Difference Gel Electrophoresis Research Articles

Proteomic analysis of rabbit fresh and cryopreserved semen provides an important insight into molecular mechanisms of cryoinjuries to spermatozoa

Cryoinjury and protein changes are a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, viability and fertilizing ability. However, potential proteomic changes in rabbit semen throughout the cryopreservation process have never been previously investigated. The aim of the present study was to compare the whole proteome of fresh and cryopreserved rabbit semen (spermatozoa and extracellular fluid), to examine the effects of freeze-thawing on proteins changes in semen. Comparative analysis and identification of proteins was carried out using 2-dimensional difference in-gel electrophoresis coupled with a matrix-assisted laser desorption/ionization mass spectrometry. Proteomic raw data are available via ProteomeXchange with identifier PXD034832 for spermatozoa and PXD034853 for extracellular fluid. Respectively, 107 and 28 proteins differed in abundance in spermatozoa and extracellular fluid between fresh and frozen groups. Most of these proteins were involved in pathways related to energy metabolism and protein quality control under stress conditions, reproductive processes and mechanisms of cell death/survival regulation, resulting in a significant decrease of motility and viability of post-thawing rabbit sperm and its potential fertilizing ability. These results broaden the understanding of the effects of cryopreservation on rabbit semen and represent a new starting point for the development of improved freezing procedures.

2D-DIGE-MS Proteomics Approaches for Identification of Gelsolin and Peroxiredoxin 4 with Lymph Node Metastasis in Colorectal Cancer.

Simple SummarySchematic presentation of the histone modification of gelsolin (GSN) and peroxiredoxin-4 (PRDX4) promoters by endogenous EGFR/RhoA/PKC/ERK signaling pathways in DLD-1 cells. Identifying lymph node metastasis (LNM)-associated proteins by two-dimensional difference gel electrophoresis MS/MS in colorectal cells (CRC) indicates the reliability of candidate markers of GSN and PRDX4 and therapeutic targets for CRC.Background/Aims: A combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry approach was used to search for potential markers for prognosis and intervention of colorectal cancer (CRC) at different stages of lymph node metastasis (LMN). This quantitative proteomic survey aimed to investigate the LNM-associated proteins and evaluate the clinicopathological characteristics of these target proteins in CRC from stage I to stage IV. Methods: Sixteen CRC cases were categorized into paired non-LNM and LNM groups, and two-dimensional difference gel electrophoresis and MS proteome analysis were performed. Differential protein expression between non-LNM and LNM CRC was further validated in a tissue microarray, including 40 paraffin-embedded samples by immunohistochemistry staining. Moreover, a Boyden chamber assay, flow cytometry, and shRNA were used to examine the epithelial–mesenchymal transition and mechanism invasiveness of the differentially expressed proteins in DLD-1 cells and in vivo xenograft mouse model. Results: Eighteen differentially expressed proteins were found between non-LNM and LNM CRC tissues. Among them, protein levels of Gelsolin (GSN) and peroxiredoxin 4 (PRDX4) were abundant in node-positive CRC. Downregulation of GSN and PRDX4 markedly suppressed migration and invasiveness and also induced cell cycle G1/S arrest in DLD-1. Mechanistically, the EGFR/RhoA/PKCα/ERK pathways are critical for transcriptional activation of histone modification of H3 lysine 4 trimethylation (H3K4me3) of GSN and PRDX4 promoters, resulting in upregulation of GSN, PRDX4, Twist-1/2, cyclinD1, proliferating cell-nuclear antigen, β-catenin, N-cadherin, and matrix metalloprotein-9. Conclusions: GSN and PRDX4 are novel regulators in CRC lymph node metastasis to potentially provide new insights into the mechanism of CRC progression and serve as a biomarker for CRC diagnosis at the metastatic stage.

