Miracle-dz/UniFAR-FTUs huggingface dataset:
In vitro emergence of rifampicin resistance in Propionibacterium acnes and molecular characterization of mutations in the rpoB gene.

OBJECTIVES Activity of rifampicin against Propionibacterium acnes biofilms was recently demonstrated, but rifampicin resistance has not yet been described in this organism. We investigated the in vitro emergence of rifampicin resistance in P. acnes and characterized its molecular background. METHODS P. acnes ATCC 11827 was used (MIC 0.007 mg/L). The mutation rate was determined by inoculation of 10(9) cfu of P. acnes on rifampicin-containing agar plates incubated anaerobically for 7 days. Progressive emergence of resistance was studied by serial exposure to increasing concentrations of rifampicin in 72 h cycles using a low (10(6) cfu/mL) and high (10(8) cfu/mL) inoculum. The stability of resistance was determined after three subcultures of rifampicin-resistant isolates on rifampicin-free agar. For resistant mutants, the whole rpoB gene was amplified, sequenced and compared with a P. acnes reference sequence (NC006085). RESULTS P. acnes growth was observed on rifampicin-containing plates with mutation rates of 2 ± 1 cfu × 10(-9) (4096× MIC) and 12 ± 5 cfu × 10(-9) (4 × MIC). High-level rifampicin resistance emerged progressively after 4 (high inoculum) and 13 (low inoculum) cycles. In rifampicin-resistant isolates, the MIC remained >32 mg/L after three subcultures. Mutations were detected in clusters I (amino acids 418-444) and II (amino acids 471-486) of the rpoB gene after sequence alignment with a Staphylococcus aureus reference sequence (CAA45512). The five following substitutions were found: His-437 → Tyr, Ser-442 → Leu, Leu-444 → Ser, Ile-483 → Val and Ser-485 → Leu. CONCLUSION The rifampicin MIC increased from highly susceptible to highly resistant values. The resistance remained stable and was associated with mutations in the rpoB gene. To our knowledge, this is the first report of the emergence of rifampicin resistance in P. acnes.
Toxicant transport by P-glycoprotein.

Toxicant Transport by P-Glycoprotein (Lisa J. Bain, University of Texas at El Paso, El Paso, Texas). This unit describes several different methods of measuring active transport of toxicants by P-glycoprotein uptake of a radiolabeled toxicant, uptake of an unlabeled toxicant with detection by HPLC or GC, uptake of radiolabeled toxicant in membrane vesicles; movement of radiolabeled toxicant from the basal to apical compartment using polarized epithelial cells grown in Transwell culture. P-glygoprotein is an important protein for limiting entry into the brain through the blood-bain barrier. It also alters the rates of uptake and elimination.
Examination of the in vitro fungicidal activity of echinocandins against Candida lusitaniae by time-killing methods.

OBJECTIVES Candida lusitaniae fungaemia, although infrequent (1%), is more common in immunocompromised patients than Candida albicans. Although infections produced by Candida spp. are therapeutic targets for treatment with echinocandins, little information is available regarding their killing kinetics against C. lusitaniae. The objectives of this study were to determine the killing kinetics of anidulafungin, micafungin and caspofungin against four blood isolates of C. lusitaniae by time-kill methodology. METHODS Time-kill studies were performed in RMPI 1640 medium (5 mL, inoculum ∼10(5) cfu/mL). The number of cfu/mL was determined at 0, 2, 4, 6 and 24 h. The anidulafungin concentrations assayed were 0.03, 0.12, 0.5, 2 and 8 mg/L, while micafungin and caspofungin concentrations were 0.25, 1, 4, 16 and 32 mg/L. RESULTS MIC ranges were 0.03-1 mg/L (anidulafungin), 0.016-0.06 mg/L (micafungin) and 0.03-1 mg/L (caspofungin). The mean maximum log decrease in cfu/mL was reached with 2 mg/L anidulafungin (1.85 ± 0.4 log), 32 mg/L caspofungin (5.5 ± 0.2 log) and 32 mg/L micafungin (2.65 ± 1.9 log). Only caspofungin and micafungin reached the fungicidal endpoint (99.9% growth reduction or a 3 log decrease) with 32 mg/L at 22.8 h (caspofungin) and 26.5 h (micafungin). Analysis of variance showed significant differences in killing activity among isolates, but not among concentrations reached in serum or echinocandins. CONCLUSIONS Anidulafungin and micafungin exhibit greater killing rates than caspofungin. Caspofungin was the only echinocandin that reached the fungicidal endpoint before 24 h, but at drug concentrations (≥ 16 mg/L) not usually reached in serum. The echinocandin killing rate was isolate dependent and concentration independent.
A Novel Hand-Held Optical Imager with Real-Time Co-Registration Facilities Towards Diagnostic Mammography

