Identification of Plasmid-Free Chlamydia muridarum Organisms Using a Pgp3 Detection-Based Immunofluorescence Assay

2015 ◽  
Vol 25 (10) ◽  
pp. 1621-1628 ◽  
Author(s):  
Chaoqun Chen ◽  
Guangming Zhong ◽  
Lin Ren ◽  
Chunxue Lu ◽  
Zhongyu Li ◽  
...  
Keyword(s):  
Immunofluorescence Assay ◽  
Chlamydia Muridarum

scholarly journals Chlamydia Genomic MinD Protein Has not the Function of Regulating Plasmid-dependent Genes as Pgp5

2018 ◽  
Vol 65 (3) ◽  
Author(s):  
Yina Sun ◽  
Jie Kong ◽  
Jingyue Ma ◽  
Manli Qi ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Shuttle Vector ◽  
Immunofluorescence Assay ◽  
Developmental Cycle ◽  
Gene Products ◽  
Similar Function ◽  
Transformation System ◽  
Transcription Level ◽  
Chlamydia Muridarum ◽  
Plaque Purification ◽  
Chlamydial Genome

Chlamydia has a unique intracellular developmental cycle which has hindered the function study of Chlamydia. Transformation system of Chlamydia developed recently has greatly advanced the chlamydial function research and has been used to find chlamydial plasmid-encoded pgp5 can down-regulate plasmid-dependent genes. It is predicted that pgp5 has similar function with MinD protein encoded by chlamydial genome. However，it is unknown whether MinD has the function of regulating these plasmid-dependent genes. The pgp5 gene in the shuttle vector pGFP::CM was replaced with MinD gene of Chlamydia trachomatis (CT0582) or Chlamydia muridarum(TC0871). The recombinant plasmid was transformed into plasmid-free organisms-CMUT3.Real time PCR was used to detect the genes transcription level in these pgp5 replacement organisms. GlgA, one of the plasmid-regulated gene products was detected by the immunofluorescence assay. After recombination, transformation and plaque purification, the stable transformants CMUT3-pGFP::CM CT0582Rpgp5 and CMUT3-pGFP::CM TC0871Rpgp5 were generated. In these transformants, the plasmid-dependent genes were up-regulated, similar with those in the pgp5 premature stop mutant and pgp5 replacement with mCherry mutant. GlgA protein was also increased in all pgp5 mutants including CT0582Rpgp5 and TC0871Rpgp5. Thus, the transformation system has allowed us to identify the function of MinD that is useful for further understanding the chlamydiae. 

Phanginin R Induces Cytoprotective Autophagy via JNK/c-Jun Signaling Pathway in Non-Small Cell Lung Cancer A549 Cells

2020 ◽  
Vol 20 (8) ◽  
pp. 982-988 ◽  
Author(s):  
Le-Le Zhang ◽  
Han Bao ◽  
Yu-Lian Xu ◽  
Xiao-Ming Jiang ◽  
Wei Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Signaling Pathway ◽  
Cell Lung Cancer ◽  
Biological Activities ◽  
A549 Cells ◽  
Immunofluorescence Assay ◽  
Small Cell ◽  
Autophagic Flux ◽  
Small Cell Lung

Background: Cassane-type diterpenoids are widely distributed in the medical plants of genus Caesalpinia. To date, plenty of cassane diterpenoids have been isolated from the genus Caesalpinia, and some of them were documented to exhibit multiple biological activities. However, the effects of these compounds on autophagy have never been reported. Objective: To investigate the effects and mechanisms of the cassane diterpenoids including Phanginin R (PR) on autophagy in Non-Small Cell Lung Cancer (NSCLC) A549 cells. Methods: Western blot analysis and immunofluorescence assay were performed to investigate the effects of the compounds on autophagic flux in A549 cells. The pathway inhibitor and siRNA interference were used to investigate the mechanism of PR. MTT assay was performed to detect cell viability. Results: PR treatment upregulated the expression of phosphatidylethanolamine-modified microtubule-associated protein Light-Chain 3 (LC3-II) in A549 cells. Immunofluorescence assay showed that PR treatment increased the production of red-fluorescent puncta in mRFP-GFP-LC3 plasmid-transfected cells, indicating PR promoted autophagic flux in A549 cells. PR treatment activated the c-Jun N-terminal Kinase (JNK) signaling pathway while it did not affect the classical Akt/mammalian Target of Rapamycin (mTOR) pathway. Pretreatment with the JNK inhibitor SP600125 or siRNA targeting JNK or c-Jun suppressed PR-induced autophagy. In addition, cotreatment with the autophagy inhibitor Chloroquine (CQ) or inhibition of the JNK/c-Jun signaling pathway increased PR-induced cytotoxicity. Conclusion: PR induced cytoprotective autophagy in NSCLC A549 cells via the JNK/c-Jun signaling pathway, and autophagy inhibition could further improve the anti-cancer potential of PR.

