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Objective To investigate the effect of Myriocin on high fat diet induced integrated stress response and further explore the mechanism. Methods A total of 18 ApoE-/-mice were fed a high⁃fat diet and randomly divided into control group (n=9, phosphate buffer solution) and Myriocin group (n=9, phosphate buffer solution+Myriocin). The drugs were administered orally for 12 weeks. Serum lipids [total cholesterol (TC), triglyceride (TG), low density lipoprotein⁃cholesterol (LDL⁃C), very low density lipoprotein⁃ cholesterol (VLDL⁃C) and high density lipoprotein⁃cholesterol (HDL⁃C)] were measured. Flow⁃cytometric analysis was used to determine the proportion of lymphocyte antigen 6 complex(Ly ⁃ 6c)high phenotype monocytes. HE staining was performed to compare the size and detailed composition of atherosclerotic plaques and immunofluorescence staining was used to observe the expression of monocyte chemotactic protein⁃1 (MCP⁃1). Real⁃time fluorescence quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression levels of inflammation related molecules [including pro⁃inflammatory factors, interleukin⁃1β and 6 (IL⁃1β and IL⁃6), tumor necrosis factor⁃α (TNF⁃α), intercellular adhesion molecule⁃1 (ICAM⁃1), vascular cell adhesion molecule⁃1 (VCAM⁃1),vascular endothelial growth factor (VEGF) and anti⁃inflammatory factor, IL⁃10].In addition, the mRNA expression levels of integrated stress response related molecules [including glucose regulated protein 78 (GRP78), protein kinase R⁃like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2 α (eIF2 α), activating transcription factor 4 and 6 (ATF4 and ATF6), endoplasmic reticulum stress ⁃ related apoptosis protein C/EBP homologous protein (CHOP) and Caspase12] were tested. The expression levels of integrated stress response related protein [including eIF2α, ATF4, inositol⁃requiring enzyme 1α (IRE1α) and phosphorylated IRE1α, p65 nuclear factor⁃κB (p65 NF⁃κB) and phosphorylated p65 NF⁃κ B, Caspase12 and cleaved Caspase12] were explored by Western blotting. Results 1) Treatment with Myriocin led to lower level of serum LDL⁃C (t=2.830, P=0.012). 2) Myriocin suppressed monocytes differentiating toward a Ly ⁃ 6chigh phenotype (t=2.866, P=0.011).3) HE staining showed less atherosclerotic lesions at 200 and 300 μm distance away from the aortic valve (t=2.281, P=0.045; t=3.506, P=0.003) and less necrotic core areas (Z=⁃2.870, P=0.004) in the Myriocin group. 4) Immunofluorescence staining showed the reduction of MCP⁃1 protein expression in the Myriocin group; real⁃time fluorescence quantitative PCR showed that IL⁃1β mRNA (t=3.968, P=0.005),TNFα mRNA (t=7.696, P=0.000), ICAM mRNA (t=3.294, P=0.013), VCAM mRNA (t=5.449, P=0.001)and VEGF mRNA (t=2.574, P=0.037) were generally decreased in the Myriocin group, while IL⁃10 mRNAwas increased (t=⁃3.132, P=0.017) in the Myriocin group. 5) Myriocin downregulated PERK mRNA (t=4.174, P=0.004）, eIF2α mRNA (Z=⁃2.692, P=0.007), ATF4 mRNA (t=3.342, P=0.012), ATF6 mRNA(t=5.841, P=0.001) and Caspase12 mRNA (t=7.270, P=0.000). 6) Western blotting showed that Myriocin suppressed the protein expression of eIF2α (t=2.175, P=0.047) and ATF4 (t=2.923, P=0.011),and the phosphorylation of p65 NF⁃ κ B (t=2.909, P= 0.011). Conclusions Myriocin could alleviate atherosclerosis progression of ApoE-/- mice by reducing integrated stress response and inflammatory response in the arterial walls. DOI:10.3969/j.issn.1672⁃6731.2020.10.003

Introduction. The diagnostic efficacy of methods for hepatitis E serodiagnostic varies over a wide range; therefore, the combined use of tests of various formats is recommended. The aim of the research was to develop a test system for the detection of IgG antibodies to hepatitis E virus (HEV) in human serum by linear immunoassay (LIA). Material and methods. Serum samples from patients with hepatitis and healthy individuals were tested using commercial enzyme-linked immunosorbent assay systems for the presence of IgG antibodies to viral agents causing hepatitis and other infections associated with liver pathology. Recombinant antigens ORF2 and ORF3 of HEV genotypes 1 and 3 were used. The “RecomLine HEV IgG/IgM” reagent kit (Mikrogen GmbH, Germany) was used as a comparison test system. Results. The first Russian diagnostic kit “Blot-HEV”, designed to detect IgG antibodies to individual HEV proteins in human serum using LIA, was developed. The antigenic base is represented by strips of a nitrocellulose membrane with immobilized recombinant antigens ORF2 (aa 406–660) and ORF3 (aa 1–113) of HEV genotypes 1 and 3, and control antigens in the form of discrete lines. The conjugate was mouse monoclonal antibodies to human class G immunoglobulins labeled with horseradish peroxidase. The chromogen solution contained the 3,3’,5,5’-tetramethylbenzidine. A visual and digital recording of results was provided. The analytical sensitivity of the test kit was 0.625 IU/ml for ORF2 antigens and 2.5 IU/ml for ORF3 antigens. The absence of the influence of endogenous interfering substances on the results of the analysis and the absence of cross-reactions with antibodies to hepatitis pathogens of the other etiologies had been shown. The sensitivity of the test system compared to the “RecomLine HEV IgG/IgM” kit was 92%, specificity 97%. Shelf life in condition of storage was determined to be 12 months. Conclusions. The developed test can be used to confirm the results of ELISA in laboratory diagnosis of hepatitis E.

Abstract Background Line blot immunoassays (LIA) for myositis-specific (MSA) and myositis-associated (MAA) autoantibodies have become commercially available. In the largest study of this kind, we evaluated the clinical performance of a widely used LIA for MSAs and MAAs. Methods Adults tested for MSA/MAA by LIA at a tertiary myositis centre (January 2016–July 2018) were identified. According to expert-defined diagnoses, true and false positive rates were calculated for strongly and weakly positive autoantibody results within three cohorts: idiopathic inflammatory myopathy (IIM), connective tissue disease (CTD) without myositis, and non-CTD/IIM. Factors associated with true positivity were determined. Results We analysed 342 cases. 67 (19.6%) had IIM, in whom 71 autoantibodies were detected (50 strong positives [70.4%], 21 weak positives [29.6%]). Of the strong positives, 48/50 (96.0%; 19 MSAs, 29 MAAs) were deemed true positives. Of the weak positives, 15/21 (71.4%; 3 MSAs, 12 MAAs) were deemed true positives. In CTD without myositis cases (n = 120), 31/61 (51.0%; 5 MSAs, 26 MAAs) autoantibodies were strongly positive, with 24/31 (77.4%; 0 MSAs, 24 MAAs) true positives. 30/61 (49.2%; 13 MSAs, 17 MAAs) were weakly positive, with 16/30 (53.3%; 0 MSAs, 16 MAAs) true positives. In non-CTD/IIM cases (n = 155), all 24 MSAs and 22 MAAs were false positives; these results included 17 (37.0%; 7 MSAs, 10 MAAs) strong positives. Individual autoantibody specificities were > 98.2 and > 97.5% for weakly and strongly positive results, respectively. True positivity was associated with high pre-test for IIM (odds ratio 50.8, 95% CI 13.7–189.2, p

Relevance. During serological diagnosis of rubella by enzyme immunoassay (detecting IgM), it is possible to obtain an indefinite or false-positive result. In this case, is needed an additional confirmatory test. It can be carried out by immune blotting, which allows detecting antibodies to specific rubella antigens. To date, in the Russian Federation, there is no such kit of reagents, which makes its development quite actual. Aim. The aim of this research was the development of a Russian test kit for detecting IgM-antibodies to individual rubella virus antigens by immune blotting (Western blot format). Materials and methods. Production of immunosorbent for a new test kit included electrophoresis of the native rubella virus lysate in a polyacrylamide gel to separate proteins by molecular weight and transfer (blotting) of separated proteins onto a nitrocellulose membrane. Investigation of sensitivity and specificity of the new test kit was carried with standard panels of serums containing and not containing IgM to the rubella virus, and with clinical samples characterized by the ELISA method. Results and its discussion. As a result of this work, was designed a Russian test kit that allowed detection IgM-antibodies to individual rubella virus antigens by immune blotting. This test kit has analytical sensitivity and specificity -100%, diagnostic sensitivity - at least 99.61% and diagnostic specificity - and at least 99-99%. The diagnostic efficiency is not less than 99.5%. Conclusion. The developed test kit has high rates of analytical and diagnostic sensitivity and specificity, as well as high diagnostic efficiency. It is intended for confirmatory research in the diagnosis of rubella infection.

