult. Here, we present a set of modifications of the standard working procedure for western blotting based on the experience working with small VCP-interacting protein and MAX-gene-associated protein, whose molecular weights are 8 and 350 kDa, respectively. We expect that these adaptations may help researchers to improve their experiments in a cost-effective manner.

BTF3 promotes proliferation and glycolysis in hepatocellular carcinoma by regulating GLUT1

ABSTRACT Hepatocellular carcinoma (HCC) is a grievous tumor with an increasing incidence worldwide. Basic transcription factor 3 (BTF3) is discovered to regulate the expression of glucose transporter 1 (GLUT1), which benefits glycolysis, a momentous signature of tumors, through transactivation of the forkhead box M1 (FOXM1) expression. BTF3 is highly expressed in HCC. However, whether BTF3 promotes GLUT1 expression through FOXM1 to modulate glycolysis in HCC remains unclear. The expression profile of BTF3 were determined by online database, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. The role and mechanism of BTF3 in the proliferation and glycolysis of HCC cells were examined by cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) incorporation, XF96 Extracellular Flux analyzer, spectrophotometry and western blot analysis. In addition, the direct interaction between BTF3 and FOXM1 was verified by dual-luciferase reporter and co-immunoprecipitation assays. Moreover, the role of BTF3 was also explored in a xenografted mice model. The expression of BTF3 was increased in HCC cells and tumor tissues. Knockdown of BTF3 reduced the cell viability, Edu positive cells, extracellular acidification rate (ECAR), glucose consumption and lactate production in both Huh7 and HCCLM3 cells. The expressions of FOXM1 and GLUT1 were increased in HCC tissues, which were positively correlated with the BTF3 expression. Moreover, a direct interaction existed between BTF3 and FOXM1 in HCC cells. Downregulation of BTF3 decreased the relative protein levels of FOXM1 and GLUT1, which were rescued with overexpression of FOXM1 in both cells. More importantly, overexpression of FOXM1 restored the cell viability, ECAR, glucose consumption and lactate production in both Huh7 and HCCLM3 cells transfected with siBTF3#1. Furthermore, inhibition of BTF3 decreased tumor weight and volume, and the relative level of BTF3, FOXM1, GLUT1 and Ki-67 in tumor tissues from mice xenografted with Huh7 cells. BTF3 enhanced the cell proliferation and glycolysis through FOXM1/GLUT1 axis in HCC.

Hesperetin attenuates cognitive dysfunction via SIRT6/NLRP3 pathway in scopolamine-induced mice.

Neuroinflammation is a critical feature in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). Hesperetin can exert anti-inflammatory, antioxidant and other neuroprotective effects. In this study, the scopolamine (SCOP)-induced cognitive dysfunction in mice model was used to evaluate the neuroprotective effects of hesperetin. Behavioral tests (Morris water maze, open field, and novel object recognition tests) were conducted to evaluate the effect of hesperetin on cognitive dysfunction behaviors. Nissl staining and Immunofluorescence were used to evaluate hippocampal neuronal damage and microglial activation in mice. The levels of proinflammatory factors, oxidant stress, and the cholinergic neurotransmitter were detected by real-time quantitative fluorescence PCR (RT-qPCR) or biochemical reagent kits. Western blotting was used to detect the relative protein expression of the sirtuin 6 (SIRT6) / NOD-like receptor thermal protein domain associated protein 3 (NLRP3) pathway. Results showed that hesperetin could ameliorate SCOP-induced cognitive impairment and neuronal damage, and regulate the levels of cholinergic neurotransmitters in the hippocampal of AD mice. Hesperetin could also enhance antioxidant defense by regulating the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT). Hesperetin exerted anti-neuroinflammation effects through inhibiting of microglia activation and down-regulating the mRNA transcript levels of inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Meanwhile, hesperetin could attenuate the expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), thioredoxin-interacting protein (TXNIP), and caspase-1 p20 and upregulate the expression of SIRT6 in SCOP-induced mice. Overall, our study suggested that hesperetin might ameliorate SCOP-induced cognitive dysfunction by improving cholinergic system dysfunction and suppressing oxidative stress and attenuating neuroinflammation via SIRT6/NLRP3 pathway in mice.

