Slot Blot Analysis Research Articles

Allergenic Potential of Common Hops (Humulus lupulus L.) in the Context of Cross-Reactions with Pollen Allergens.

Common hops (Humulus lupulus L.) play a key role in brewing, providing the bitterness, flavor, and aroma of beer, and are widely used in supplements for their antibacterial, anti-inflammatory, and antioxidant properties. However, despite their broad applications, the allergenic potential of common hops remains underexplored, particularly when compared to the closely related Humulus japonicus. This preliminary study aimed to investigate the allergenic potential of common hops and their potential cross-reactivity with common pollen allergens. The immunoreactivity of hop stalks, leaves, and cones was assessed using antibodies against major allergens from birch (Bet v1a), mugwort (Art v1), and timothy grass (Phl p5b), as well as three sera from pollen-allergic patients. Slot Blot analysis was performed using phosphate-buffered saline extracts from the stalks, leaves, and cones of three hop cultivars, while Western Blotting followed SDS-PAGE protein separation. The results revealed significant immunoreactivity in native hop proteins, with diminished reactivity observed in denatured proteins. Cross-reactivity between hop proteins and major pollen allergens was confirmed, indicating that hop proteins may contribute to allergic sensitization in pollen-sensitive individuals. These findings underscore the potential allergenic risks associated with the consumption or exposure to hop-containing products.

Slot Blot Analysis of Intracellular Glyceraldehyde-Derived Advanced Glycation End Products Using a Novel Lysis Buffer and Polyvinylidene Difluoride Membrane.

Advanced glycation end products (AGEs) are formed through the reaction/modification of proteins by saccharides (e.g., glucose and fructose) and their intermediate/non-enzymatic products [e.g., methylglyoxal and glyceraldehyde (GA)]. In 2017, Dr. Takanobu Takata et al. developed the novel slot blot method to quantify intracellular GA-derived AGEs (GA-AGEs). Although the original method required nitrocellulose membranes, we hypothesized that the modified proteins contained in the AGEs may be effectively probed on polyvinylidene difluoride (PVDF) membranes. Because commercial lysis buffers are unsuitable for this purpose, Dr. Takata developed the slot blot method using an in-house-prepared lysis buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) that effectively probes AGEs onto PVDF membranes. The slot blot method also entails the calculation of Tris, urea, thiourea, and CHAPS concentrations, as well as protein and mass to be probed onto the PVDF membranes. GA-AGE-modified bovine serum albumin (BSA, GA-AGEs-BSA) is used to draw a standard curve and perform neutralization against a non-specific combination of anti-GA-AGEs antibodies, thereby enabling the quantification of GA-AGEs in cell lysates. This paper presents the detailed protocol for slot blot analysis of intracellular GA-AGE levels in C2C12 cells. Key features • This protocol leverages the idea that advanced glycation end products are modified proteins. • The lysis buffer containing Tris, urea, thiourea, and CHAPS enables probing proteins onto PVDF membranes. • Intracellular GA-AGE levels may be quantified for some cell types using polyclonal anti-GA-AGE antibodies and standard GA-AGE-modified BSA. • The lysis buffer may be simultaneously prepared with the cell lysate. • There is no limit to the type of cultured cells used in the preparation of cell lysate.

Potential of the Novel Slot Blot Method with a PVDF Membrane for Protein Identification and Quantification in Kampo Medicines.

Kampo is a Japanese traditional medicine modified from traditional Chinese medicine. Kampo medicines contain various traditional crude drugs with unknown compositions due to the presence of low-molecular-weight compounds and proteins. However, the proteins are generally rare and extracted with high-polarity solvents such as water, making their identification and quantification difficult. To develop methods for identifying and quantifying the proteins in Kampo medicines, in the current study we employ previous technology (e.g., column chromatography, electrophoresis, and membrane chromatography), focusing on membrane chromatography with a polyvinylidene difluoride (PVDF) membrane. Moreover, we consider slot blot analysis based on the principle of membrane chromatography, which is beneficial for analyzing the proteins in Kampo medicines as the volume of the samples is not limited. In this article, we assess a novel slot blot method developed in 2017 and using a PVDF membrane and special lysis buffer to quantify advanced glycation end products-modified proteins against other slot blots. We consider our slot blot analysis superior for identifying and quantifying proteins in Kampo medicines compared with other methods as the data obtained with our novel slot blot can be shown with both error bars and the statistically significant difference, and our operation step is simpler than those of other methods.