2D-DIGE based urinary proteomics and functional enrichment studies to reveal novel Mycobacterium tuberculosis and human protein biomarker candidates for pulmonary tuberculosis

In the absence of a sensitive and specific diagnostic modality capable of detecting all forms of tuberculosis (TB), proteomics may identify specific Mycobacterium tuberculosis (M.tb) proteins in urine, with a potential as biomarkers. To identify candidate biomarkers for TB, proteome profile of urine from pulmonary TB patients was compared with non-disease controls (NDC) and disease controls (DC, Streptococcus pneumonia infected patients) using a combination of two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LCMS/MS). Eleven differentially expressed host proteins and Eighteen high abundant M.tb proteins were identified. Protein-protein interactome (PPI) and functional enrichment analyses like Gene Ontologies, Reactome pathway etc. demonstrated that the human proteins mainly belong to extracellular space and show physiological pathways for immune response and hematological disorders. Whereas, M.tb proteins belong to the cell periphery, plasma membrane and cell wall, and demonstrated catalytic, nucleotide binding and ATPase activities along with other functional processes. The study findings provide valuable inputs about the biomarkers of TB and shed light on the probable disease consequences as an outcome of the bacterial pathogenicity.

Variations in Blood Platelet Proteome and Transcriptome Revealed Altered Expression of Transgelin-2 in Acute Coronary Syndrome Patients.

Proteomic analyses based on mass spectrometry provide a powerful tool for the simultaneous identification of proteins and their signatures. Disorders detection at the molecular level delivers an immense impact for a better understanding of the pathogenesis and etiology of various diseases. Acute coronary syndrome (ACS) refers to a group of heart diseases generally associated with rupture of an atherosclerotic plaque and partial or complete thrombotic obstruction of the blood flow in the infarct-related coronary artery. The essential role in the pathogenesis of ACS is related to the abnormal, pathological activation of blood platelets. The multifactorial and complex character of ACS indicates the need to explain the molecular mechanisms responsible for thrombosis. In our study, we performed screening and comparative analysis of platelet proteome from ACS patients and healthy donors. Two-dimensional fluorescence difference gel electrophoresis and nanoscale liquid chromatography coupled to tandem mass spectrometry showed altered expressions of six proteins (i.e., vinculin, transgelin-2, fibrinogen β and γ chains, apolipoprotein a1, and tubulin β), with the overlapping increased expression at the mRNA level for transgelin-2. Dysregulation in protein expression identified in our study may be associated with an increased risk of thrombotic events, correlated with a higher aggregability of blood platelets and induced shape change, thus explaining the phenomenon of the hyperreactivity of blood platelets in ACS.

Proteomics analysis of pyrene biodegradation using strain Mycobacterium sp. 16F

ABSTRACT Mycobacterium sp. 16 F can degrade 94% of pyrene (20 ppm) in 4 days. To investigate its pyrene degradation mechanism, proteomic changes were analyzed using two-dimensional differential gel electrophoresis (2DE-DIGE). Comparative analysis of differential proteins revealed 91 differentially expressed protein spots after pyrene exposure. Among these, 65 spots were identified as 57 proteins. Further analysis revealed that 13 spots were involved in the pyrene degradation pathway, and most of these were dioxygenases and dehydrogenases. Further, 16 up-regulated expression protein spots were associated with four pathways that may be related to pyrene degradation. Bioinformatics analysis further revealed that the pentose phosphate and glycolytic pathways led to the production of amino acids and nucleotide precursors in pyrene-induced cells. The metabolites from these processes then entered the shikimate pathway via the β-ketoadipate pathway in conjunction with the pyrene degradation pathway. This study provides a new model for the pyrene degradation pathway in Mycobacteria. Abbreviations: PPP, pentose phosphate pathway; ED-EMP, Entner-Doudoroff pathway and Embden-Meyerhof-Parnas pathway; E4P, erythrose-4-phosphate; PEP, phosphoenol-pyruvate; PAL, phenylalanine ammonia-lyase; SucCoA, succinyl-CoA; AcCoA, acetyl-CoA; TCA, tricarboxylic acid cycle.

HSPA4 Is a Biomarker of Placenta Accreta and Enhances the Angiogenesis Ability of Vessel Endothelial Cells.