Epstein-Barr virus (EBV) plays the most important role in various types of human cancers. Patients receiving chemotherapy have low white blood cell (WBC) counts that elevate the risk of infection and is the first public health priority in Iraq. WBCs are capable of tumor destruction and aid in combatting infection; they also promote tumor growth, metastasis and chemotherapy resistance. Low WBC count makes the body more predisposed to infection. Our study sought to investigate the relationship between low WBC count and EBV infection in cancer patients. A cross-sectional study was performed using 120 samples obtained from cancer patients, who had a low WBC count and were receiving chemotherapy. The study included samples from 60 males and 60 females aged between 7 and 80 years from Najaf / Iraq. Among the different cancer types, breast cancer cases were high (24 patients; 20%); additionally, a high number of cancer patients (52; 43.3%) also had a low WBC count (≤2 to ≥4 in 109 / L of cells). There were no significant differences in the serological detection of positive cases for viral capsid protein IgM and EBNA-1 and EBNA-2 nuclear genes (P˃0.05) between the cancer patients of different age groups and genders. EBV infection is associated with a low WBC count in cancer patients receiving chemotherapy. No significant differences for patients was observed with respect to age and gender.

Allergen protein is a protein that could triggers allergy reaction. This study purposes to examine the characteristic of allergen protein of unripe, boiled, and fried nila baby fish. Characteristic of allergen protein were observed by electrophoresis and immunoblotting method, while the determination of protein concentration was observed by Bradford method. Identification of the used nila baby fish was accomplished in Faculty of Fishery and Marine Science of IPB. The result of identification showed that the used nila baby fish was Oreochromis niloticus. The proximate analysis of unripe, boiled, and fried nila baby fish resulted water content ranged from 19.16%-23.68%, fat content ranged from 1.03%-20.44% and carbohydrate content ranged from 0.16%-20.27%. Protein concentration of unripe, boiled, and fried nila baby fish extract respectively were 1963.45 mg/L; 699.82 mg/L; 607.79 mg/L. The band amount of allergen protein of unripe, boiled, and fried nila baby fish which was detected by electrophoresis, respectively were 16 of protein band, 26 of protein band and 16 of protein band. The immunoblotting showed that the sum of respondent who contained specific IgE that can bind with allergen protein of boiled and fried nila baby fish were more than allergen protein of unripe nila baby fish. It indicated that the processing process by boiling and frying would increase allergenicity toward nila baby fish.

Abstract Background STAG3 is the meiotic component of cohesin and a member of the Cancer Testis Antigen (CTA) family. This gene has been found to be overexpressed in many types of cancer, and recently, its variants have been implicated in other disorders and many human diseases. Therefore, this study aimed to analyze the major variants of STAG3. Western blot (WB) and immunoprecipitation (IP) assays were performed using two different anti-STAG3 antibodies that targeted the relevant protein in MCF-7, T-47D, MDA-MB-468, and MDA-MB-231 breast cancer cells with Jurkat and MCF-10A cells as positive and negative controls, respectively. In silico analyses were searched to study the major isoforms. Results WB and IP assays revealed two abundant polypeptides

Abstract Background Haemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. Results Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21–103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 μg/well at 37 °C 1 h and overnight at 4 °C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. Conclusion These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

Primary infection during pregnancy by the protozoan Toxoplasma gondii can be worrisome because transmission to the fetus may lead to congenital toxoplasmosis (CT). Neonatal diagnosis is usually performed by serological profile comparison of the mother and newborn. As previously reported in 2012 by C. L’Ollivier et al., three IgM bands at 75, 90 and 100 kDa called the “IgM triplet” has caught our attention and seems to be pathognomonic of CT. This retrospective multicenter study involved nine reference laboratories included in the French National Reference Center for Toxoplasmosis network and concerned determining the specificity and sensitivity of this IgM triplet. On this basis, we were able to propose a new read of the comparison of IgG and IgM immunoblot profiles of mother and infant to increase the sensitivity of this diagnostic marker. The effect of the trimester of pregnancy at the time of infection, but also of maternal treatment with pyrimethamine/sulfadiazine/folinic acid on the presence of this IgM triplet in the infant, could be studied. The presence of the triplet appears pathognomonic for the diagnosis of CT, and it increased the sensitivity of the immunoblot assay from 55.04% to 72.48%. As a result, it would be wise to enhance conventional immunoblot reading by adding the presence of the three IgM bands in the infant pattern for neonatal diagnosis of CT.

Background: Anti-p200 pemphigoid is a rare autoimmune subepidermal blistering disease. Although the phenomenon of epitope spreading has been reported to be common in anti-p200 pemphigoid, the association between its clinical and immunoserological features has yet to be elucidated., Objectives: Our aim was to compare the clinical and immunoserological characteristics of anti-p200 pemphigoid patients with and without epitope spreading., Methods: We performed a retrospective cohort study encompassing 30 patients with anti-p200 pemphigoid between January 2015 and December 2022. The clinical and immunoserological characteristics of anti-p200 pemphigoid were analyzed using combined immunoserological assays., Results: Epitope spreading was observed in 11 of 30 patients (36.7%) with anti-p200 pemphigoid. Compared with patients in the non-epitope spreading group, patients in the epitope spreading group showed more heterogeneous clinical presentations (P = 0.018), a higher proportion of mucosal involvement (P = 0.003), higher Bullous Pemphigoid Disease Area Index (BPDAI) scores for skin erosions/blisters (P = 0.018), mucosal erosions/blisters (P = 0.001), activity (P = 0.017) and total scores (P = 0.022), and required a higher initial dose of prednisone for disease control (P = 0.040)., Conclusions: This study supported the idea that anti-p200 pemphigoid was prone to epitope spreading. Anti-p200 pemphigoid patients with epitope spreading are more likely to present heterogeneous clinical phenotypes, frequent mucosal involvement, and a more severe and recalcitrant disease course., (© 2024 the International Society of Dermatology.)

Objective: Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles., Results: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFβ1, β-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies., (© 2024. The Author(s).)

The adaption of plants to stressful environments depends on long-distance responses in plant organs, which themselves are remote from sites of perception of external stimuli. Jasmonic acid (JA) and its derivatives are known to be involved in plants' adaptation to salinity. However, to our knowledge, the transport of JAs from roots to shoots has not been studied in relation to the responses of shoots to root salt treatment. We detected a salt-induced increase in the content of JAs in the roots, xylem sap, and leaves of pea plants related to changes in transpiration. Similarities between the localization of JA and lipid transfer proteins (LTPs) around vascular tissues were detected with immunohistochemistry, while immunoblotting revealed the presence of LTPs in the xylem sap of pea plants and its increase with salinity. Furthermore, we compared the effects of exogenous MeJA and salt treatment on the accumulation of JAs in leaves and their impact on transpiration. Our results indicate that salt-induced changes in JA concentrations in roots and xylem sap are the source of accumulation of these hormones in leaves leading to associated changes in transpiration. Furthermore, they suggest the possible involvement of LTPs in the loading/unloading of JAs into/from the xylem and its xylem transport.