Abstract : Hand-held based optical imaging devices using near-infrared (NIR) light are currently developed toward clinical translation of the technology. However, none of the NIR devices developed to date have attempted 3D tomography since they are not able to coregister the image to the tissue geometry. The objective for the work described herein is the clinical translation of a hand-held optical imager with automated coregistration facilities toward 3D tomography. Studies were performed in vivo with normal human subjects to demonstrate fast (near real-time) 2D fluorescence imaging for target detection prior to 3D tomography. The results showed that 0.23 cm3 and 0.45 cu cm fluorescent targets placed behind the breast tissue were detected through ~2.5 cm deep tissue. Parallely, studies were performed on phantoms composed of minced chicken breast and 1% Liposyn solution to demonstrate coregistered imaging in vitro. The results showed that the 3D tracking system was able to track the position of the probe in real-time and accurately coregister the image to the geometry of the object. Additionally, deeper targets can be detected upon summation of multiple coregistered images. These results demonstrate the potential of the device to perform 3D tomographic imaging in human subjects via coregistered imaging on complex breast geometries.
Detection of Biliverdin Reductase Activity

The conversion of biliverdin to the bile pigment bilirubin is catalyzed by biliverdin reductase, a cytosolic enzyme with two pH optima with different cofactors. The enzyme is assayed at pH 8.7 with NADPH as a cofactor and at 6.75 with NADH. The production of bilirubin is detected as described in this unit with a spectrophotometric assay. The enzyme can be qualitatively assayed by immunoblotting and changes in reductase isoform expression can be detected by two‐dimensional electrophoresis.
Methods for Distinguishing Nitrosative and Oxidative Chemistry of Reactive Nitrogen Oxide Species Derived from Nitric Oxide

NO‐derived intermediates formed under aerobic conditions may engage in complex chemical reactions with biologically important molecules. The outcomes of these reactions and their ultimate effect on biological systems depend on the selectivity of the species and the concentrations of different substances present and whether the reaction takes place in the gas or aqueous phase. In this unit conversion of two different compounds to fluorescent products is used to distinguish between oxidative and nitrosative chemistry of different reactive nitrogen oxide species.
Methods to Assess Neuroinflammation

Neuroinflammation is observed in many brain pathologies: in neurodegenerative diseases and multiple sclerosis as well as in chemically induced lesions. It is characterized by the reactivity of microglial cells and astrocytes, activation of inducible NO‐synthase (i‐NOS), and increased expression and/or release of cytokines and chemokines. Clearly, cell‐to‐cell signaling between the different brain cell types plays an important role in the initiation and propagation of neuroinflammation, but despite the growing list of known molecular actors, the underlying pathways and the sequence of events remain to be fully elucidated. The present chapter presents an example of how to assess neuroinflammation in complex brain tissues, using aggregating brain cell cultures as an in vitro model. This three‐dimensional cell culture system provides optimal cell‐to‐cell interactions crucial for histotypic cellular maturation and control of neuroinflammatory processes. The techniques described here comprise immunocytochemistry to assess the reactivity of microglia and astrocytes and the expression of cytokines; quantitative RT‐PCR to measure the mRNA expression of cytokines (TNF‐α, IL‐1β, IL‐6, IL‐1ra, TGF‐β, IL‐15, IFN‐γ), chemokines (ccl5, cxcl1, cxcl2), and i‐NOS; and immunoblotting to assess MAP kinase pathway activation (phosphorylation of p38 and p44/42 MAP kinases). Curr. Protoc. Toxicol. 50:12.19. 1‐12.19. 20. © 2011 by John Wiley & Sons, Inc.
Internally displaced people: a global survey