scholarly journals Pt(II)-Thiocarbohydrazone Complex as Cytotoxic Agent and Apoptosis Inducer in Caov-3 and HT-29 Cells through the P53 and Caspase-8 Pathways

2021 ◽  
Vol 14 (6) ◽  
pp. 509
Author(s):  
Abeer A. Ibrahim ◽  
Mohanad M. Kareem ◽  
Taghreed H. Al-Noor ◽  
Tahani Al-Muhimeed ◽  
Abeer A. AlObaid ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Immunofluorescence Assay ◽  
Cell Cycle Distribution ◽  
Caspase 8 ◽  
Ovarian Adenocarcinoma ◽  
Fragmentation Test ◽  
Cycle Arrest ◽  
Apoptosis Inducer ◽  
Test Flow ◽  
Ht 29

In this study, a platinum(II) complex ([Pt(H2L)(PPh3)] complex) containing a thiocarbohydrazone as the ligand was tested as an anti-proliferative agent against ovarian adenocarcinoma (Caov-3) and human colorectal adenocarcinoma (HT-29) through MTT assays. Apoptotic markers were tested by the AO/PI double staining assay and DNA fragmentation test. Flow cytometry was conducted to measure cell cycle distribution, while the p53 and caspase-8 pathways were tested via immunofluorescence assay. Results demonstrated that the cytotoxic effect of the Pt(II)-thiocarbohydrazone complexes against Caov-3 and HT-29 cells was highly significant, and this effect triggered the activation of the p53 and caspase-8 pathways. Besides, apoptosis stimulated by the Pt(II)-thiocarbohydrazone complex was associated with cell cycle arrest at the G0/G1 phase. These findings suggest that the target complex inhibited the proliferation of Caov-3 and HT-29 cells, resulting in the arrest of the cell cycle and induction of apoptosis via the stimulation of the p53 and caspase-8 pathways. The present data suggests that the Pt(II)-thiocarbohydrazone complex could also be a promising chemotherapeutic agent for other types of cancer cells.

scholarly journals Rheumatologist perspective of the Brazilian consensus for detection of auto antibodies in HEp-2 CELLS

2021 ◽  
Vol 61 (1) ◽  
Author(s):  
Isadora Carvalho Medeiros Francescantonio ◽  
Leandro Augusto Rodrigues dos Santos ◽  
Paulo Luiz Carvalho Francescantonio ◽  
Luiz Eduardo Coelho Andrade ◽  
Wilson de Melo Cruvinel
Keyword(s):  
Clinical Practice ◽  
Immunofluorescence Assay ◽  
Chi Square ◽  
Dichotomous Data ◽  
Level Of Satisfaction ◽  
Significant Difference ◽  
Chi Square Test ◽  
Geographic Regions ◽  
Relevant Pattern ◽  
Structured Questionnaire