Background: Isoelectric focusing (IEF) is a method with an exquisite resolution, and coupled with affinity immunoblotting (AIB), it can provide superior sensitivity to detect monoclonal free light chains (FLC)., Methods: We tested the hypothesis that IEF/AIB is more sensitive and specific for monoclonal FLC detection in serum and urine samples than conventional methods, that is, electrophoresis (ELP), immunofixation (IF) and serum FLC ratio assessment. Investigation included 107 samples of 68 patients, among which 21 multiple myeloma patients were recently tested for minimal residual disease and 18 patients with AL amyloidosis., Results: Monoclonal FLC were detected by IEF/AIB in 37% of serum samples negative for monoclonal FLC on ELP/IF. As for urine samples, significant advantage of the IEF/AIB over ELP/IF was not demonstrated. Considering both serum and urine results, IEF/AIB definitely revealed monoclonal FLC in 20/83 (24%) of ELP/IF-negative samples. FLC ratio was abnormally high (>1.65) in all 11 patients definitely positive for monoclonal FLC kappa by IEF/AIB but also in 16/47 (34%) IEF/AIB-negative samples. Abnormally low values (<0.26) were found only in 10/28 samples (36%) positive for monoclonal FLC lambda. Appropriate use of renal FLC ratio reference range reduced the number of presumably false positives (6/47, i.e. 13%) but not false negatives (17/28, i.e. 61%)., Conclusions: The IEF/AIB method is more sensitive than IF and might be used in patients with negative IF results before deciding whether to proceed to minimal residual disease testing., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Literature reports suggest that the presence of proteins in pomegranate seeds is responsible for sensitization and IgE-mediated allergic reactions. The objective of this study was the analysis of a pomegranate seed extract and the isolation and characterization of proteins contained in high amounts. The extract characterization showed a protein profile with main bands at about 18 kDa and below 10 kDa upon SDS-PAGE, and molecules were recognized by specific IgEs upon immunoblotting. Then, two new 2S albumins, a monomeric and a heterodimeric one, were isolated by using classical biochemical methods. They were identified via direct protein sequencing and mass spectrometry, and their primary structure was analyzed and compared with homologous allergenic proteins via bioinformatics. In an Italian population of 703 suspected allergic patients, analyzed by using the FABER ® test, the frequency of sensitization to the monomeric and heterodimeric 2S albumins was 1.7% and 0.28%, respectively. This study reports for the first time the isolation and characterization of two 2S albumins from pomegranate seeds. The clinical relevance of these molecules needs further investigation, for instance in populations having different exposures and allergy profiles.

The article discusses the association between cancer and systemic sclerosis (SSc), focusing on the role of specific serum autoantibodies. It confirms previous findings regarding the predictive value of certain autoantibodies for cancer risk in SSc patients and highlights new observations regarding the timing of cancer onset and the influence of concurrent autoantibodies.

Background: Recently, we published an article retrospectively summarizing the results in 55 anti-laminin 332 (LM332)-type mucous membrane pemphigoid (MMP) cases examined at Kurume University, which were diagnosed by strict inclusion criteria, including positive reactivity in direct immunofluorescence and absence of antibodies to non-LM332 autoantigens. However, indirect immunofluorescence using 1M-NaCl-split normal human skin (ssIIF) is also valuable for diagnosis of anti-LM332-type MMP. Methods: In this second study, we selected 133 anti-LM332-type MMP cases, which were diagnosed by our different inclusion criteria: (i) immunoglobulin G (IgG) deposition to basement membrane zone (BMZ) by direct immunofluorescence or IgG reactivity with dermal side of split skin by ssIIF, (ii) positivity for at least one of the three subunits of LM332 by immunoblotting of purified human LM332, and (iii) the presence of mucosal lesions. Clinical, histopathological, and immunological findings were summarized and analyzed statistically. Although these cases included the 55 previous cases, the more detailed study for larger scale of patients was conducted for further characterization. Results: Clinically, among the 133 patients, 89% and 43% patients had oral and ocular mucosal lesions, respectively, 71% had cutaneous lesions, and 17% had associated malignancies. Histopathologically, 93% patients showed subepidermal blisters. The sensitivities of ssIIF and direct immunofluorescence are similar but are significantly higher than indirect immunofluorescence using non-split human skin (both p < 0.001). In immunoblotting of purified LM332, patient IgG antibodies most frequently reacted with LMγ2 subunit (58%), followed by LMα3 (49%) and LMβ3 (36%). Thirty-four percent patients recognized additional non-LM332 autoantigens. Statistical analysis revealed that autoantibodies against non-LM332 autoantigens might stimulate the production of anti-LMγ2 antibodies. Conclusions: This retrospective study further characterized in more detail the clinical and immunological features of 133 cases of anti-LM332-type MMP, in which the new diagnostic criteria without positive direct immunofluorescence reactivity were useful for the diagnosis. Higher frequency with anti-LMγ2 antibodies suggested more significant pathogenic role of this subunit. Additional autoantibodies to non-LM332 autoantigens detected in one-third of the patients may contribute to complexity in anti-LM332-type MMP, including the induction of anti-LMγ2 antibodies. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) is an abrupt deterioration of renal function often caused by severe clinical disease such as sepsis, and patients require intensive care. Acute-phase parameters for systemic inflammation are well established and used in routine clinical diagnosis, but no such parameters are known for AKI and inflammation at the local site of tissue damage, namely the nephron. Therefore, we sought to investigate complement factors C3a/C3 in urine and urinary sediment cells. After the development of a C3a/C3-specific mouse monoclonal antibody (3F7E2), urine excretion from ICU sepsis patients was examined by dot blot and immunoblotting. This C3a/C3 ELISA and a C3a ELISA were used to obtain quantitative data over 24 hours for 6 consecutive days. Urine sediment cells were analyzed for topology of expression. Patients with severe infections (n = 85) showed peak levels of C3a/C3 on the second day of ICU treatment. The majority (n = 59) showed C3a/C3 levels above 20 μg/ml at least once in the first 6 days after admission. C3a was detectable on all 6 days. Peak C3a/C3 levels correlated negatively with peak C-reactive protein (CRP) levels. No relationship was found between peak C3a/C3 with peak leukocyte count, age, or AKI stage. Analysis of urine sediment cells identified C3a/C3-producing epithelial cells with reticular staining patterns and cells with large-granular staining. Opsonized bacteria were detected in patients with urinary tract infections. In critically ill sepsis patients with AKI, urinary C3a/C3 inversely correlated with serum CRP. Whether urinary C3a/C3 has a protective function through autophagy, as previously shown for cisplatin exposure, or is a by-product of sepsis caused by pathogenic stimuli to the kidney must remain open in this study. However, our data suggest that C3a/C3 may function as an inverse acute-phase parameter that originates in the kidney and is detectable in urine. [ABSTRACT FROM AUTHOR]

Background: Cystic echinococcosis (CE) affects predominantly young patients in highly endemic areas. Improved serological methods are needed for the follow-up of CE cases, especially given the high rates of post-surgical relapse that require detection as soon as possible. Methods: We designed a study to investigate the value of antigenic proteins extracted from Echinococcus granulosus (E. granulosus) protoscoleces, and of recombinant B2t and 2B2t proteins, for assessing the efficacy of surgical treatment carried out on CE-affected children. This study was performed on 278 plasma samples collected from 59 Tunisian children surgically treated for CE and monitored for 3 years post-surgery. The patients were classified according to post-surgical outcomes into a "non-relapsed" (NRCE) and a "relapsed" (RCE) group. We performed in-house ELISAs to measure anti-B2t and anti-2B2t IgG and immunoblotting for the detection of IgG against SDS-PAGE-resolved E. granulosus protoscoleces-specific antigens. The Wilcoxon test was applied to assess anti-B2t and anti-2B2t IgG levels. We applied the Cochran Q test to compare the distribution of immunoblotting antigenic bands between 1-month and 1-year post-surgery. Results: The probability of being "relapse-free" when a decrease in antibody titers occurred between 1 month and 1 year post-surgery was 81% and 75%, respectively, for anti-B2t and anti-2B2t IgG. We identified five protoscolex protein bands of 20, 26/27, 30, 40 and 46 kDa as highly immunoreactive by immunoblot for both RCE and NRCE patients at 1 month post-surgery, and significantly lower immunoreactivity after 1 year (p < 10–4) for NRCE compared to RCE patients. The proteins at 26/27 and 40 kDa displayed the best performance in predicting the outcome, with an 84% probability of being relapse-free when the reactivity against the 40 kDa antigen, the doublet at 26/27 kDa, or both was absent or disappeared between 1 month and 1 year post-surgery, and a 93% probability of being relapsed when both bands remained reactive or increased in intensity between the two time points. Conclusions: The B2t protein could be useful for the prediction of CE early post-surgical outcomes. The proteins of E. granulosus protoscoleces, especially the doublet P26/27 and P40, could be promising predictive biomarkers for the post-surgical follow-up of CE cases as well. [ABSTRACT FROM AUTHOR]