Rev-erbα agonists suppresses TGFβ1-induced fibroblast-to-myofibroblast transition and pro-fibrotic phenotype in human lung fibroblasts

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by excessive scarring of the lungs that can lead to respiratory failure and death. Lungs of patients with IPF demonstrate excessive deposition of extracellular matrix (ECM) and an increased presence of pro-fibrotic mediators such as transforming growth factor-beta 1 (TGFβ1), which is a major driver of fibroblast-to-myofibroblast transition (FMT). Current literature supports that circadian clock dysfunction plays an essential role in the pathophysiology of various chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and IPF. The circadian clock transcription factor Rev-erbα is encoded by Nr1d1 that regulates daily rhythms of gene expression linked to immunity, inflammation, and metabolism. However, investigations into the potential roles of Rev-erbα in TGFβ-induced FMT and ECM accumulation are limited. In this study, we utilized several novel small molecule Rev-erbα agonists (GSK41122, SR9009, and SR9011) and a Rev-erbα antagonist (SR8278) to determine the roles of Rev-erbα in regulating TGFβ1-induced FMT and pro-fibrotic phenotypes in human lung fibroblasts. WI-38 cells were either pre-treated/co-treated with or without Rev-erbα agonist/antagonist along with TGFβ1. After 48 h, the following parameters were evaluated: secretion of COL1A1 (Slot-Blot analysis) and IL-6 (ELISA) into condition media, expressions of α-smooth muscle actin (αSMA: immunostaining and confocal microscopy), and pro-fibrotic proteins (αSMA and COL1A1 by immunoblotting), as well as gene expression of pro-fibrotic targets (qRT-PCR: Acta2, Fn1, and Col1a1). Results revealed that Rev-erbα agonists inhibited TGFβ1-induced FMT (αSMA and COL1A1), and ECM production (reduced gene expression of Acta2, Fn1, and Col1a1), and decreased pro-inflammatory cytokine IL-6 release. The Rev-erbα antagonist promoted TGFβ1-induced pro-fibrotic phenotypes. These findings support the potential of novel circadian clock-based therapeutics, such as Rev-erbα agonist, for the treatment and management of fibrotic lung diseases and disorders.

Is the Novel Slot Blot a Useful Method for Quantification of Intracellular Advanced Glycation End-Products?

Various types of advanced glycation end-products (AGEs) have been identified and studied. I have reported a novel slot blot analysis to quantify two types of AGEs, glyceraldehyde-derived AGEs, also called toxic AGEs (TAGE), and 1,5-anhydro-D-fructose AGEs. The traditional slot blot method has been used for the detection and quantification of RNA, DNA, and proteins since around 1980 and is one of the more commonly used analog technologies to date. However, the novel slot blot analysis has been used to quantify AGEs from 2017 to 2022. Its characteristics include (i) use of a lysis buffer containing tris-(hydroxymethyl)-aminomethane, urea, thiourea, and 3-[3-(cholamidopropyl)-dimetyl-ammonio]-1-propane sulfonate (a lysis buffer with a composition similar to that used in two-dimensional gel electrophoresis-based proteomics analysis); (ii) probing of AGE-modified bovine serum albumin (e.g., standard AGE aliquots); and (iii) use of polyvinylidene difluoride membranes. In this review, the previously used quantification methods of slot blot, western blot, immunostaining, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry (MS), matrix-associated laser desorption/ionization-MS, and liquid chromatography-electrospray ionization-MS are described. Lastly, the advantages and disadvantages of the novel slot blot compared to the above methods are discussed.

Intracellular Toxic Advanced Glycation End-Products May Induce Cell Death and Suppress Cardiac Fibroblasts.