Placenta accreta spectrum (PAS) accounts for 7% of maternal mortality and is associated with intraoperative and postoperative morbidity caused by massive blood loss, infection, and adjacent organ damage. The aims of this study were to identify the protein biomarkers of PAS and to further explore their pathogenetic roles in PAS. For this purpose, we collected five placentas from pregnant subjects with PAS complications and another five placentas from normal pregnancy (NP) cases. Then, we enriched protein samples by specifically isolating the trophoblast villous, deeply invading into the uterine muscle layer in the PAS patients. Next, fluorescence-based two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-TOF/MS were used to identify the proteins differentially abundant between PAS and NP placenta tissues. As a result, nineteen spots were determined as differentially abundant proteins, ten and nine of which were more abundant in PAS and NP placenta tissues, respectively. Then, specific validation with western blot assay and immunohisto/cytochemistry (IHC) assay confirmed that heat shock 70 kDa protein 4 (HSPA4) and chorionic somatomammotropin hormone (CSH) were PAS protein biomarkers. Further tube formation assays demonstrated that HSPA4 promoted the in vitro angiogenesis ability of vessel endothelial cells, which is consistent with the in vivo scenario of PAS complications. In this study, we not only identified PAS protein biomarkers but also connected the promoted angiogenesis with placenta invasion, investigating the pathogenetic mechanism of PAS.

2D-DIGE based proteome analysis of wheat-Thinopyrum intermedium 7XL/7DS translocation line under drought stress

BackgroundDrought stress is the most limiting factor for plant growth and crop production worldwide. As a major cereal crop, wheat is susceptible to drought. Thus, discovering and utilizing drought-tolerant gene resources from related species are highly important for improving wheat drought resistance. In this study, the drought tolerance of wheat Zhongmai 8601-Thinopyrum intermedium 7XL/7DS translocation line YW642 was estimated under drought stress, and then two-dimensional difference gel electrophoresis (2D-DIGE) based proteome analysis of the developing grains was performed to uncover the drought-resistant proteins.ResultsThe results showed that 7XL/7DS translocation possessed a better drought-tolerance compared to Zhongmai 8601. 2D-DIGE identified 146 differential accumulation protein (DAP) spots corresponding to 113 unique proteins during five grain developmental stages of YW642 under drought stress. Among them, 55 DAP spots corresponding to 48 unique proteins displayed an upregulated expression, which were mainly involved in stress/defense, energy metabolism, starch metabolism, protein metabolism/folding and transport. The cis-acting element analysis revealed that abundant stress-related elements were present in the promoter regions of the drought-responsive protein genes, which could play important roles in drought defense. RNA-seq and RT-qPCR analyses revealed that some regulated DAP genes also showed a high expression level in response to drought stress.ConclusionsOur results indicated that Wheat-Th. intermedium 7XL/7DS translocation line carried abundant drought-resistant proteins that had potential application values for wheat drought tolerance improvement.

Application of two-dimensional difference gel electrophoresis to identify protein changes between center, margin, and adjacent non-tumor tissues obtained from non-small-cell lung cancer with adenocarcinoma or squamous cell carcinoma subtype.

Lung cancer is responsible for the most cancer-related mortality worldwide and the mechanism of its development is poorly understood. Proteomics has become a powerful tool offering vital knowledge related to cancer development. Using a two-dimensional difference gel electrophoresis (2D-DIGE) approach, we sought to compare tissue samples from non-small-cell lung cancer (NSCLC) patients taken from the tumor center and tumor margin. Two subtypes of NSCLC, adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were compared. Data are available via ProteomeXchange with identifier PXD032736 and PXD032962 for ADC and SCC, respectively. For ADC proteins, 26 significant canonical pathways were identified, including Rho signaling pathways, a semaphorin neuronal repulsive signaling pathway, and epithelial adherens junction signaling. For SCC proteins, nine significant canonical pathways were identified, including hypoxia-inducible factor-1α signaling, thyroid hormone biosynthesis, and phagosome maturation. Proteins differentiating the tumor center and tumor margin were linked to cancer invasion and progression, including cell migration, adhesion and invasion, cytoskeletal structure, protein folding, anaerobic metabolism, tumor angiogenesis, EMC transition, epithelial adherens junctions, and inflammatory responses. In conclusion, we identified several proteins that are important for the better characterization of tumor development and molecular specificity of both lung cancer subtypes. We also identified proteins that may be important as biomarkers and/or targets for anticancer therapy.