Alternaria alternata is the most important allergenic fungus, with up to 20% of allergic patients affected. The sensitization profile of patients sensitized to A. alternata and how it changes when treated with immunotherapy is not known. Our objective is to determine the allergen recognition pattern of allergic patients to A. alternata and to study its association to the parameters studied in a clinical trial recently published. Sera of 64 patients from the clinical trial of immunotherapy with native major allergen Alt a 1 were analyzed by immunoblotting; 98. 4% of the patients recognized Alt a 1. The percentage of recognition for Alt a 3, Alt a 4, and/or Alt a 6, Alt a 7, Alt a 8, Alt a 10 and/or Alt a 15 was 1.6%, 21.9%, 12.5%, 12.5%, and 12.5% respectively. Of the 64 patients, 45 (70.3%) only recognized Alt a 1 among the allergens present in the A. alternata extract. Immunotherapy with Alt a 1 desensitizes treated patients, reducing their symptoms and medication consumption through the elimination of Alt a 1 sensitization, which is no longer present in the immunoblotting of some patients. There may be gender differences in the pattern of sensitization to A. alternata allergens, among others. [ABSTRACT FROM AUTHOR]

The novel anti-neoplastic glycopeptide T11TS retards glioma both in in-vitro clinical samples and in-vivo models. This study investigates the correlation between altering the glioma microenvironment with glioma arrest and death. Flow cytometry, immunoblotting, ELISA, and co-immunoprecipitation were employed to investigate glioma cell arrest and death. Results include a decline in phosphorylation of Akt and attenuation of p21 phosphorylation (Thr145,Ser146) and disassociation of p-Akt-Mdm2 and p-Akt-BAD facilitating death by Akt>BAD. T11TS influence phosphorylation patterns in two focal axes Akt>p21 and Akt>Mdm2>p53. The current article provides crucial insight in deciphering the mechanism of T11TS induced glioma cell arrest and death. [ABSTRACT FROM AUTHOR]

Objective(s): Mouse breast cancer cell line 4T1 can accurately mimic the response to immune receptors and targeting therapeutic agents. Combined therapy has emerged as an important strategy with reduced side effects and maximum therapeutic effect. Mocetinostat (MGCD0103) is one of the members of Class I Histone Deacetylase Inhibitors (HDACi) and its mechanism of action has not been defined, yet. Capecitabine (Xeloda) is an antimetabolite and currently is widely utilized to treat a wide range of solid tumors. The aim of this study was to investigate the effects of the capecitabine, mocetinostat and their combined application on the 4T1 cell line. Materials and Methods: The effects of combined administration of mocetinostat and capecitabine on 4T1 cells were investigated by cell viability and migration assays, apoptosis analysis, and Western blotting technique. Results: The concentrations of drugs that give a half-maximal response (IC50) were detected for capecitabine (1700 µM), mocetinostat (3,125 µM), and 50 µM Capecitabine+1,5 µM Mocetinostat for 48 hr. In capecitabine+mocetinostat combine group, we observed that cell migration decreased, DNA fragmentation increased compared to the control group. capecitabine + mocetinostat group induced apoptosis by decreasing Bcl-2, PI3K, Akt, c-myc protein levels, while increasing Bax, Caspase-3, PTEN, cleaved-PARP, Caspase-7, Caspase-9, p53, cleaved-Cas-9 protein levels in 4T1 cells. Conclusion: Capecitabine and mocetinostat played a toxic role through inducing apoptosis on 4T1 cancer cells in a time- and concentration-dependent manner. These results showed that combined therapy with low concentrations were detected to be more effective than that with high-concentration alone drug treatment. [ABSTRACT FROM AUTHOR]

Background: The pemphigoid group of diseases may present clinically and immunologically in a very similar fashion. Indirect immunofluorescence microscopy with readily available salt-split human skin in a BIOCHIP™ helps to classify these conditions as those with either with roof binding or floor binding of immunoreactants. Epidermolysis bullosa acquisita, anti-laminin 332 pemphigoid and anti-p200 pemphigoid show floor binding, while in the most frequent type of pemphigoid disease, bullous pemphigoid, epidermal side staining pattern is seen on salt-split skin Aims: The aim of the study was to detect the target antigens in sub-epidermal bullous diseases.Methods: Forty patients with bullous pemphigoid diagnosed by lesional histopathology and direct immunofluorescence microscopy were re-evaluated by a BIOCHIP™ mosaic containing both tissue substrates and recombinant target antigens. Sera with floor pattern staining on salt-split skin were further evaluated by immunoblotting with dermal extract.Results: Five patients with floor staining had anti-p200 pemphigoid.Limitations: We could not perform serration pattern analysis of direct immunofluorescence in our patients.Conclusion: Histopathology and direct immunofluorescence microscopy cannot differentiate between various entities of pemphigoid diseases. A multivariant approach using a BIOCHIP™ mosaic including salt-split skin followed by immunoblotting with dermal extract helps to identify the target antigen. [ABSTRACT FROM AUTHOR]

Precise classification of periodontal disease has been the objective of concerted efforts and has led to the introduction of new consensus-based and data-driven classifications. The purpose of this study was to characterize the microbiological signatures of a latent class analysis (LCA)–derived periodontal stratification system, the Periodontal Profile Class (PPC) taxonomy. We used demographic, microbial (subgingival biofilm composition), and immunological data (serum IgG antibody levels, obtained with checkerboard immunoblotting technique) for 1,450 adult participants of the Dental Atherosclerosis Risk in Communities (ARIC) study, with already generated PPC classifications. Analyses relied on t tests and generalized linear models with Bonferroni correction. Men and African Americans had higher systemic antibody levels against most microorganisms compared to women and Caucasians (P < 0.05). Healthy individuals (PPC-I) had low levels of biofilm bacteria and serum IgG levels against most periodontal pathogens (P < 0.05). Subjects with mild to moderate disease (PPC-II to PPC-III) showed mild/moderate colonization of multiple biofilm pathogens. Individuals with severe disease (PPC-IV) had moderate/high levels of biofilm pathogens and antibody levels for orange/red complexes. High gingival index individuals (PPC-V) showed moderate/high levels of biofilm Campylobacter rectus and Aggregatibacter actinomycetemcomitans. Biofilm composition in individuals with reduced periodontium (PPC-VI) was similar to health but showed moderate to high antibody responses. Those with severe tooth loss (PPC-VII) had significantly high levels of multiple biofilm pathogens, while the systemic antibody response to these microorganisms was comparable to health. The results support a biologic basis for elevated risk for periodontal disease in men and African Americans. Periodontally healthy individuals showed a low biofilm pathogen and low systemic antibody burden. In the presence of PPC disease, a microbial-host imbalance characterized by higher microbial biofilm colonization and/or systemic IgG responses was identified. These results support the notion that subgroups identified by the PPC system present distinct microbial profiles and may be useful in designing future precise biological treatment interventions. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed. [ABSTRACT FROM AUTHOR]

Background: Fluoxetine (FLX) represents the antidepressant of choice for the management of pediatric mood-related illnesses. Accumulating preclinical evidence suggests that ontogenic FLX exposure leads to deregulated affect-related phenotypes in adulthood. Mood-related symptomatology constitutes a risk-factor for various neurological disorders, including Alzheimer's disease (AD), making it possible for juvenile FLX history to exacerbate the development of neurodegenerative diseases.Objective: Because AD is characterized by the pathological accumulation of hyperphosphorylated tau, which can result from impaired function of protein degradation pathways, such as autophagy and the ubiquitin-proteasome system (UPS), we evaluated the long-term effects of adolescent FLX exposure on these pathways, using mice as a model system.Methods: We subjected C57BL/6 adolescent male mice to FLX (20 mg/kg/day) from postnatal day (PD) 35 to PD49. Twenty-one days after the last FLX injection (i.e., adulthood; PD70), mice were euthanized and, using immunoblotting analysis, we evaluated protein markers of autophagy (Beclin-1, LC3-II, p62) and the UPS (K48-pUb), as well as AD-associated forms of phosphorylated tau, within the hippocampus and prefrontal cortex.Results: Juvenile FLX pre-exposure mediated long-term changes in the expression of protein markers (increased LC3-II and decreased p62) that is consistent with autophagy activation, particularly in the prefrontal cortex. Furthermore, FLX history induced persistent accumulation of AD-associated variants of tau in both the hippocampus and prefrontal cortexConclusion: Adolescent FLX treatment may have enduring effects in the neuronal protein degradation machinery, which could adversely influence clearance of abnormal proteins, potentially predisposing individuals to developing AD in later life. [ABSTRACT FROM AUTHOR]