Abstract Objective To evaluate the perception of rheumatologists regarding the recommendations of the Brazilian Consensus for detection of Autoantibodies (BCA) on HEp-2 Cells by Indirect Immunofluorescence assay (IFA) and how BCA recommendations help in clinical practice. Methodology A structured questionnaire regarding the BCA recommendations for detection and interpretations of autoantibodies in HEp-2 cells was applied to randomly selected rheumatologists. The results were tabulated using the Microsoft® Excel program, expressed as a simple percentage and the dichotomous data were analyzed using the Chi-square test and the Epi Info® program. Results Four hundred fuorteen rheumatologists participated in the study: 70% of them considered their knowledge of the HEp-2 IFA test satisfactory or excellent, and 43% said they knew the BCA recommendations in general, without distinguishing the edition of the BCA to which they refer. The Revista Brasileira de Rheumatologia/Advances in Rheumatology was the means of dissemination most consulted by specialists (50%). According to the rheumatologists’ opinion, the most relevant pattern was the homogeneous nuclear (78%) and 65% stated they were satisfied with the BCA recommendations at a level of satisfaction greater than or equal to 80%. There was no significant difference in the perception of rheumatologists from the several Brazilian geographic regions. Conclusion Brazilian rheumatologists are aware of the BCA guidelines and most are satisfied with the content published, considering that the BCA recommendations assist positively in the clinical practice. Most rheumatologists recognize the patterns associated with rheumatic autoimmune diseases and have used BCA recommendations to interpret the results of the HEp-2 IFA test.

scholarly journals Etiology, clinical characteristics and coinfection status of bronchiolitis in Suzhou

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiahong Tan ◽  
Jinfeng Wu ◽  
Wujun Jiang ◽  
Li Huang ◽  
Wei Ji ◽  
...  
Keyword(s):  
Virus Infection ◽  
Clinical Characteristics ◽  
Immunofluorescence Assay ◽  
Clinical Syndrome ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Immunofluorescence ◽  
Increased Risk ◽  
Syncytial Virus ◽  
Direct Immunofluorescence Assay ◽  
Mixed Virus Infection

Abstract Background Bronchiolitis is a clinical syndrome commonly encountered in practice, particularly among infants and young children. To investigate the prevalence of pathogens in hospitalized children with bronchiolitis and study the clinical characteristics of bronchiolitis with or without coinfections. Methods We investigated the respiratory specimens and clinical data of 1012 children with bronchiolitis who were treated at the Children’s Hospital of Soochow University between November 2011 and December 2018. The nasopharyngeal aspirates were examined to detect viruses by direct immunofluorescence assay or polymerase chain reaction (PCR). Mycoplasma pneumoniae (MP) was tested by PCR and enzyme-linked immunosorbent assay. Results Of the 1134 children less than 2 years with bronchiolitis, 122 were excluded by exclusion criteria. Causative pathogen was detected in 83.2% (842 of 1012). The majority of these (614 [72.9%] of 842) were single virus infection. The most common pathogens detected were respiratory syncytial virus (RSV) (44.4%), MP (15.6%), and human rhinovirus (HRV) (14.4%). Coinfection was identified in 13.5% (137 of 1012) of the patients. Coinfection included mixed virus infection and virus infection with MP infection. Children with single virus infection had a higher rate of oxygen therapy compared with single MP infection. Conclusions The most common pathogen detected in children with bronchiolitis is RSV, followed by MP and HRV. Coinfection leads to a longer period of illness, increased severity of the symptoms and increased risk of hypoxemia.

miR-125b enhances metastasis and progression of cancer via the TXNIP and HIF1α pathway in pancreatic cancer

2021 ◽  
pp. 1-12
Author(s):  
Pengli Wang ◽  
Dan Zheng ◽  
Hongyang Qi ◽  
Qi Gao
Keyword(s):  
Pancreatic Cancer ◽  
Hypoxia Inducible Factor ◽  
Immunofluorescence Assay ◽  
Luciferase Assay ◽  
Interacting Protein ◽  
Over Expression ◽  
Thioredoxin Interacting Protein ◽  
Cellular Processes ◽  
And Migration