Some 25 million persons across the world today are counted as internally displaced. Because they have not crossed an international border, they are not protected by the international regulations which concern refugees. They are people whose states are unable or unwilling to see that their needs are recognized andmet. This book indicates the scope of the problem and the responses to it in the year 2002. The Global IDP Project data base (www.idpproject.org) provides updates and additional information. What is the significance of the problem? In numbers it is relatively large: 25 million is twice the number of refugees — five times as many as in the 1970s. In terms of governance, internal displacement indicates unsolved fundamental problems. A seven-page chapter on the Global IDP Project’s training programme near the beginning of the book, and the fundamental 10 pages ofGuiding principles at the end give the reader an idea of the strategies and principles that exist for dealing with the problems which are so starkly documented in the regional profiles. But the real aim of this publication is to convince us of the enormity of the problem, and in this it succeeds all too well. Focusing on 48 countries, the survey looks at four factors which lead to displacement: armed conflict, generalized violence, the systematic violation of human rights, and forced displacement as a military or political objective. In country after country, variations on these same themes are reported. The difficulty of a response from humanitarian agencies to the needs of the displaced is cited again and again. The website www.globalproject. org, besides giving updates on particular countries, contains reports on a training module used with government and nongovernmental organizations. There are also guides to these modules, written jointly by the Norwegian Refugee Council and the United Nations High Commissioner for Refugees. The report entitled ‘‘Recipients as resources — a community based approach’’ should be required reading for anyone developing programmes for (or, more correctly, with) displaced populations. The Global IDP Project team hopes that its information services will help to improve the nowvery inadequate and still ad hoc response to internal displacement. In 1992 Dr Francis M. Deng was appointed representative to the UN Secretary-General on internally displaced persons. The Global IDP Project began in 1998 with its first global survey, and established an internet data base in 1999. That year the United Nations adopted the UN guiding principles on internal displacement, drafted at the request of the UN High Commission on Human Rights. These guiding principles, though not legally binding, represent a consensus on how countries and agencies ought to act in relation to these populations. In 2002, the UN created an IDP unit in the UN Office for the Coordination of Humanitarian Affairs. n
Association of GRACE Risk Score with Angiographic Severity of Coronary Artery Disease in patients with ST Elevation Myocardial Infarction

Background: Clinical guidelines recommend that optimal management of acute coronary syndrome should include patient risk stratification. Predicting the anatomical extension of coronary artery disease is also potentially useful for clinical decision. The objective of our study is to determine whether the GRACE risk score correlates with the angiographic extent and severity of coronary artery disease in patients with ST elevation myocardial infarction. Methodology: 50 patients diagnosed with Acute Myocardial Infarction were included as sample by purposive sampling method. GRACE risk score for each patient was calculated and the patients were divided into groups according to the GRACE risk score: low risk (<108); intermediate risk (109-140). The severity of the coronary artery disease was assessed by vessel score and Gensini score. Relation between Grace score and Gensini score was evaluated. Results: Mean GRACE score of study population was 128.3±22.7. Mean Gensini score was 23.88±17. Mean Gensini score were 15.47±10.4, 27.75±9.26 and 31.52±16.91 in low GRACE risk group, intermediate group and high risk group respectively and the difference of mean Gensini score was statistically significant (p=0.006). In our study correlation co-efficient between GRACE risk score and Gensini score was r=0.17 (p=0.04). Multiple regression analysis showed that age more than 50 years (p=0.02), ST segment deviation (p=0.01), smoking (p=0.02), hypertension (p=0.01) were able to independently predict patients with severe CAD. Conclusion: Our study demonstrates that the GRACE risk score carries a significant positive correlation with the coronary artery disease severity in patients with STEMI. Cardiovasc. j. 2015; 8(1): 30-34
Overexpression of Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is associated with tumor aggressiveness and poor prognosis in patients with invasive ductal breast carcinoma