Cardiovascular disease (CVD) is a lifestyle-related disease (LSRD) induced by the dysfunction and cell death of cardiomyocytes. Cardiac fibroblasts are activated and differentiate in response to specific signals, such as transforming growth factor-β released from injured cardiomyocytes, and are crucial for the protection of cardiomyocytes, cardiac tissue repair, and remodeling. In contrast, cardiac fibroblasts have been shown to induce injury or death of cardiomyocytes and are implicated in the pathogenesis of diseases such as cardiac hypertrophy. We designated glyceraldehyde-derived advanced glycation end-products (AGEs) as toxic AGEs (TAGE) due to their cytotoxicity and association with LSRD. Intracellular TAGE in cardiomyocytes decreased their beating rate and induced cell death in the absence of myocardial ischemia. The TAGE levels in blood were elevated in patients with CVD and were associated with myocardial ischemia along with increased risk of atherosclerosis in vascular endothelial cells in vitro. The relationships between the dysfunction or cell death of cardiac fibroblasts and intracellular and extracellular TAGE, which are secreted from certain organs, remain unclear. We examined the cytotoxicity of intracellular TAGE by a slot blot analysis, and TAGE-modified bovine serum albumin (TAGE-BSA), a model of extracellular TAGE, in normal human cardiac fibroblasts (HCF). Intracellular TAGE induced cell death in normal HCF, whereas TAGE-BSA did not, even at aberrantly high non-physiological levels. Therefore, only intracellular TAGE induced cell death in HCF under physiological conditions, possibly inhibiting the role of HCF.

Polyblend Nanofibers to Regenerate Gingival Tissue: A Preliminary In Vitro Study

Aim: The regeneration of small periodontal defects has been considered an important divide and challenging issue for dental practitioners. The aim of this preliminary in vitro study was to analyze the effects of polycaprolactone (PCL) nanofibers enriched with hyaluronic acid and vitamin E vs. nude nanofibers on gingival fibroblasts activity, an innovative graft for periodontal soft tissue regeneration purposes.Methods: Nanofibers were produced in PCL (NF) or PCL enriched with hyaluronic acid and vitamin E (NFE) by electrospinning technique. NF and NFE were stereologically and morphologically characterized by scanning electron microscope (SEM), and composition was analyzed by infrared spectroscopy. Human fibroblasts were obtained from one gingival tissue fragment (HGF) and then seeded on NF, NFE, and plastic (CT). Cell adhesion and morphology were evaluated using SEM at 24 h and cell viability after 24, 48, and 72 h by alamarBlue® assay. Gene expression for COL-I, LH2b, TIMP-1, PAX, and VNC was analyzed by real-time RT-PCR in samples run in triplicate and GAPDH was used as housekeeping gene. Slot blot analysis was performed and immunoreactive bands were revealed for MMP-1 and COL-I. YAP and p-YAP were analyzed by Western blot and membranes were reprobed by α-tubulin. Statistical analysis was performed.Results: IR spectrum revealed the presence of PCL in NF and PCL and vitamin E and hyaluronic acid in NFE. At 24 h, HGF adhered on NF and NFE conserving fibroblast like morphology. At 72 h from seeding, statistically significant differences were found in proliferation of HGF cultured on NF compared to NFE. Expression of genes (LH2b, TIMP-1, and MMP-1) and proteins (COL-I) related to collagen turnover revealed a reduction of COL-1 secretion in cells cultured on NF and NFE compared to CT; however, NFE stimulated cross-linked collagen deposition. Mechanosensor genes (PAX, VNC, and YAP) were upregulated in HGF on NF while they were decreased in cells grown on NFE.Conclusion: Preliminary data suggest that PCL-enriched nanofibers could represent a support to induce HGF proliferation, adhesion, collagen cross-linking, and to reduce collagen degradation, therefore favoring collagen deposition in gingival connective tissue.

Periplasmic expression of EGFRvIII extracellular domain as a fusion protein in Escherichia coli NiCo21(DE3)

Epidermal growth factor receptor variant III is a mutant variant of EGFR that has a deletion on its DNA encoding extracellular ligan-binding domain. EGFRvIII is only found in cancer cells and not in normal cells, make it an ideal target as a biomarker for antibody-based cancer therapy. This study performed the expression and characterization of EGFRvIII extracellular domain conjugated with a blue fluorescent protein (BFP) as a fusion protein in Escherichia coli periplasmic space. Endoxylanase signal peptide was employed to guide the recombinant protein through the membrane. IPTG as inducer was added into expression medium with various concentrations of 0; 0.1; 0.25; 0.5; 1 mM, followed by periplasmic extraction using the hypertonic solution. Total proteins and periplasmic proteins were characterized using SDS-PAGE and slot blot analyses. Extracellular domain of EGFRvIII-BFP fusion protein was detected using confocal fluorescence microscopy. This study showed that the ∼43 kDa target protein was successfully expressed on E. coli NiCo21(DE3) periplasmic space with optimum IPTG concentration of 0.1 mM and and showed a blue fluorescence color under the microscope..