Identification of heat shock protein70-2 and protamine-1 mRNA, proteins, and analyses of their association with fertility using frozen-thawed sperm in Madura bulls.

This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers. The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) (79.04%; n = 4), and low fertility (LF) (65.84%; n = 4). mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate. Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality. HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.

Blockade of P2X7 receptor-mediated purinergic signaling with A438079 protects against LPS-induced liver injury in rats.

The study aimed to investigate the hepatoprotective effects of purinergic receptor (P2X7R) antagonism by A438079 in liver damage. An experimental model of inflammation was performed by intraperitoneal (i.p.) lipopolysaccharide (LPS) administration in the rat. The groups were Control, A438079, dimethyl sulfoxide (DMSO), LPS, LPS + DMSO, and LPS + A438079. Following LPS (8 mg/kg) injection, A438079 (15 mg/kg) and DMSO (0.1 mL) were administrated (i.p) in the study groups. The blood and the liver tissues were removed for histological, biochemical, and western blot analyses. In the biochemical analysis, serum aspartate transaminase (AST) and alanine transaminase (ALT) concentrations, the tissue glutathione (GSH) level, and superoxide dismutase (SOD) activity dramatically decreased and malondialdehyde (MDA) level increased in the LPS and LPS + DMSO groups compared to the LPS + A438079 group. In the histological analysis, severe sinusoidal dilatation, necrotic hepatocytes, and inflammatory cell infiltration were observed in the LPS and LPS + DMSO groups while these effects were lessened in the LPS + A438079 group. The relative protein expression levels of P2X7R, Nf-kB-p65, IL-6, and Caspase-3 were significantly higher in the LPS and the LPS + DMSO groups than in the LPS + A438079 group. On the other hand, these protein expressions were considerably lower in the Control, A438079, and DMSO groups compared to the LPS + A438079 group. In addition, Bcl-2 protein expression was significantly lower in the LPS and the LPS + DMSO groups and higher in the LPS + A438079 group compared to the other groups. The protective effect of A438079 against LPS-induced hepatic inflammation may be related to P2X7R antagonism, inflammatory mediators, and apoptotic cell death.

Resveratrol induces the growth inhibition of CDX-deficient gastric cancer cells using CDX2 and RUNX3 via the β-catenin/TCF4 signaling pathway

This study aimed to determine the expression levels of runt-related transcription factor 3 (RUNX3) and caudal-related homeobox 2 (CDX2) in patients with chronic gastritis, intestinal metaplasia, atypical hyperplasia, and gastric cancer (GC). To analyze the overexpression of CDX2 and the effects of resveratrol (Res) on MKN7 and TMK1 cells, immunohistochemical staining was performed to determine the protein expression levels in tissue samples. The biological activity of MKN7 and TMK1 cells was determined. Relative mRNA and protein expression levels were also determined. RUNX3 expression was positively correlated with CDX2 expression and negatively correlated with β-catenin and transcription factor 4 (TCF-4) levels in GC tissues. Interestingly, RUNX3 expression was negatively correlated with CDX2 expression in other tissues. CDX2 overexpression or Res treatment inhibited cell proliferation, migration, and invasion, while inducing cell apoptosis. Furthermore, RUNX3 and B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax) expression levels were increased, while those of of β-catenin, TCF-4, and Bcl-2 were decreased in the CDX2 group. Upon treatment with lithium chloride (LiCl), the proliferation, migration, and invasion of CDX2-overexpressing MKN7 and TMK1 cells were enhanced. Our results indicate that Res inhibits the growth of MKN7 and TMK1 cells by increasing RUNX3 and CDX2 expression levels, with the potential involvement of the β-catenin/TCF-4 signaling pathway.