The attenuation effect of potassium 2-(1-hydroxypentyl)-benzoate in a mouse model of diabetes-associated cognitive decline: The protein expression in the brain.

Aims dl‐PHPB (potassium 2‐(1‐hydroxypentyl)‐benzoate) has been shown to have neuroprotective effects against acute cerebral ischemia, vascular dementia, and Alzheimer's disease. The aim of this study was to investigate the effects of dl‐PHPB on memory deficits and preliminarily explore the underlying molecular mechanism.MethodsBlood glucose and behavioral performance were evaluated in the KK‐Ay diabetic mouse model before and after dl‐PHPB administration. Two‐dimensional difference gel electrophoresis (2D‐DIGE)‐based proteomics was used to identify differentially expressed proteins in brain tissue. Western blotting was used to study the molecular mechanism of the related signaling pathways.ResultsThree‐month‐old KK‐Ay mice were given 150 mg/kg dl‐PHPB by oral gavage for 2 months, which produced no effect on the level of serum glucose. In the Morris water maze test, KK‐Ay mice treated with dl‐PHPB showed significant improvements in spatial learning and memory deficits compared with vehicle‐treated KK‐Ay mice. Additionally, we performed 2D‐DIGE to compare brain proteomes of 5‐month KK‐Ay mice treated with and without dl‐PHPB. We found 14 altered proteins in the cortex and 11 in the hippocampus; two of the 25 altered proteins and another four proteins that were identified in a previous study on KK‐Ay mice were then validated by western blot to further confirm whether dl‐PHPB can reverse the expression levels of these proteins. The phosphoinositide 3‐kinase/protein kinase B/glycogen synthase kinase‐3β (PI3K/Akt/GSK‐3β) signaling pathway was also changed in KK‐Ay mice and dl‐PHPB treatment could reverse it.ConclusionsThese results indicate that dl‐PHPB may play a potential role in diabetes‐associated cognitive impairment through PI3K/Akt/GSK‐3β signaling pathway and the differentially expressed proteins may become putative therapeutic targets.

Proteomic Analysis of Endometrial Cancer Tissues from Patients with Type 2 Diabetes Mellitus.

Endometrial cancer (EC) is the most common form of gynecological cancer. Type 2 diabetes mellitus is associated with an increased risk of EC. Currently, no proteomic studies have investigated the role of diabetes in endometrial cancers from clinical samples. The present study aims to elucidate the molecular link between diabetes and EC using a proteomic approach. Endometrial tissue samples were obtained from age-matched patients (EC Diabetic and EC Non-Diabetic) during surgery. Untargeted proteomic analysis of the endometrial tissues was carried out using a two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF). A total of 53 proteins were identified, with a significant difference in abundance (analysis of variance (ANOVA) test, p ≤ 0.05; fold-change ≥ 1.5) between the two groups, among which 30 were upregulated and 23 downregulated in the EC Diabetic group compared to EC Non-Diabetic. The significantly upregulated proteins included peroxiredoxin-1, vinculin, endoplasmin, annexin A5, calreticulin, and serotransferrin. The significantly downregulated proteins were myosin regulatory light polypeptide 9, Retinol dehydrogenase 12, protein WWC3, intraflagellar transport protein 88 homolog, superoxide dismutase [Cu-Zn], and retinal dehydrogenase 1. The network pathway was related to connective tissue disorder, developmental disorder, and hereditary disorder, with the identified proteins centered around dysregulation of ERK1/2 and F Actin signaling pathways. Cancer-associated protein alterations such as upregulation of peroxiredoxin-1, annexin 5, and iNOS, and downregulation of RDH12, retinaldehyde dehydrogenase 1, SOD1, and MYL 9, were found in the EC tissues of the diabetic group. Differential expression of proteins linked to cancer metastasis, such as the upregulation of vinculin and endoplasmin and downregulation of WWC3 and IFT88, was seen in the patients with diabetes. Calreticulin and alpha-enolase, which might have a role in the interplay between diabetes and EC, need further investigation.

Chromium [Cr(VI)] Exposure Causes Cytotoxicity of Human Bronchial Epithelial Cells (16-HBE) and Proteomic Alterations.