Novel antibodies detect additional α-synuclein pathology in synucleinopathies: potential development for immunotherapy

BackgroundAlpha-synuclein (α-Syn) aggregation is the primary characteristic of synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Immunotherapy targeting α-Syn has shown promising results in animal models of the disease. This study investigates the target specificity of three different active vaccines for pathological α-Syn aggregates found in human brain tissue from synucleinopathies.MethodsGuinea pigs were immunised with 3 vaccines developed by United Neuroscience, and IgG fractions purified from the resulting immune sera (IGG-1, IGG-2 or IGG-3) were used to perform immunohistochemical staining of human cases of PD, DLB and MSA. The resulting immunoreactivity was compared to a commercially available α-Syn antibody from Novacastra (NOV) commonly used for diagnostic purposes. Images were captured from the substantia nigra (SN), temporal lobe, internal capsule, insular cortex and putamen and quantified for the percentage area with α-Syn immunoreactivity. Lewy bodies (LB) and Lewy neurites (LN) were further analysed in PD and DLB cases.ResultsVaccine-generated antibodies detected more α-Syn pathology compared to NOV. The levels of α-Syn immunoreactivity varied between brain region and disease type with IGG-3 recognising the highest levels of α-Syn in most cases and in all brain regions that are affected early in disease progression. IGG-3 had a high recognition for glial inclusions found in MSA which are known to have a more compact conformation. Slot blot analysis confirmed the specificity of IGG-3 for native oligomers and fibrillar α-Syn. Higher levels of α-Syn were recognised by IGG-2 in cortical regions, and by IGG-3 in SN of PD and DLB cases. This was due to increased immunolabelling of LNs in these brain regions suggesting that IGG-2 and IGG-3 recognised additional α-Syn pathology compared to IGG-1 and NOV. Whether the unique binding properties of the antibodies produced in guinea pigs will translate in the clinic remains to be addressed, which is the main limitation of this study.ConclusionsThese vaccines induce antibodies that bind α-Syn oligomers and aggregates in the human brain and specifically support the choice of the vaccine generating IGG-3 (i.e. UB-312) as a candidate for clinical trials for synucleinopathies.

Detection of trifluridine in tumors of patients with metastatic colorectal cancer treated with trifluridine/tipiracil.

Trifluridine (FTD) is the active component of the nucleoside chemotherapeutic drug trifluridine/tipiracil (FTD/TPI), which is approved worldwide for the treatment of patients with metastatic gastrointestinal cancer. FTD exerts cytotoxic effects via its incorporation into DNA, but FTD has not been detected in the tumor specimens of patients. The purpose of this study was to detect FTD in tumors resected from metastatic colorectal cancer (mCRC) patients who were administered FTD/TPI. Another purpose was to investigate the turnover rate of FTD in tumors and bone marrow in a mouse model. Tumors and normal tissue specimens were obtained from mCRC patients who were administered FTD/TPI or placebo at Kyushu University Hospital. Tumors and bone marrow were resected from mice with peritoneal dissemination treated with FTD/TPI. To detect and quantitate FTD incorporated into DNA, immunohistochemical staining of paraffin-embedded specimens (IHC-p staining) and slot-blot analysis of DNA purified from these tissues were performed using an anti-BrdU antibody. IHC-p staining of proliferation and apoptosis markers was also performed. FTD was detected in metastatic tumors obtained from mCRC patients who were administered FTD/TPI, but who had discontinued the treatment several weeks before surgery. In a peritoneal dissemination mouse model, FTD was still detected in tumors 13days after the cessation of FTD/TPI treatment, but had disappeared from bone marrow within 6days. These results indicate that FTD persists longer in tumors than in bone marrow, which may cause a sustained antitumor effect with tolerable hematotoxicity.