Background: Current therapy of chronic hepatitis C virus (HCV) with direct-acting antivirals (DAAs) has dramatically improved the sustained virologic response (SVR) of affected patients; however, treatment with DAAs remains expensive, and drug-resistant HCV variants remain a threat. As a result, there is still a need to continue to develop affordable and effective drugs for the treatment of HCV. Previously, we have demonstrated that a crude extract from Artocarpus heterophyllus leaves is a potential anti-HCV candidate. In this study, we have further purified this crude extract, examined which sub-fraction possesses the highest antiviral activity, and then explored its efficacy at different HCV life cycle stages. We also assessed synergistic antiviral effects between the A. heterophyllus extract and commercially available anti-HCV drugs. Methods: We used vacuum liquid chromatography (VLC) and high-performance liquid chromatography (HPLC) to fractionate a dichloromethane extract of A. heterophyllus leaves. We then examined the anti-HCV activity of the fractions using HCV genotype 2a, JFH1a; the antiviral mode of action was determined by exploring adding the treatments at different times. We examined the antiviral effects on the viral entry stage through a virucidal activity test, viral adsorption examination, and pretreatment of cells with the drug. The effects on the post-viral entry stage were determined by the levels of HCV protein expression and HCV RNA expression in infected cells. Results: Through activity guided purification, we identified the sub-fraction FR3T3 as possessing the most robust anti-HCV activity with an IC50 value of 4.7 ± 1.0 μg/mL. Mode-of-action analysis revealed that FR3T3 inhibited post-viral entry stages such as HCV NS3 protein expression and HCV RNA replication with marginal effects on the viral entry stage. Thin-layer Chromatography (TLC) indicated that FR3T3 contained terpenoids and chlorophyll-related compounds. We also found a synergistic antiviral activity when the DCM extract of A. heterohyllus was used in combination therapy with commercial anti-HCV drugs; Ribavirin, Simeprevir, Cyclosporin A. Conclusions: The extract of A. heterophyllus and its sub-fraction, FR3T3, presented here have anti-HCV activities and could be candidate drugs for add-on-therapy for treatment of chronic HCV infections. [ABSTRACT FROM AUTHOR]

Objective: Axial spondyloarthritis (SpA) is a chronic autoinflammatory disease with new bone formation, which is controlled by Wnt/β‐catenin signaling. Dkk‐1 is an inhibitor of the Wnt pathway, and in humans, platelets represent a major source of Dkk‐1. This study was undertaken to investigate whether levels of Dkk‐1 in serum and platelet expression of DKK1 messenger RNA (mRNA) and Dkk‐1 protein are affected in patients with axial SpA compared to healthy controls. Methods: Forty‐one patients with axial SpA and 35 healthy controls were enrolled in the study. Total serum Dkk‐1 levels in all patients and healthy controls were measured by quantitative enzyme‐linked immunosorbent assay. Platelet DKK1 mRNA was analyzed by quantitative reverse transcriptase–polymerase chain reaction in 20 patients with axial SpA and 20 controls, and Dkk‐1 protein levels were measured by immunoblotting in 20 patients with axial SpA and 18 controls. Results: We found a lower concentration of Dkk‐1 in the serum from patients with axial SpA compared to the serum from healthy controls (P < 0.0001). Furthermore, the expression of Dkk‐1 was significantly reduced both at the transcriptional level (P < 0.04) and at the protein level (P < 0.007) in platelets isolated from the blood of patients with axial SpA. Conclusion: Our preliminary observations suggest that dysfunction of the megakaryocyte/platelet axis might be responsible for reduced serum Dkk‐1 levels in patients with axial SpA. Dkk‐1 is down‐regulated in the platelets of patients with axial SpA, a mechanism that might play a role in new bone formation. [ABSTRACT FROM AUTHOR]

With the drastic spread of COVID-19 and mass mortality of people globally, detection of the progression of this disease has stood out to be a necessity. Hence, we set out to identify the prevalence of COVID-19 antibodies in Bangladesh using the in-house rapid pan-immunoglobulin dot-blot test kit and evaluate the performance of this kit. Methods: In this cross-sectional study, we tested serum collected between mid-May and mid-June 2020 for COVID-19 antibodies by using the in-house rapid pan-immunoglobulin dot-blot test kit in RTPCR confirmed patients with symptoms for 1-7 days (Group Ia; n =100) and 8-14 days (Group Ib; n = 100); symptomatic RT-PCR negative patients (Group II; n = 100) and convalescent patients (Group III; n = 109) while comparing with pre-pandemic sera samples collected prior two years to December-2019 (Group IV; n = 100). Results: Our kit detected that almost 70% of the convalescent patients produced antibodies against COVID-19 compared to other groups. However, the group with individuals at the end phase of COVID-19 exhibited the second-highest percentage of seroprevalence (41%). We also observed that though Group II was RT-PCR negative, 20% of them showed COVID-19 antibodies. Conclusion: With a specificity of 96% in our kit, we can say that our kit will be a potential device for the detection of SARS-CoV-2 antibodies and to understand herd immunity in Bangladesh. [ABSTRACT FROM AUTHOR]

Objective: Autoimmune diseases occur as a result of the immune response to self antigens and tissues of the organism and the detection of autoantibodies is very important in the diagnosis of these disorders. Antinuclear antibodies (ANA) autoantibodies generated against to nuclear/cytoplasmic components of the cell are important diagnostic criteria for connective tissue diseases. Currently, a number of methods are available for the detection of ANA. The gold standard method for the detection of ANA is Indirect Immunofluorescence Antibody (IIFA) assay using Hep2 (human laryngeal epidermoid carcinoma). When positive results are observed, Extractable Nuclear Antigen(ENA) tests follow as a means of confirming the diagnosis. Identification of the specific extractable nuclear antigens is warranted because this may further differentiate between the distinct types of autoimmune connective tissue diseases. In this study, were analyzed retrospectively that ENA test results in patients with positive ANA IIFA test. Methods: Antinuclear antibodies were tested for a total of 3000 patients admitted to various clinics of Ondokuz Mayıs University Medical Faculty. Each serum sample was studied at 1: 100 dilution in accordance with the manufacturer's recommendations (Euroimmun AG, Lübeck, Germany) and staining pattern with fluorescence intensity was evaluated with immunofluorescence microscope. ENA was investigated by immunoblotting method (Euroimmun AG, Lübeck, Germany) in 640 ANA positive sera. Results: Distribution of the patients included in the study, 2192 (73.07%) were female and 808 (26.93%) were male. 640 samples detected as ANA positive. When we look at the distribution of ANA patterns in positive samples; granular 173 (27.03%), granular and cytoplasmic granular 98 (15.31%), homogenous and granular 67 (10.47%) were the most common. When the ENA profiles of the ANApositive samples were examined, 557 (83.7%) were found to be positive and 83 (12.97%) were negative. According to our study, SSA (26.88%), SSB (17.81%), Sm / RNP (17.66%) were the first three places in the ENA positivite. Conclusion: Our ANA positivity rate was found to be compatible with the literature. The pattern distribution is similar to the data of our region. After the first screening with IIFA, looking for different antigens with the immunoblot test; not only it is a cost effective approach;but also facilitate the diagnosis of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Objective: Sterol regulatory element binding protein 1c (SREBP1c) is one of the major transcription factors that is involved in nonalcoholic fatty liver disease (NAFLD) development by increasing hepatic fatty acid and triglyceride synthesis. Our study aimed to investigate the interaction of SREBP1c with endoplasmic reticulum (ER) stress in oleic acid (OA) induced lipid accumulation. Material and Methods: Optimum lipid droplet (LD) formation and SREBP-1c induction were determined in alpha mouse liver 12 (AML12) hepatocytes following the incubation with different OA concentrations. To determine the effect of SREBP-1c, cells were transfected with siRNA specific for SREBP-1c. LD formation and SREBP-1c induction were determined via Oil Red O and immunblotting, respectively. Phospho IRE1, GRP78, CHOP, ATF6 and JNK levels were determined with immunofluorescence staining. Results: Optimum LD formation and SREBP-1c induction were achieved at 0.5 mM oleat concentration. While SREBP-1c silencing decreased LD formation in non-OA treated cells, no significant effect of silencing was determined following OA administration. On the other hand, SREBP-1c silencing in OA treated cells reduced phospho IRE1, ATF6, JNK and CHOP expressions. Conclusion: Our results suggest that the novel function of SREBP-1c can regulate ER stress response in OA induced lipid accumulation. [ABSTRACT FROM AUTHOR]