Hexavalent chromium [Cr(VI)] is a common industrial pollutant, and exposure may cause toxic effects in multiple organ systems and carcinogenesis, including lung cancer. However, the toxic effect of Cr(VI) on the respiratory system is poorly understood. In the present study, it was demonstrated that Cr(VI) exposure significantly decreased the viability of human bronchial epithelial cells (16-HBE) in a dose-dependent manner. Flow cytometry demonstrated that Cr(VI) enhanced the transition of 16-HBE cells from G1 to S phase and arrested S-phase progression. Reverse transcription-quantitative polymerase chain reaction analysis revealed a significant alteration in the expression of apoptosis-associated genes in Cr(VI)-treated 16-HBE cells. In addition, using two-dimensional fluorescence differential gel electrophoresis with mass spectrometry, 15 differentially expressed proteins (1 upregulated and 14 downregulated) were identified in 16-HBE cells with Cr(VI) treatment compared with controls. Functional classification revealed that these differentially expressed proteins were involved in apoptosis, cytoskeletal structure, and energy metabolism. In conclusion, these data suggested that Cr(VI) caused toxic effects in bronchial epithelial cells and the mechanisms may involve the abnormal expression of apoptosis-associated proteins, cytoskeletal proteins, and energy metabolism-associated proteins.

A Pilot Proteomic Study of Vestibular Fluid From Patients With Vulvodynia.

ObjectiveMany women are affected by vulvodynia, but medical therapies to date have proven ineffective. We performed a pilot study using gel-based proteomics to develop a map of proteins present in vaginal/vestibular secretions and identify proteins that could be considered for future evaluation as potential therapeutic targets.Materials and MethodsWe collected vestibular fluid from 4 controls and 4 patients with vulvodynia by placing a cotton swab in the vestibule and extracting the absorbed proteins. The proteins underwent 2-dimensional difference gel electrophoresis and mass spectrometry to develop a protein map. Immunohistochemistry was used to validate proteomic findings.ResultsA map was constructed of 32 of the more abundant proteins in vestibular fluid and their levels compared in control subjects and vulvodynia patients. Among these were annexin A1, interleukin 1 receptor antagonist, protein S100 A9, and a number of antiproteases and proteases. Many of these proteins differed by at least 50% between groups, but only annexin A1, one of the protease inhibitors, and immunoglobulin G κ chain were significantly different. The results with annexin A1 were validated by similar findings with immunohistochemistry.ConclusionsThe findings of this pilot study demonstrate a set of vestibule mucosa proteins that differ significantly—either increasing or decreasing—in vulvodynia patients compared with controls, and several others that exhibited greater than 1.5-fold change but did not reach statistical significance. This study constitutes a proof-of-principle that an open, unbiased proteomic approach can identify molecular participants in vulvodynia, some of which had not been identified to date by hypothesis-driven studies.

Gamma-tocotrienol, a radiation countermeasure, reverses proteomic changes in serum following total-body gamma irradiation in mice

Radiological incidents or terrorist attacks would likely expose civilians and military personnel to high doses of ionizing radiation, leading to the development of acute radiation syndrome. We examined the effectiveness of prophylactic administration of a developmental radiation countermeasure, γ-tocotrienol (GT3), in a total-body irradiation (TBI) mouse model. CD2F1 mice received GT3 24 h prior to 11 Gy cobalt-60 gamma-irradiation. This dose of radiation induces severe hematopoietic acute radiation syndrome and moderate gastrointestinal injury. GT3 provided 100% protection, while the vehicle control group had 100% mortality. Two-dimensional differential in-gel electrophoresis was followed by mass spectrometry and Ingenuity Pathway Analysis (IPA). Analysis revealed a change in expression of 18 proteins in response to TBI, and these changes were reversed with prophylactic treatment of GT3. IPA revealed a network of associated proteins involved in cellular movement, immune cell trafficking, and inflammatory response. Of particular interest, significant expression changes in beta-2-glycoprotein 1, alpha-1-acid glycoprotein 1, alpha-2-macroglobulin, complement C3, mannose-binding protein C, and major urinary protein 6 were noted after TBI and reversed with GT3 treatment. This study reports the untargeted approach, the network, and specific serum proteins which could be translated as biomarkers of both radiation injury and protection by countermeasures.