Monitoring plasma protein aggregation during aging using conformation-specific antibodies and FTIR spectroscopy

The loss of proteostasis during aging has been well described using different models, however little is known with respect to protein aggregation levels in biofluids with aging. Therefore, the aim of this study was to assess the pattern of age-related protein aggregation in human plasma using two distinct approaches: analysis with conformation-specific antibodies and FTIR spectroscopy. The latter has been widely used in biomedical research to study protein conformational changes in health and disease.Samples from a primary care based-cohort from the Aveiro region, Portugal, were used for slot-blot analyses followed by immunodetection with conformation-specific antibodies and for the acquisition of FTIR spectra. Immunoblot analyses revealed an age-dependent evolution of the protein conformational profile in human plasma, towards a decrease in prefibrillar oligomers and an increase in fibrillar structures. This finding was also supported by PLS-R multivariate analysis of FTIR data, where a positive correlation between the age of the donors and secondary structure of plasma proteins could be observed. Samples from younger donors are characterized by antiparallel β-sheet-containing structures while intermolecular β-sheets characterized older samples. Exclusion of age-associated co-morbidities improved the correlation between protein conformational profiles and aging. The results reveal structural changes in human plasma proteins from middle to old age, confirming the age-associated changes in protein aggregation, and support the applicability of FTIR as a reliable approach to study proteostasis during aging.

Differences in the immunoreactivity of milk from local farms and from points of purchase

Cow’s milk proteins belong to food allergens. The composition of milk is influenced by many factors, among others by cows’ feeding. Thus, the aim of the study was to check if introducing legumes seeds (soy meal and ground lupine) into fodder changes the composition of milk immunoreactive protein. 18 milk samples were compared, (UHT milks and samples obtained from local farms). The differences in the total protein content were not statistically significantly different, but the studied milk varied in the content of histidine, serine, aspartic acid, glutamic acid, lysine, phenylalanine, and tyrosine. To control immunoreactivity of milk proteins, extracts in PBS were prepared. Thus, the samples were deprived of most of casein—the main milk allergen. Immunoreactivity of the milk from the local farms and obtained from the cows fed with a fodder containing soy (FS) differed from the immunoreactivity of UHT milk and the milk from the cows fed with a fodder containing soy and lupine (FL) and without the addition of legumes (NL). It was proved by a slot-blot analysis, both with sera of the children allergic solely to milk, as well the children allergic solely to soy (not to milk). SDS-PAGE analysis of the prepared extracts showed the presence of additional peptide bands: ~ 18 kDa in case of the most of immunoreactive milks (FS).

Acrolein Is Involved in the Synergistic Potential of Cigarette Smoking- and Betel Quid Chewing-Related Human Oral Cancer.

Cigarette smoking (CS) and betel quid (BQ) chewing are two known risk factors and have synergistic potential for the development of oral squamous cell carcinoma (OSCC) in Taiwan. The p53 mutation characteristics in OSCC (G to A or G to T mutations) are similar to that of acrolein-induced DNA damage. Acrolein is a major cigarette-related carcinogen that preferentially causes p53 mutations and inhibits DNA repair function in lung cancer. We hypothesize that acrolein is associated with OSCC carcinogenesis. A total of 97 patients with OSCC and 230 healthy subjects with CS and/or BQ chewing histories were recruited. Slot blot analysis of Acr-dG adducts, an indicator of acrolein-induced DNA damage in buccal DNA, and LC/MS-MS analysis of 3-HPMA levels, urinary Acr metabolites, were performed. Our results showed that the level of Acr-dG adducts in buccal cells was 1.4-fold higher in patients with OSCC than in healthy subjects with CS and/or BQ chewing histories (P < 0.001). In addition, in healthy subjects, CS and BQ chewing were associated with significantly higher levels of 3-HPMA, indicating that CS and BQ chewing promotes acrolein absorption. However, 3-HPMA levels in patients with OSCC were significantly lower than those in healthy subjects, indicating impaired acrolein metabolism. In this study, we provide a novel mechanism by which increased acrolein uptake and impaired metabolism may contribute to the synergistic potential of CS and BQ-induced OSCC. Elevated acrolein-induced DNA damage (Acr-dG adducts) detected in buccal swabs may serve as an early indicator to identify patients at risk of developing OSCC.