Interleukin 2 (IL-2) treatment is associated with proteinuria. Materials and Methods: A conditionally immortalized human podocyte cell line was used to investigate expression of the podocyte specific marker podocin, IL-2R alpha (IL-2Rα), apoptosis marker Bax, and autophagy markers LC3I AND LC3II, determined by quantitative immunoblotting, following 24, 48, and 72 hours of IL-2 stimulation, comparing them to unstimulated cells. Results: Podocin was expressed at all time points. IL-2Rα expression was increased after 24 and 72 hrs (p = 0.0014, p = 0.0139) and decreased after 48 hours (p = 0.0445). Bax, LC3I, and LC3II were increased after 24 hrs (p = 0.0094, p = 0.0016, p = 0.0004) and 48 hrs (p = 0.0072, p = 0.0024, p = 0.0087). Conclusion: Human podocytes express the IL-2R and activation results in increased autophagy and apoptosis. [ABSTRACT FROM AUTHOR]

Objective To investigate the value of multiplex bead-based flow fluorescent immunoassay (MBFFI) in the diagnosis of primary biliary cholangitis (PBC) by analyzing its results in detecting antimitochondrial antibody M2 subtype (AMA-M2), gp210 antibody, and sp100 antibody. Methods A total of 340 patients who attended The Affiliated Hospital of Qingdao University from August 2018 to June 2020 and were diagnosed with PBC were enrolled as PBC group, and 143 patients with other diseases and 117 individuals undergoing physical examination were also enrolled. MBFFI and immunoblotting test (IBT) were used to detect AMA-M2, gp210, and sp100 autoantibodies in serum, and the Kappa-test was used to compare the consistency of the two methods in detecting the same autoantibody; the receiver operating characteristic (ROC) curve was used to evaluate the value of MBFFI detection of three antibodies in the diagnosis of PBC; the chi-square test was used to compare positive. Results In the PBC group, the two methods showed the best consistency in detecting AMA-M2(Kappa=0.874) and showed relatively good consistency in detecting gp210 and sp100 antibodies (Kappa=0.713 and 0.749). MBFFI had the highest sensitivity of 72.06% in detecting AMA-M2; it had a sensitivity of 44.71% in the combined detection of gp210 and sp100 antibodies, which was higher than its sensitivity in detecting each antibody alone; it had a sensitivity of 82.65% in the combined detection of AMA-M2, gp210, and sp100 antibodies, which was higher than its sensitivity in detecting each antibody alone. Combined detection of the three antibodies had the largest area under the ROC curve of 0.907 0 in the diagnosis of PBC. Conclusion MBFFI has good consistency with IBT in detecting autoantibodies associated with PBC, and the combined detection of AMA-M2, gp210, and sp100 antibodies by MBFFI has higher sensitivity and value in the diagnosis of PBC. [ABSTRACT FROM AUTHOR]

Fast, cheap, and simple separation of lipids and hydrocarbons can currently be achieved using thin‐layer chromatography. Here, we describe an alternative planar chromatographic method using polyvinylidene difluoride membranes as the stationary phase. The procedure has the same advantages of thin‐layer chromatography over other expensive and time‐consuming techniques, such as high‐performance liquid chromatography or gas chromatography. Polyvinylidene difluoride membranes, however, also provide an immediate support for analyte development via immunodetection, are easy to manipulate, and potentially increase the performance of other detection methods. We show that polyvinylidene difluoride membranes are compatible with a variety of solvents that can migrate by capillarity and redistribute analytes between the membrane and the solvent according to their relative affinities, providing a chromatographic separation. We directly test the developed membranes by immunoblotting using anti‐squalene antibodies that cross‐react with acyclic isoprenoids. Separations of crude oils and plant extracts under different solvent conditions show the potential to resolve hydrocarbon group types and also to provide characteristic fingerprints of plant pigments and squalene degradation products. Polyvinylidene difluoride membranes prove useful as a stationary phase for planar chromatography and for the subsequent immunodetection of the separated compounds, providing a new and simple chromatographic technique to analyze lipids and hydrocarbons. [ABSTRACT FROM AUTHOR]

The identification of proteins that are altered following nicotine/tobacco exposure can facilitate and positively impact the investigation of related diseases. In this report, we investigated the effects of chronic (–)‐menthol exposure in 14 murine brain regions for changes in total β2 subunit protein levels and changes in epibatidine binding levels using immunoblotting and radioligand binding assays. We identified the habenula as a region of interest due to the region's marked decreases in β2 subunit and nAChR levels in response to chronic (–)‐menthol alone. Thus, we further examined the habenula, a brain region associated with both the reward and withdrawal components of addiction, for additional protein level alterations using mass spectrometry. A total of 552 proteins with altered levels were identified after chronic (–)‐menthol exposure. Enriched in the proteins with altered levels after (–)‐menthol exposure were proteins associated with signaling, immune systems, RNA regulation, and protein transport. The continuation and expansion of the brain region‐specific protein profiling in response to (–)‐menthol will provide a better understanding of how this common flavorant in tobacco and e‐liquid products may affect addiction and general health. [ABSTRACT FROM AUTHOR]

Objective: Non‐small‐cell lung cancer (NSCLC) is one of the most common fatal cancers in the world. Although the treatment of NSCLC has been significantly improved, there is still an unmet need to identify novel targets for developing therapeutic agents and diagnostic/prognostic markers. The aim of this study is explore the role and underlying mechanism of the epithelial splicing regulatory protein (ESRP1) in the development and progression of NSCLC. Methods: A total of 115 participants, 65 cases of NSCLC, 20 cases of precancerous lesions, and 30 cases of benign lung nodules, were included in this study. The expressions of ESRP1 and related transcription factor Twist in enrolled lung tissues were evaluated by histochemistry and immunohistochemistry assay. The survival analysis and related prognosis factors were evaluated by the Kaplan–Meier curve and Cox regression. In addition, the expression of ESRP1 and epithelial‐mesenchymal transition (EMT)related transcription factor Twist and EMT markers E‐cadherin and N‐cadherin were ascertained by immunohistochemical and immunoblotting assay on A549 lung adenocarcinoma cell lines that were exposed to transforming growth factor β1 (TGFβ1). Results: Compared with normal lung tissues, the abundance of ESRP1 protein was significantly increased in precancerous lesions and lung cancer. Correlation analysis demonstrated that ESRP1 was an independent prognostic factor in NSCLC. The expression of ESRP1 and Twist was positively correlated in lung tissues (r = 0.285, p < 0.001). In vitro analysis further showed that TGFβ1 could upregulate the expression of EMT transcription factor Twist while downregulating ESRP1. Conclusions: Our data suggest that the aberrant expression of ESRP1 is an early event in the development of NSCLC. The ESRP1 could serve as a prognostic biomarker for NSCLC, particularly when combined with Twist. The Twist negatively regulated the expression of ESRP1, emphasizing the role of the TGFβ/ESRP1 pathway in the development of NSCLC, which warrants further investigation. [ABSTRACT FROM AUTHOR]

Purpose: Lifelong exercise is known to attenuate sarcopenia (age-associated reduction in muscle mass and function); however, the underlying molecular mechanisms remain unclear. As microRNAs are widely involved in the regulation of skeletal muscle growth and development, we aimed to evaluate the effects of lifelong regular exercise on age-related alterations in muscle microRNA expression profiles as well as on skeletal muscle atrophy, apoptosis, and mitochondria and autophagy dysfunction. Methods: Female 8-month-old Sprague-Dawley rats were divided into four groups; 1) 18 months of moderate-intensity continuous training (MICT) initiated at 8 months (adult-MICT, n = 12), 2) 8 months of MICT initiated at 18 months (presarcopenia-MICT, n = 12), 3) 8-month-old adult sedentary controls (adult-SED), and 4) 26-month-old aging sedentary controls (old- SED). Age skeletal muscles were then subjected to quantitative reverse transcription-polymerase chain reaction, Kyoto Encyclopedia of Genes and Genomes, immunoblotting, and miR-486 3' untranslated region luciferase reporter gene analyses. Results: Age-related loss of miR-486 expression was improved, skeletal muscle atrophy and apoptosis were downregulated, and mitochondrial activity and autophagy were upregulated in the adult-MICT group. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the PI3K/Akt pathway was upregulated in adult-MICT rats compared with that in old-SED. In vitro analyses in rat skeletal muscle L6 cells further confirmed that miR-486 targets PTEN, not SAV1, thereby activating the PI3K/Akt pathway and indirectly inhibiting HIPPO signaling. Conclusions: Compared with presarcopenia-MICT rats, adult-MICT rats experienced greater beneficial effects regarding ameliorated age-related alterations in muscle miRNA expression profile, skeletal muscle atrophy, apoptosis, and mitochondria and autophagy dysfunction, which is potentially associated with the increased miR-486 expression and concomitant targeting of the PTEN/Akt signaling pathway. [ABSTRACT FROM AUTHOR]