Identification of Serum Biomarkers in Patients with Alzheimer’s Disease by 2D-DIGE Proteomics

Aim: The aim of this study is to identify potential serum biomarkers of Alzheimer’s disease (AD) for early diagnosis and to evaluate these markers on a large cohort. Methods: We performed two-dimensional difference gel electrophoresis to compare the serum of AD patients and normal controls. Western blot or enzyme-linked immunosorbent assay (ELISA) was used to identify the expression levels of proteins. Results: In this study, a total of 13 differentially expressed proteins were identified. Among them, 2 proteins (inter-alpha-trypsin inhibitor heavy chain H4 [ITI-H4], Apolipoprotein A-IV) were validated by Western blot and 4 proteins (Cofilin 2, Tetranectin, Zinc-alpha-2-glycoprotein [AZGP1], Alpha-1-microglobulin/bikunin precursor [AMBP]) were validated by ELISA, respectively. Western blot results showed that the full size of the ITI-H4 protein was increased, while a fragment of ITI-H4 was decreased in AD patients. In contrast, 1 fragment of Apo A-IV was mainly found in control group and rare to be detected in AD patients. On the other hand, ELISA results showed that Cofilin 2, Tetranectin, AZGP1, and AMBP were significantly increased in AD patients, and Cofilin 2 is strongly correlated with the Mini-Mental State Examination scores of the AD patients. Serum Cofilin 2 was unchanged in Parkinson disease patients as compared to the control group, indicating a specific correlation of serum Cofilin 2 with AD. Moreover, Cofilin 2 was increased in both the serum and brain tissue in the APP/PS1 transgenic mice. Conclusion: Our study identified several potential serum biomarkers of AD, including: ITI-H4, ApoA-IV, Cofilin 2, Tetranectin, AZGP1, and AMBP. Cofilin 2 was upregulated in different AD animal models and might play important roles in AD pathology.

Host Plant Adaptation Drives Changes in Diaphorina citri Proteome Regulation, Proteoform Expression, and Transmission of 'Candidatus Liberibacter asiaticus', the Citrus Greening Pathogen.

The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, 'Candidatus Liberibacter asiaticus' (CLas), which is associated with citrus greening disease. The citrus relative Murraya paniculata (orange jasmine) is a host plant of D. citri but is more resistant to CLas compared with all tested Citrus genotypes. The effect of host switching of D. citri between Citrus medica (citron) and M. paniculata plants on the acquisition and transmission of CLas was investigated. The psyllid CLas titer and the proportion of CLas-infected psyllids decreased in the generations after transfer from CLas-infected citron to healthy M. paniculata plants. Furthermore, after several generations of feeding on M. paniculata, pathogen acquisition (20 to 40% reduction) and transmission rates (15 to 20% reduction) in psyllids transferred to CLas-infected citron were reduced compared with psyllids continually maintained on infected citron. Top-down (difference gel electrophoresis) and bottom-up (shotgun MS/MS) proteomics methods were used to identify changes in D. citri protein expression resulting from host plant switching between Citrus macrophylla and M. paniculata. Changes in expression of insect metabolism, immunity, and cytoskeleton proteins were associated with host plant switching. Both transient and sustained feeding on M. paniculata induced distinct patterns of protein expression in D. citri compared with psyllids reared on C. macrophylla. The results point to complex interactions that affect vector competence and may lead to strategies to control the spread of citrus greening disease.

High Expression of G6PD Increases Doxorubicin Resistance in Triple Negative Breast Cancer Cells by Maintaining GSH Level.