Fucosyltransferase gene expression in goat endometrium during the estrous cycle and early pregnancy

Regulation of the expression of the alpha(1,2)fucosyltransferase (FUT) genes and their enzymatic products, including the H-type 1 antigen (HT1), on the luminal surface of the uterus is believed to be critical for establishment of pregnancy in mammals. The FUT1 gene is a marker for conception rates in dairy cows and HT1 is a marker for uterine receptivity in rodents. To determine the spatiotemporal expression patterns of FUT1 and FUT2 genes in goats, endometrial tissues were obtained on six days spanning the estrous cycle (Days 5, 11, 13, 15, 17 and 19) and seven days spanning early pregnancy (Days 5, 11, 13, 15, 17, 19 and 25). In all data, we found no effect of status (cyclic or pregnant; P > 0.1) and pooled data where appropriate. We cloned FUT1 cDNA from goat endometrium and made probes from it for Northern and slot blot analyses. The analyses indicated that FUT1 gene expression was high until Day 13, and then declined. In situ hybridization revealed a change in the cell-specificity of FUT1 gene expression over the estrous cycle and early pregnancy. In situ hybridization signal intensity scores indicated that FUT1 expression by uterine epithelium was high on Day 5, moderate on Day 11, and minimal on subsequent days. In situ hybridization signals in uterine glandular epithelial cells remained high from Day 5 to Day 13, with weaker signals thereafter. Quantitative reverse transcription-PCR (RT-qPCR) assays were used for quantitation of FUT1 and FUT2 mRNAs. Quantitative RT-qPCR data were generated from endometrium collected from cyclic and pregnant animals on Days 5, 11 and 17. Relative levels of FUT1 mRNA were high on Days 5 and 11, but then fell 5-fold by Day 17 (P < 0.01). FUT2 mRNA concentrations were below the accurate detectable limit of the assay. High levels of HT1 were observed on the apical surface of uterine luminal epithelia on Days 5, 15, 17 and 19, with much lower levels on Days 11 and 13. Thus, data suggests that FUT1 is the primary enzyme responsible for the high levels of HT1 antigen present on the uterine luminal epithelium between Days 5 and 11 of the estrous cycle and early pregnancy. But changes in the expression of the FUT1 gene does not directly correlate to HT1 staining, which increased from Day 13–15. Future studies are required to understand the regulation of the HT1 antigen on the luminal surface of endometrium.

High Levels of Soluble Triggering Receptor Expressed on Myeloid Cells-Like Transcript (TLT)-1 Are Associated With Acute Respiratory Distress Syndrome.

We have previously demonstrated that elevated levels of soluble triggering receptor expressed on myeloid cells–like transcript 1 (sTLT-1) modulate sepsis-induced inflammation and positively correlate with disseminated intravascular coagulation (DIC). Here, we evaluate the clinical implications of plasma sTLT-1 in acute respiratory distress syndrome (ARDS), which is common in sepsis patients. Soluble TLT-1 levels in the plasma of ARDS patients (n = 20) were determined by slot blot analysis and were compared with clinical parameters to identify significant associations. For comparisons to ARDS, we also measured sTLT-1 levels in matched healthy controls (n = 20). Of the 20 plasma samples evaluated from patients with ARDS, 60% were diagnosed with sepsis and 40% were diagnosed with septic shock. The white blood cells (WBCs) of patients with ARDS were found to be significantly elevated over healthy controls with a mean of 13 k/µL over 6.2 k/µL, respectively. The mean plasma levels of sTLT-1 were 148.4 pg/mL ± 16.52 in the patient cohort and 92.45 pg/mL ± 17.12 in the control group (P = .02). No statistically significant correlations were found between plasma levels of sTLT-1 and WBCs, sepsis, septic shock or acute physiologic, and chronic health evaluation II scores. A statistically significant inverse correlation (r2 = .25, P < .05) was found between plasma sTLT-1 and peripheral platelet counts in patients with ARDS. Increased levels of sTLT-1 in ARDS patients suggest that TLT-1 may mediate the pathobiology of ARDS. Moreover, our data are the first to demonstrate a specific platelet marker in the development of ARDS due to sepsis.