Current antiepileptic strategies aim to normalize the interaction of the excitatory and inhibitory systems, which is ineffective in treating patients with drug-resistant epilepsy. Neuroinflammatory processes in the epileptic focus and its perifocal area can trigger apoptosis and also contribute to the development of drug resistance. The level of pro- and anti-apoptotic proteins (p-NF-kB, TNF-α, p53, FAS, caspase-3, caspase-9) was analyzed in intraoperative biopsies of the temporal lobe gray and white matter in the brain of patients with drug-resistant epilepsy. An increased level of pro-apoptotic proteins was revealed in the cortex and perifocal area's white matter against the background of an imbalance of protective anti-apoptotic proteins. It appears that the activation of the extrinsic pathway of apoptosis occurs in the perifocal area, while in the epileptic focus, there are proteins responsible for the activation of the anti-apoptotic survival pathways. Active neuroinflammation in the epileptic focus and perifocal area of the temporal lobe may contribute to the development of the resistance to antiepileptic drugs and the progression of neurodegeneration in such patients. [ABSTRACT FROM AUTHOR]

Fasciolosis, caused by liver fluke species of the genus Fasciola, are well recognized because of its high veterinary impact. Stool examination for Fasciola eggs is not a sensitive method, and limited efforts to find a reliable and cheaper means of detection are available. The present study aimed to develop rapid diagnostic ELISA test against fasciolosis. The excretory/secretory (ES) and somatic (SA) products of Fasciola helminths were analyzed using polyacrylamide gel electrophoresis (PAGE). Immunogenicity was evaluated by immunoblotting using hyperimmune sera raised in rabbits and seroprevalence was determined by indirect ELISA. The results of SA antigen of Fasciola species showed polypeptide bands ranged from 10kDa-100kDa, while ES antigen of Fasciola showed bands of 15kDa-55kDa. The immunoblotting results showed the most prominent bands against ES antibodies were 25, 35, 55-70, 100 and 250 kDa and SA antigens showed 10, 15-25, 35, 70, 100 and 250 kDa polypeptide bands. The sensitivity and specificity of developed indirect ELISA for SA antigens was 95.45% and 87.1%, while for ES antigens was 100% and 77.42% respectively. The overall seroprevalence recorded for fascioliasis based on SA antigen was 39.8% and 29.8% for ES antigen. The fasciolosis did not show significant association with host type, sex, and age groups of examined animals, however significantly higher infection was found in months of September and October. The result provides sensitive in house immunodetection assay for diagnosis of fasciolosis alternative to commercial kits with high import cost. [ABSTRACT FROM AUTHOR]

Objective: To study the mechanism of the anti-tumor effect of Morinda citrifolia (noni). Methods: The influences of noni juice on cell proliferation, apoptosis, invasion, migration and the activity of AKT/nuclear factor- κ B (NF- κ B) signaling pathway in A549 human lung cancer cells were detected by MTT, cell counting kit-8, colony formation, Annexin V/PI double labeling, transwell, scratch test and immunoblotting assay, respectively. A549 cells were inoculated into the right axilla of nude mice, followed by noni juice treatment. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell proliferation and expression of apoptosis-related proteins were measured by immunohistochemistry, and the activity of NF- κ B signaling pathway was measured by immunoblotting. Results: The in vitro studies showed that noni juice inhibited the A549 cells proliferation, migration and invasion. Noni juice also promoted cells apoptosis in A549 cells. Immunoblotting assay showed that the phosphorylation level of AKT, p50, and STAT3 proteins was inhibited to different extents after noni juice treatment. The in vivo studies showed that noni juice effectively suppressed tumor formation of A549 cells in nude mice. Noni juice treatment inhibited the expression of Ki67, PCNA, and Bcl-2 protein in the tumor; while promoted the expression of caspase-3 protein. Additionally, we also found that noni juice treatment could restrain the activity of AKT/NF- κ B signaling pathway in the tumor tissue. Conclusion: Noni juice inhibited the proliferation of A549 lung cancer cells, induced apoptosis, and inhibited cell invasion and migration via regulating AKT/NF- κ B signaling pathway. [ABSTRACT FROM AUTHOR]

Objective: To examine the role of carvacrol in modulating PI3K/AKT signaling involved in human breast cancer pathogenesis using in vitro experimental model MCF-7 cells. Methods: MTT and lactate dehydrogenase assays were performed with cells treated with different doses of carvacrol (0-250 p mol/L) at different time points (24 and 48 h). The nuclear morphology was assessed in MCF-7 cells with propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) staining and analyzed by fluorescence microscopy. Events like cell cycle arrest, apoptosis was observed by flow cytometric analysis and expressions of p-Rb, cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6, Bax, Bcl-2, PI3K/p-AKT was analyzed by immunoblot. Results: Carvacrol significantly reduced cell viability with the half maximal inhibitory concentration value of 200 µmol/L at 24 and 48 h (P<0.05). importantly, there was a significant increase in the accumulation of the G0/G1 phase upon treatment with carvacrol in MCF-7 cells (P<0.05 or P<0.01). A remarkable decrease in protein expressions of p-Rb, cyclin D1, CDK4 and CDK6 denotes cell cycle arrest (P<0.05 or P<0.01). In addition, carvacrol treatment significantly inhibited PI3K/p-AKT protein expressions leading to induction of apoptosis mediated by decreased Bcl2 and increased Bax protein expressions. Further, Annexin V/PI staining by FACS analysis, dual staining by AO/EB and PI staining studies suggests induction of apoptosis by carvacrol through PI3K/Akt signaling pathway in MCF-7 cells. Conclusion: Carvacrol significantly inhibited the breast cancer MCF-7 cell proliferation and induced apoptosis via suppressing PI3/AKT signaling pathway. [ABSTRACT FROM AUTHOR]

Due to their immunosuppressive properties, mesenchymal stem cells (MSC) have been evaluated for the treatment of immunological diseases. However, the animal-derived growth supplements utilized for MSC manufacturing may lead to clinical complications. Characterization of alternative media formulations is imperative for MSC therapeutic application. Human BMMSC and AdMSC were expanded in media supplemented with either human platelet lysates (HPL), serum-free media/xeno-free FDA-approved culture medium (SFM/XF), or fetal bovine serum (FBS) and the effects on their properties were investigated. The immunophenotype of resting and IFN-γ primed BMMSC and AdMSC remained unaltered in all media. Both HPL and SFM/XF increased the proliferation of BMMSC and AdMSC. Expansion of BMMSC and AdMSC in HPL increased their differentiation, compared to SFM/XF and FBS. Resting BMMSC and AdMSC, expanded in FBS or SFM/XF, demonstrated potent immunosuppressive properties in both non-primed and IFN-γ primed conditions, whereas HPL-expanded MSC exhibited diminished immunosuppressive properties. Finally, IFN-γ primed BMMSC and AdMSC expanded in SFM/XF and HPL expressed attenuated levels of IDO-1 compared to FBS. Herein, we provide strong evidence supporting the use of the FDA-approved SFM/XF medium, in contrast to the HPL medium, for the expansion of MSC towards therapeutic applications.