Resistance to doxorubicin (DOX) remains a big challenge to breast cancer treatment especially for triple negative breast cancer (TNBC). Our previous study revealed that the antioxidant system plays an important role in conferring metastasis derived DOX resistance. In this study, we used two-dimensional difference gel electrophoresis (2D-DIGE) proteomics to compare the expression profiles of two generations of TNBC cell lines which have increased metastatic ability in nude mice and exhibited resistance to DOX. Through careful analyses, one antioxidant protein: glucose-6-phosphate dehydrogenase (G6PD) was identified with 3.2-fold higher level in metastatic/DOX-resistant 231-M1 than its parental 231-C3 cells. Analyses of clinical data showed that TNBC patients with higher G6PD levels exhibited lower overall survival than patients with lower G6PD level. Reducing G6PD expression by siRNA or inhibiting its activity with dehydroepiandrosterone (DHEA) significantly increased DOX's cytotoxicity in both cell lines. Importantly, inhibiting G6PD's activity with DHEA dramatically increased the apoptotic rate of 1.25 µM DOX from 2% to 54%. Our results suggest that high level of G6PD can help TNBC to resist DOX-induced oxidative stress. Thus, inhibiting G6PD shall be a good strategy to treat DOX-resistant TNBC.

Association of 'Klotho' gene polymorphism with cerebral infarction.

BackgroundWe aimed to investigate the expression of Klotho gene in peripheral blood of patients with cerebral infarction (CI) and the association of its polymorphisms with the occurrence of CI.MethodsA total of 60 CI patients (CI group) and 20 healthy people receiving physical examination (control group) were enrolled as the research subjects. The expression of Klotho gene in CI group and control group was determined using enzyme-linked immunosorbent assay kit. Single nucleotide polymorphisms (rs192031, rs200131 and rs102312) in the promoter region of the Klotho gene were typed via conformational difference gel electrophoresis. Besides, whether the distribution frequencies of Klotho genotypes conformed to Hardy-Weinberg equilibrium was evaluated by chi-square test. Meanwhile, the associations of Klotho alleles and gene polymorphisms with CI occurrence were analyzed.ResultsThe protein expression level of Klotho in the peripheral blood was remarkably lower in patients in CI group than that in control group (P<0.05).HardyWeinberg equilibrium analysis revealed that Klotho gene polymorphisms (rs192031, rs200131 and rs102312) conformed to the genetic equilibrium distribution (P>0.05). Gene-based association analysis manifested that only rs192031 polymorphism and alleles were correlated with CI occurrence (P<0.05). Systolic blood pressure and highdensity lipoprotein cholesterol were notably higher in CI patients with TT genotype of Klotho gene polymorphism rs192031 than those in control group (P<0.05). Furthermore, there were no associations of rs200131 and rs102312 polymorphisms and alleles with the occurrence of CI (P>0.05).ConclusionsThe expression level of Klotho is evidently reduced in the peripheral blood of CI patients. Rs192031 in the promoter region of the Klotho gene is associated with the occurrence of CI, while rs200131 and rs102312 have no relations with CI.

Single nucleotide polymorphisms rs102313, rs118231 and rs201832 of CTEP TaqIB gene correlated with lipid metabolism abnormalities and cerebral infarction in patients with atherosclerosis.

The purpose of this study was to investigate the correlations of cholesterol ester transfer protein (CTEP) TaqIB gene polymorphism with lipid metabolism abnormalities and cerebral infarction (CI) in patients with atherosclerosis (AS). A case-control study was conducted on 100 AS patients complicated with (CI) as AS+CI group, and 200 AS patients with matched age, gender and race as controls (AS group). Single nucleotide polymorphisms (SNPs) rs102313, rs118231 and rs201832 in the promoter region of CTEP TaqIB gene were classified by conformational differential gel electrophoresis. Then, Chi-square test was carried out to determine whether the distribution frequency of CTEP TaqIB genotypes conforms to the law of genetic equilibrium. In the meantime, the correlations of gene polymorphisms and allelotypes in the promoter region of CTEP TaqIB with CI and lipid metabolism abnormalities in AS patients were analyzed. Hardy-Weinberg genetic equilibrium analysis showed that the three polymorphisms of CTEP TaqIB gene were in accordance with the genetic equilibrium distribution (p>0.05). Moreover, the results of gene association analysis revealed that the polymorphisms rs102313 and rs118231 and allelotypes in the promoter region of CTEP TaqIB gene were correlated with CI in AS patients (p<0.05). Specifically, AS patients with GG genotype and allele G at rs102313 and those with TT genotype and allele T at rs118231 had a higher risk of CI (p<0.05). Besides, the polymorphism rs102313 in the promoter region of CTEP TaqIB gene was markedly related to lipid metabolism abnormalities in AS patients (p<0.05). The polymorphisms rs102313 and rs118231 in the promoter region of CTEP TaqIB gene are associated with CI in AS patients, and the polymorphism rs102313 is remarkably correlated with lipid metabolism abnormalities in AS patients.