HIV-1 tat expression and sulphamethoxazole hydroxylamine mediated oxidative stress alter the disulfide proteome in Jurkat T cells

BackgroundAdverse drug reactions (ADRs) are a significant problem for HIV patients, with the risk of developing ADRs increasing as the infection progresses to AIDS. However, the pathophysiology underlying ADRs remains unknown. Sulphamethoxazole (SMX) via its active metabolite SMX-hydroxlyamine, when used prophylactically for pneumocystis pneumonia in HIV-positive individuals, is responsible for a high incidence of ADRs. We previously demonstrated that the HIV infection and, more specifically, that the HIV-1 Tat protein can exacerbate SMX-HA-mediated ADRs. In the current study, Jurkat T cell lines expressing Tat and its deletion mutants were used to determine the effect of Tat on the thiol proteome in the presence and absence of SMX-HA revealing drug-dependent changes in the disulfide proteome in HIV infected cells.Protein lysates from HIV infected Jurkat T cells and Jurkat T cells stably transfected with HIV Tat and Tat deletion mutants were subjected to quantitative slot blot analysis, western blot analysis and redox 2 dimensional (2D) gel electrophoresis to analyze the effects of SMX-HA on the thiol proteome.ResultsRedox 2D gel electrophoresis demonstrated that untreated, Tat-expressing cells contain a number of proteins with oxidized thiols. The most prominent of these protein thiols was identified as peroxiredoxin. The untreated, Tat-expressing cell lines had lower levels of peroxiredoxin compared to the parental Jurkat E6.1 T cell line. Conversely, incubation with SMX-HA led to a 2- to 3-fold increase in thiol protein oxidation as well as a significant reduction in the level of peroxiredoxin in all the cell lines, particularly in the Tat-expressing cell lines.ConclusionSMX-HA is an oxidant capable of inducing the oxidation of reactive protein cysteine thiols, the majority of which formed intermolecular protein bonds. The HIV Tat-expressing cell lines showed greater levels of oxidative stress than the Jurkat E6.1 cell line when treated with SMX-HA. Therefore, the combination of HIV Tat and SMX-HA appears to alter the activity of cellular proteins required for redox homeostasis and thereby accentuate the cytopathic effects associated with HIV infection of T cells that sets the stage for the initiation of an ADR.

Impact of β‐adrenergic receptors on UV‐induced skin damage, inflammation and photoprotective effect of carvedilol

Our previous studies demonstrated that the β‐blocker drug carvedilol prevents ultraviolet (UV)‐induced skin cancer in mice. However, how carvedilol interferes with UV‐induced skin carcinogenesis is unknown. Since carvedilol is a β‐adrenergic receptor subtype nonselective antagonist, we hypothesized that carvedilol prevents skin cancer via binding to the β‐adrenergic receptors and inhibiting the adrenergic signaling. The present study has two aims: (1) to elucidate the role of the β‐adrenergic receptors (Adrb1 and 2) on UV‐induced skin damages and inflammation, which are the initial hallmarks driving skin carcinogenesis; and (2) to examine whether the preventive effects of carvedilol against UV‐induced detrimental events is dependent on the β‐adrenergic receptors. We utilized Adrb1/2 double knockout mice (Adrb1tm1BkkAdrb2tm1Bkk/J) on a mixed background (129S1/Sv*129X1/SvJ*C57BL/6J*DBA/2*FVB/N) and wild‐type mice on C57BL/6 background, exposed these mice to single or multiple doses of UV (300 mJ/cm2), and examined parameters relevant to skin damages and inflammation. Physical parameters measured were skin thickness and Sunburn Index. Molecular parameters were expression of COX2 and pro‐inflammatory cytokines as well as cyclobutane pyrimidine dimers (CPD). 10 uM Carvedilol dissolved in acetone was applied immediately after each UV irradiation. In the single dose UV study, in wild‐type mice, UV irradiation increased skin thickness by an average of 4.5% relative to control; in knock‐out mice, UV irradiation increased skin thickness by 3.5% relative to control. In wild‐type mice, UV increased the skin mRNA expression of IL‐1β and IL‐6 by 20 folds. However, interestingly, the increase of IL‐1β and IL‐6 was not observed in knock‐out mice. Similarly, in wild‐type mice, UV increased the skin protein expression of COX‐2 and total P53 both by 2 folds; however, the increase of COX‐2 and P53 was not observed in knock‐out mice. Slot blot analysis indicated UV irradiation greatly induced CPD formation in both wild‐type and knock‐out mice to the same degree. However, different from the results we obtained from SKH‐1 hairless mice, in both wild‐type and knock‐out mice, carvedilol showed modest effect on UV‐induced COX‐2, IL‐1β, IL‐6 and CPD formation. Since the knockout mice may be less sensitive to the UV, we repeated the experiment by exposing mice to four higher doses of UV (400 mJ/cm2) and mice received topical treatment of carvedilol or acetone immediately after each UV dose. In the multiple UV study, in both wild‐type and knock‐out mice, UV strongly increased skin thickness, Sunburn Index, expression of IL‐1β and IL‐6. Carvedilol treatment improved Sunburn Index in both wild‐type and knock‐out mice but did not show benefit for other parameters. Therefore, we conclude that the observed difference between wild‐type and knock‐out mice may be partly due to inter‐strain difference in sensitivity to UV‐induced skin inflammation. We cannot exclude the possibility that Adrb1/2 plays a role in UV‐induced skin events. Adrb1/2 may be critical factor for regulating UV‐induced apoptosis and inflammation. The effects of carvedilol on these events, although modest, did not show difference between the wild‐type and knock‐out mice, indicating β‐adrenergic receptor independent. Future studies should repeat these experiments using wild‐type and knock‐out mice on congenic background.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