Pancreatic acinar cells possess very high protein synthetic rates as they need to produce and secrete large amounts of digestive enzymes. Acinar cell damage and dysfunction cause malnutrition and pancreatitis, and inflammation of the exocrine pancreas that promotes development of pancreatic ductal adenocarcinoma (PDAC), a deadly pancreatic neoplasm. The cellular and molecular mechanisms that maintain acinar cell function and whose dysregulation can lead to tissue damage and chronic pancreatitis are poorly understood. It was suggested that autophagy, the principal cellular degradative pathway, is impaired in pancreatitis, but it is unknown whether impaired autophagy is a cause or a consequence of pancreatitis. To address this question, we generated Atg7(Δpan) mice that lack the essential autophagy-related protein 7 (ATG7) in pancreatic epithelial cells. Atg7(Δpan) mice exhibit severe acinar cell degeneration, leading to pancreatic inflammation and extensive fibrosis. Whereas ATG7 loss leads to the expected decrease in autophagic flux, it also results in endoplasmic reticulum (ER) stress, accumulation of dysfunctional mitochondria, oxidative stress, activation of AMPK, and a marked decrease in protein synthetic capacity that is accompanied by loss of rough ER. Atg7(Δpan) mice also exhibit spontaneous activation of regenerative mechanisms that initiate acinar-to-ductal metaplasia (ADM), a process that replaces damaged acinar cells with duct-like structures.

Although a variety of genetic alterations have been found across cancer types, the identification and functional characterization of candidate driver genetic lesions in an individual patient and their translation into clinically actionable strategies remain major hurdles. Here, we use whole genome sequencing of a prostate cancer tumor, computational analyses, and experimental validation to identify and predict novel oncogenic activity arising from a point mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor protein. We demonstrate that this mutation (p.A126G) produces an enzymatic gain-of-function in PTEN, shifting its function from a phosphoinositide (PI) 3-phosphatase to a phosphoinositide (PI) 5-phosphatase. Using cellular assays, we demonstrate that this gain-of-function activity shifts cellular phosphoinositide levels, hyperactivates the PI3K/Akt cell proliferation pathway, and exhibits increased cell migration beyond canonical PTEN loss-of-function mutants. These findings suggest that mutationally modified PTEN can actively contribute to well-defined hallmarks of cancer. Lastly, we demonstrate that these effects can be substantially mitigated through chemical PI3K inhibitors. These results demonstrate a new dysfunction paradigm for PTEN cancer biology and suggest a potential framework for the translation of genomic data into actionable clinical strategies for targeted patient therapy.

The efficiency of gene expression in all organisms depends on the nucleotide composition of the coding region. GC content and codon usage are the two key sequence features known to influence gene expression, but the underlying molecular mechanisms are not entirely clear. Here we have determined the relative contributions of GC content and codon usage to the efficiency of nuclear gene expression in the unicellular green alga Chlamydomonas reinhardtii. By comparing gene variants that encode an identical amino acid sequence but differ in their GC content and/or codon usage, we show that codon usage is the key factor determining translational efficiency and, surprisingly, also mRNA stability. By contrast, unfavorable GC content affects gene expression at the level of the chromatin structure by triggering heterochromatinization. We further show that mutant algal strains that permit high-level transgene expression are less susceptible to epigenetic transgene suppression and do not establish a repressive chromatin structure at the transgenic locus. Our data disentangle the relationship between GC content and codon usage, and suggest simple strategies to overcome the transgene expression problem in Chlamydomonas.

RationaleEndothelial cells have the ability to undergo endothelial-mesenchymal transitions (EndMTs), by which they acquire a mesenchymal phenotype and stem cell-like characteristics. We previously found that EndMTs occurred in the endothelium deficient in matrix γ-carboxyglutamic acid protein enabling endothelial cells to contribute cells to vascular calcification. However, the mechanism responsible for initiating EndMTs is not fully understood.ObjectiveTo determine the role of specific serine proteases and sex determining region Y-box 2 (Sox2) in the initiation of EndMTs.Methods and resultsIn this study, we used in vivo and in vitro models of vascular calcification to demonstrate that serine proteases and Sox2 are essential for the initiation of EndMTs in matrix γ-carboxyglutamic acid protein-deficient endothelium. We showed that expression of a group of specific serine proteases was highly induced in endothelial cells at sites of vascular calcification in Mgp null aortas. Treatment with serine protease inhibitors decreased both stem cell marker expression and vascular calcification. In human aortic endothelial cells, this group of serine proteases also induced EndMTs, and the activation of proteases was mediated by Sox2. Knockdown of the serine proteases or Sox2 diminished EndMTs and calcification. Endothelial-specific deletion of Sox2 decreased expression of stem cell markers and aortic calcification in matrix γ-carboxyglutamic acid protein-deficient mice.ConclusionsOur results suggest that Sox2-mediated activation of specific serine proteases is essential for initiating EndMTs, and thus, may provide new therapeutic targets for treating vascular calcification.

Nav channels are essential for metazoan membrane depolarization, and Nav channel dysfunction is directly linked with epilepsy, ataxia, pain, arrhythmia, myotonia, and irritable bowel syndrome. Human Nav channelopathies are primarily caused by variants that directly affect Nav channel permeability or gating. However, a new class of human Nav channelopathies has emerged based on channel variants that alter regulation by intracellular signaling or cytoskeletal proteins. Fibroblast growth factor homologous factors (FHFs) are a family of intracellular signaling proteins linked with Nav channel regulation in neurons and myocytes. However, to date, there is surprisingly little evidence linking Nav channel gene variants with FHFs and human disease. Here, we provide, to our knowledge, the first evidence that mutations in SCN5A (encodes primary cardiac Nav channel Nav1.5) that alter FHF binding result in human cardiovascular disease. We describe a five*generation kindred with a history of atrial and ventricular arrhythmias, cardiac arrest, and sudden cardiac death. Affected family members harbor a novel SCN5A variant resulting in p.H1849R. p.H1849R is localized in the central binding core on Nav1.5 for FHFs. Consistent with these data, Nav1.5 p.H1849R affected interaction with FHFs. Further, electrophysiological analysis identified Nav1.5 p.H1849R as a gain-of-function for INa by altering steady-state inactivation and slowing the rate of Nav1.5 inactivation. In line with these data and consistent with human cardiac phenotypes, myocytes expressing Nav1.5 p.H1849R displayed prolonged action potential duration and arrhythmogenic afterdepolarizations. Together, these findings identify a previously unexplored mechanism for human Nav channelopathy based on altered Nav1.5 association with FHF proteins.

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

Plant damage promotes the interaction of lipoxygenases (LOXs) with fatty acids yielding 9-hydroperoxides, 13-hydroperoxides, and complex arrays of oxylipins. The action of 13-LOX on linolenic acid enables production of 12-oxo-phytodienoic acid (12-OPDA) and its downstream products, termed "jasmonates." As signals, jasmonates have related yet distinct roles in the regulation of plant resistance against insect and pathogen attack. A similar pathway involving 9-LOX activity on linolenic and linoleic acid leads to the 12-OPDA positional isomer, 10-oxo-11-phytodienoic acid (10-OPDA) and 10-oxo-11-phytoenoic acid (10-OPEA), respectively; however, physiological roles for 9-LOX cyclopentenones have remained unclear. In developing maize (Zea mays) leaves, southern leaf blight (Cochliobolus heterostrophus) infection results in dying necrotic tissue and the localized accumulation of 10-OPEA, 10-OPDA, and a series of related 14- and 12-carbon metabolites, collectively termed "death acids." 10-OPEA accumulation becomes wound inducible within fungal-infected tissues and at physiologically relevant concentrations acts as a phytoalexin by suppressing the growth of fungi and herbivores including Aspergillus flavus, Fusarium verticillioides, and Helicoverpa zea. Unlike previously established maize phytoalexins, 10-OPEA and 10-OPDA display significant phytotoxicity. Both 12-OPDA and 10-OPEA promote the transcription of defense genes encoding glutathione S transferases, cytochrome P450s, and pathogenesis-related proteins. In contrast, 10-OPEA only weakly promotes the accumulation of multiple protease inhibitor transcripts. Consistent with a role in dying tissue, 10-OPEA application promotes cysteine protease activation and cell death, which is inhibited by overexpression of the cysteine protease inhibitor maize cystatin-9. Unlike jasmonates, functions for 10-OPEA and associated death acids are consistent with specialized roles in local defense reactions.

Although oncology therapy regimens commonly include radiation and genotoxic drugs, tumour cells typically develop resistance to these interventions. Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance. CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response. Accordingly, a phospho-mimetic mutant of CRAF (S338D) is sufficient to induce the CRAF/CHK2 association enhancing tumour radioresistance, while an allosteric CRAF inhibitor sensitizes tumour cells to ionizing radiation or genotoxic drugs. Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.