Tumor necrosis factor-α-induced protein 8 (TNFAIP8), a transcription factor NF-κB-inducible, antiapoptotic and oncogenic molecule, is associated with prognosis of several human malignancies. However, the relationship between TNFAIP8 and the prognosis of the invasive ductal carcinoma (IDC) of the breast remains unclear. TNFAIP8 expression was evaluated using real-time PCR and Western blot analysis in 20 fresh IDC tissues, and immunohistochemical analysis in 351 paraffin-embedded IDC tissues. Real-time PCR and Western blot analysis demonstrated that both TNFAIP8 mRNA and protein were upregulated in IDC tissues compared with the paired adjacent noncancerous tissues. Immunohistochemistry revealed that TNFAIP8 expression was significantly correlated with some clinicopathological factors, including axillary lymph node metastasis (P=0.001), advanced TNM stage (P<0.001), high histological grade (P<0.001), molecular subtype (P<0.001), and postoperative recurrence (P<0.001). Univariate and multivariate logistic regression analyses demonstrated that TNFAIP8 overexpression was strongly associated with axillary lymph node metastasis (odds ratio, 1.818; 95%CI, 1.167–2.832; P=0.008). Moreover, Kaplan–Meier analysis indicated that IDC patients with high TNFAIP8 expression had a shorter survival time than those with low TNFAIP8 expression, and multivariate analysis indicated that TNFAIP8 was an independent prognostic for overall survival and disease-free survival in IDC (P=0.041 and P=0.020, respectively). Therefore, TNFAIP8 overexpression may contribute to tumor progression, and it may be a novel prognostic biomarker for the patients with IDC.
Gene Expression Using the Vaccinia Virus/ T7 RNA Polymerase Hybrid System

This unit describes a transient cytoplasmic expression system that relies on the synthesis of the bacteriophage T7 RNA polymerase in the cytoplasm of mammalian cells. A gene of interest is inserted into a plasmid such that it comes under the control of the T7 RNA polymerase promoter (pT7). Using liposome‐mediated transfection, this recombinant plasmid is introduced into the cytoplasm of cells infected with vTF7‐3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the gene of interest is transcribed with high efficiency by T7 RNA polymerase. For large‐scale work, protocols are provided for insertion of the pT7‐regulated gene into a second recombinant vaccinia virus by homologous recombination and subsequent coinfection with vTF7‐3 into cells grown in suspension or for direct transfection into OST7‐1 cells (a stable cell line that constitutively expresses the T7 RNA polymerase). Expressed protein is then analyzed by pulse‐labeling and purified. One new development to this vaccinia virus/T7 RNA polymerase hybrid expression system described here is the VOTE inducible expression system, which eliminates the need to use two recombinant viruses or a special cell line.
Imaging Protein‐Protein Interactions by Förster Resonance Energy Transfer (FRET) Microscopy in Live Cells

This unit describes an acceptor‐sensitized emission FRET method using a confocal microscope for image acquisition. In contrast to acceptor photobleaching, which is an end‐point assay that destroys the acceptor fluorophore, the sensitized emission method is amenable for FRET measurements in live cells and can be used to measure changes in FRET efficiency over time. The purpose of this unit is to provide a basic starting point for understanding and performing the sensitized emission method with a simple teaching tool for live‐cell imaging. References that discuss the vagaries of acquiring and analyzing FRET between individually tagged molecules are provided. Curr. Protoc. Protein Sci. 56:19.5. 1‐19.5. 12. © 2009 by John Wiley & Sons, Inc.
Purification and Characterization of Proteasomes from Saccharomyces cerevisiae

The proteasome plays a central role in eukaryotic cells since it is responsible for the degradation of specific proteins involved in a large range of cellular processes. Analysis of proteasome mechanisms of action, or in vitro reconstitution, or dissection of the complex biological pathways in which it partakes, requires a reliable source of pure active proteasome. Although the biologically relevant form of the proteasome is usually considered to be the 26S proteasome, this unit describes different methods for purification and study of both 26S and 20S proteasomes from Saccharomyces cerevisiae cells.
Handling Metalloproteinases