Comparison of Gene Expression Between Two Types of Anti-EGFRvIII ScFv Antibodies Having Different Variable Domain Orders in &lt;i&gt;Escherichia coli&lt;/i&gt;

Several studies reported that the expression of various kinds of Single-chain variable fragment (scFv) antibodies in Escherichia coli are significantly influenced by the order of their variable domains. To date, the effect of the order of variable domains in the expression of scFv antibodies against epidermal growth factor receptor variant III (EGFRvIII) has not been reported. This study aimed to compare the expression between VH-linker-VL and VL-linker-VH domain orders of the anti-EGFRvIII scFv antibodies in E. coli expression system. Recombinant plasmids inserted with DNA encoding scFv proteins were transformed into E. coli NiCo21(DE3) competent cells and characterized by colony PCR. The expression of scFv proteins was done by using optimum concentration of inducer. Total proteins, soluble periplasmic and cytoplasmic proteins, also extracellular proteins were isolated, subsequently characterized by SDS-PAGE, Slot Blot, and ImageJ software analyses. The antigen-binding activity of both scFvs proteins against EGFRvIII was observed. The results showed that the relative percentage of scFv expression with VH-linker-VL domain order is higher than that of VL-linker-VH in each compartment. Moreover, both of scFvs proteins have antigen-binding activity against EGFRvIII.

Redox Proteomic Profiling of Specifically Carbonylated Proteins in the Serum of Triple Transgenic Alzheimer's Disease Mice.

Oxidative stress is a key event in the onset and progression of neurodegenerative diseases, including Alzheimer’s disease (AD). To investigate the role of oxidative stress in AD and to search for potential biomarkers in peripheral blood, serums were collected in this study from the 3-, 6-, and 12-month-old triple transgenic AD mice (3×Tg-AD mice) and the age- and sex-matched non-transgenic (non-Tg) littermates. The serum oxidized proteins were quantified by slot-blot analysis and enzyme-linked immunosorbent assay (ELISA) to investigate the total levels of serum protein carbonyl groups. Western blotting, in conjunction with two-dimensional gel electrophoresis (2D-Oxyblot), was employed to identify and quantify the specifically-carbonylated proteins in the serum of 3×Tg-AD mice. The results showed that the levels of serum protein carbonyls were increased in the three month old 3×Tg-AD mice compared with the non-Tg control mice, whereas no significant differences were observed in the six and 12 months old AD mice, suggesting that oxidative stress is an early event in AD progression. With the application of 2D-Oxyblot analysis, (immunoglobin) Ig gamma-2B chain C region (IGH-3), Ig lambda-2 chain C region (IGLC2), Ig kappa chain C region (IGKC), and Ig kappa chain V-V region HP R16.7 were identified as significantly oxidized proteins compared with the control. Among them IGH-3 and IGKC were validated via immunoprecipitation and Western blot analysis. Identification of oxidized proteins in the serums of 3×Tg-AD mice can not only reveal potential roles of those proteins in the pathogenesis of AD but also provide potential biomarkers of AD at the early stage.