BACKGROUND: MicroRNAs (miRNAs) play potential role in the development of various types of cancer conditions including pancreatic cancer (PC) targeting several cellular processes. Present study was aimed to evaluate function of miR-125b and the mechanism involved in PC. METHODS: Cell migration, MTT and BrdU study was done to establish the migration capability, cell viability and cell proliferation respectively. Binding sites for miR-125b were recognized by luciferase assay, expression of protein by western blot and immunofluorescence assay. In vivo study was done by BALB/c nude xenograft mice for evaluating the function of miR-125b. RESULTS: The study showed that expression of miR-125b was elevated in PC cells and tissues, and was correlated to proliferation and migration of cells. Also, over-expression of miR-125b encouraged migration, metastasis and proliferation of BxPC-3 cells, the suppression reversed it. We also noticed that thioredoxin-interacting protein (TXNIP) was the potential target of miR-125b. The outcomes also suggested that miR-125b governed the expression of TXNIP inversely via directly attaching to the 3′-UTR activating hypoxia-inducible factor 1α (HIF1α). Looking into the relation between HIF1α and TXNIP, we discovered that TXNIP caused the degradation and export of HIF1α by making a complex with it. CONCLUSION: The miR-125b-TXNIP-HIF1α pathway may serve useful strategy for diagnosing and treating PC.

SAT0323 MYOSITIS-SPECIFIC AND MYOSITIS-ASSOCIATED AUTOANTIBODIES IN A LARGE INDIAN COHORT OF INFLAMMATORY MYOSITIS REVEAL NOVEL CLINICO-PHENOTYPIC PATTERNS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1107.1-1107
Author(s):  
L. Gupta ◽  
P. Gaur ◽  
V. Agarwal ◽  
R. Aggarwal ◽  
R. Misra
Keyword(s):  
Disease Duration ◽  
Unique Feature ◽  
Diagnostic Value ◽  
Immunofluorescence Assay ◽  
Major Influence ◽  
Antibody Prevalence ◽  
Research Support ◽  
Inflammatory Myositis ◽  
Idiopathic Inflammatory Myositis ◽  
Physician Diagnosis

Background:Idiopathic Inflammatory Myositis (IIM) are heterogenous, with distinct autoantibodies reflecting upon possible clinical evolution and outcomes. Ethnicity has major influence on both antibody prevalence patterns as well as phenotypic behaviours linked to them.Objectives:Thus we sought prevalence and co-existence of myositis specific autoantibodies (MSAs) and myositis associated autoantibodies (MAAs) and associated clinical characteristics in a large cohort of patients with IIM.Methods:Adult patients with a physician diagnosis of IIM as per ACR/EULAR classification criteria were investigated for the presence of MSAs/MAAs by Line immunoassay (G4, Euro-Immune, Lubeck, Germany). Anti-Nuclear Antibody (ANA) was tested by Immunofluorescence assay (IFA), and patterns in various antibody subsets explored. Prevalence and associations of different antibodies were assessed in disease subsets and clinical phenotypes.Results:MSA and MAAs were tested in 250 IIM patients (F:M 3.8:1) of median age 37 (25-47) and disease duration 6 (3-17) years. Dermatomyositis (DM) was seen in most patients 83 (33.2%) followed by overlap myositis (OM), juvenile DM, Anti-synthetase syndrome (ASS), polymyositis (PM), and cancer associated myositis (CAM). MSAs/MAAs were found in 148 (59.2%) of patients, of which 95 (64.2%) had an MSA and 53 (35.8%) had MAAs (Fig, 1A). 93 (62.8%) of autoantibody positive patients were positive for a single antibody, and only 2 (0.8%) of total had more than one MSA (Table 1).Table 1.Multiple antibodies positive upon testing for MSA and MAA by the LIANote: ** PL-7 co-exists with Ku + Pm/Scl, **PL-12 co-exists with Pm/Scl + Ro52, **SRP co-exists with Pm/Scl + Ro52The most frequently detected MSA was anti-Jo-1 (8%), with a further 9 specificities each found in 0.5–7.0% of patients. Amongst the autoantibody positive patients, 21% (n=53) had isolated MAA positivity, anti-Ro52 (33, 62.3%) being the most common, followed by anti-Pm/Scl (11, 20.8%) and anti-Ku (9, 17.0%) (Fig. 1B).Figure 1.A. MSA and MAA in Indian cohort of myositis B. ANA patterns in myositis C. MSA in ANA negative IIMOn ANA, 76.0% (172 of 226) were positive, with speckled being the most common pattern (37%,Fig. 1C). Of those ANA negative (n=54), 61% had either MSA or MAA (Fig 1D). 18 (54.6%) had autoantibodies associated with cytoplasmic patterns suggesting that cytoplasmic ANA may be underreported.Clinical presentation akin to DM was seen with all MSA except anti-SRP. PM group was heterogenous, and included ASS, OM and necrotizing phenotype (Fig. 2A). On occasion, anti-SRP, anti-Mi-2 and anti-MDA5 presented with clinical phenotype of ASS. (Fig 2A,C). Patients with ARS or anti-SAE were often clinically amyopathic (Figure 2B,C)Figure 2.A. Phenotypic associated with various antibody subsets B,C,and D. MSA/MAA in muscle weakness, rash and ILD phenotype. E. Unique feature of eye-lid edema in some patients with MDA-5 positive myositisARS were associated with mechanic’s hand (p<0.0001,OR 7.6), ILD (p<0.0001,OR 4.4), and arthritis (p=.002,OR 2.6) though there was no difference between Jo-1 and non-Jo-1 ASS. Anti-MDA-5 associated with fever (p=0.003,OR 12) and weight loss (p=0.008,OR 10.2) and unique phenotype of eye-lid edema in some adults (Figure 2E) and arthritis in children (p=0.01, OR 11.5). Anti-TIF-1ɣ associated with alopecia (p=0.007,OR 5.9) and malignancy (p= <0.0001,OR 34) in adults but not children.Conclusion:Myositis autoantibodies are seen in two-thirds IIM and identify distinct clinical subsets as well as unique phenotypes. MSA/MAA are positive in two-thirds of those negative on ANA, adding diagnostic value. MSAs are nearly always mutually exclusive and thus useful as biomarkers for diagnosis.Acknowledgments:MSA testing supported by grants from APLAR and Association of Physicians of India.Disclosure of Interests:Latika Gupta: None declared, Priyanka Gaur: None declared, Vikas Agarwal: None declared, Rohit Aggarwal Grant/research support from: Pfizer, Genentech, BMS, Mallinckrodt, Consultant of: Pfizer, Genentech, BMS, Mallinckrodt, Bristol Myers-Squibb, octapharma, CSL Behring, AstraZeneca, Corbus, Kezar, Abbvie, Ramnath Misra: None declared