Substrate cleavage by metalloproteinases involves nucleophilic attack on the scissile peptide bond by a water molecule that is polarized by a catalytic metal, usually a zinc ion, and a general base, usually the carboxyl group of a glutamic acid side chain. The zinc ion is most often complexed by imidazole nitrogens of histidine side chains. This arrangement suggests that the physiological pH optimum of most metalloproteinases is in the neutral range. In addition to their catalytic metal ion, many metalloproteinases contain additional transition metal or alkaline earth ions, which are structurally important or modulate the catalytic activity. As a consequence, these enzymes are generally sensitive to metal chelators. Moreover, the catalytic metal can be displaced by adventitious metal ions from buffers or biological fluids, which may fundamentally alter the catalytic function. Therefore, handling, purification, and assaying of metalloproteinases require specific precautions to warrant their stability. © 2016 by John Wiley & Sons, Inc.
Proteomic Identification of Cellular Protease Substrates Using Isobaric Tags for Relative and Absolute Quantification (iTRAQ)

Identification of protease substrates is essential to identify and understand the functional consequences of normal and dysregulated proteolysis in disease on the proteome. Isobaric tags for relative and absolute quantification (iTRAQ) can be used to identify novel protease substrates in the cellular context. An amine‐targeted iTRAQ tag labels tryptic peptides generated from the proteins and protease cleavage products of secreted proteins, as well as protein domains shed from the cell membrane or pericellular matrix of protease‐transfected cells that have accumulated in conditioned medium; a second iTRAQ tag is used for control cells. MS/MS fragmentation enables sequencing of the pooled pairs of differently labeled but identical peptides and generates a low mass signature ion peak unique for each label. This signature ion peak identifies the peptides originating from the protease‐transfected or control cells; comparison of the peak areas enables relative quantitation of the peptide between the samples. Curr. Protoc. Protein Sci. 49:21.18. 1‐21.18. 12. © 2007 by John Wiley & Sons, Inc.
The CLIP‐CHIP Oligonucleotide Microarray: Dedicated Array for Analysis of All Protease, Nonproteolytic Homolog, and Inhibitor Gene Transcripts in Human and Mouse

The CLIP‐CHIP oligonucleotide microarray allows the analysis of mRNA transcript levels in a tissue sample for all proteases, nonproteolytic homologs, and protease inhibitors of the human and mouse genome. In the protocol presented in this unit, total RNA is extracted from a tissue, and the resulting mRNA is reverse transcribed into cDNA and dsDNA and then amplified in an in vitro transcription reaction. The amplified antisense RNA is labeled with a fluorescent dye and hybridized to the CLIP‐CHIP, which contains unique oligonucleotides that are specifically designed for the protease, nonproteolytic homologs, protease inhibitors, and control samples. After hybridization, the fluorescence intensity of each spot is measured, thus identifying mRNA transcripts that are expressed and allowing basic quantification of expressed transcripts. Curr. Protoc. Protein Sci. 49:21.19. 1‐21.19. 16. © 2007 by John Wiley & Sons, Inc.
Post‐Translational Modification Profiling—a High‐Content Assay for Identifying Protein Modifications in Mammalian Cellular Systems

Protein microarrays are extremely useful for detecting substrates of phosphorylation, substrates of ubiquitylation, or other post‐translational modifications. The ability to screen binding interactions as well as post‐translational modifications of thousands of proteins at once has improved our ability to identify their targets. Utilizing such systems in combination with functional mammalian cell extracts that preserve enzymatic activity offers advantages in identifying semi‐quantitative changes of these interactions in the context of specific cellular conditions. This unit provides a detailed procedure for setting up an extract‐based activity assay for high content detection of protein post‐translation modifications. It also provides basic guidelines for data analysis. Curr. Protoc. Protein Sci. 77:27.8. 1‐27.8. 13 © 2014 by John Wiley & Sons, Inc.