Platelet Phenotype Analysis of COVID-19 Patients Reveals Progressive Changes in the Activation of Integrin αIIbβ3, F13A1, the SARS-CoV-2 Target EIF4A1 and Annexin A5.

Background: The fatal consequences of an infection with severe acute respiratory syndrome coronavirus 2 are not only caused by severe pneumonia, but also by thrombosis. Platelets are important regulators of thrombosis, but their involvement in the pathogenesis of COVID-19 is largely unknown. The aim of this study was to determine their functional and biochemical profile in patients with COVID-19 in dependence of mortality within 5-days after hospitalization.Methods: The COVID-19-related platelet phenotype was examined by analyzing their basal activation state via integrin αIIbβ3 activation using flow cytometry and the proteome by unbiased two-dimensional differential in-gel fluorescence electrophoresis. In total we monitored 98 surviving and 12 non-surviving COVID-19 patients over 5 days of hospital stay and compared them to healthy controls (n = 12).Results: Over the observation period the level of basal αIIbβ3 activation on platelets from non-surviving COVID-19 patients decreased compared to survivors. In line with this finding, proteomic analysis revealed a decrease in the total amount of integrin αIIb (ITGA2B), a subunit of αIIbβ3, in COVID-19 patients compared to healthy controls; the decline was even more pronounced for the non-survivors. Consumption of the fibrin-stabilizing factor coagulation factor XIIIA (F13A1) was higher in platelets from COVID-19 patients and tended to be higher in non-survivors; plasma concentrations of the latter also differed significantly. Depending on COVID-19 disease status and mortality, increased amounts of annexin A5 (ANXA5), eukaryotic initiation factor 4A-I (EIF4A1), and transaldolase (TALDO1) were found in the platelet proteome and also correlated with the nasopharyngeal viral load. Dysregulation of these proteins may play a role for virus replication. ANXA5 has also been identified as an autoantigen of the antiphospholipid syndrome, which is common in COVID-19 patients. Finally, the levels of two different protein disulfide isomerases, P4HB and PDIA6, which support thrombosis, were increased in the platelets of COVID-19 patients.Conclusion: Platelets from COVID-19 patients showed significant changes in the activation phenotype, in the processing of the final coagulation factor F13A1 and the phospholipid-binding protein ANXA5 compared to healthy subjects. Additionally, these results demonstrate specific alterations in platelets during COVID-19, which are significantly linked to fatal outcome.

Manganese exposure causes movement deficit and changes in the protein profile of the external globus pallidus in Sprague Dawley rats

Manganese (Mn) is required for normal brain development and function. Excess Mn may trigger a parkinsonian movement disorder but the underlying mechanisms are incompletely understood. We explored changes in the brain proteomic profile and movement behavior of adult Sprague Dawley (SD) rats systemically treated with or without 1.0 mg/mL MnCl2 for 3 months. Mn treatment significantly increased the concentration of protein-bound Mn in the external globus pallidus (GP), as demonstrated by inductively coupled plasma mass spectrometry. Behavioral study showed that Mn treatment induced movement deficits, especially of skilled movement. Proteome analysis by two-dimensional fluorescence difference gel electrophoresis coupled with mass spectrometry revealed 13 differentially expressed proteins in the GP of Mn-treated versus Mn-untreated SD rats. The differentially expressed proteins were mostly involved in glycolysis, metabolic pathways, and response to hypoxia. Selected pathway class analysis of differentially expressed GP proteins, which included phosphoglycerate mutase 1 (PGAM1), primarily identified enrichment in glycolytic process and innate immune response. In conclusion, perturbation of brain energy production and innate immune response, in which PGAM1 has key roles, may contribute to the movement disorder associated with Mn neurotoxicity.