scholarly journals A rapid point-of-care assay accurately measures vitamin D

2021 ◽  
Author(s):  
K. Albrecht ◽  
J. Lotz ◽  
L. Frommer ◽  
K. J. Lackner ◽  
G. J. Kahaly
Keyword(s):  
Vitamin D ◽  
Metabolic Pathways ◽  
Point Of Care ◽  
Immunofluorescence Assay ◽  
Laboratory Method ◽  
Controlled Study ◽  
Mean Deviation ◽  
Serum Samples ◽  
Assay Validation ◽  
Accuracy And Precision

Abstract Purpose Vitamin D (VitD) is a pleiotropic hormone with effects on a multitude of systems and metabolic pathways. Consequently, the relevance of a sufficiently high VitD serum level becomes self-evident. Methods A rapid immunofluorescence assay designed for the point-of-care measurement of serum VitD3 solely was tested. Inter- and intra-assay validation, double testing and result comparison with a standardized laboratory method were performed. Results An overall linear correlation of r = 0.89 (Pearson, 95% CI 0.88–0.92, p < 0.01) between the point of care and the conventional reference assay was registered. Accuracy and precision were of special interest at cut-points (10 ng/ml [mean deviation 1.7 ng/ml, SD 1.98 ng/ml, SE 0.16 ng/ml], 12 ng/ml [MD 0.41, SD 1.89, SE 0.19] and 30 ng/ml [MD − 1.11, SD 3.89, SE 0.35]). Only a slight deviation was detected between the two assays when using fresh (r = 0.91, 95% CI 0.86–0.94, p < 0.01) and frozen serum samples (r = 0.86, 0.82–0.89, p < 0.01). Results remained steady when samples were frozen several times. Inter- and intra-assay validation according to the CLSI protocol as well as multiuser testing showed stable results. Conclusion This novel, innovative, and controlled study indicates that the evaluated rapid point of care VitD assay is reliable, accurate, and suited for clinical practice.

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