scholarly journals Molecular Tools for the Detection of Nitrogen Cycling Archaea

Archaea ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Antje Rusch
Keyword(s):  
Nitrogen Cycling ◽  
High Sensitivity ◽  
The Novel ◽  
Degenerate Primers ◽  
Culture Studies ◽  
Metabolic Genes ◽  
Nitrite Reductases ◽  
Genes Encoding ◽  
Moderate Sensitivity

Archaea are widespread in extreme and temperate environments, and cultured representatives cover a broad spectrum of metabolic capacities, which sets them up for potentially major roles in the biogeochemistry of their ecosystems. The detection, characterization, and quantification of archaeal functions in mixed communities require Archaea-specific primers or probes for the corresponding metabolic genes. Five pairs of degenerate primers were designed to target archaeal genes encoding key enzymes of nitrogen cycling: nitrite reductases NirA and NirB, nitrous oxide reductase (NosZ), nitrogenase reductase (NifH), and nitrate reductases NapA/NarG. Sensitivity towards their archaeal target gene, phylogenetic specificity, and gene specificity were evaluated in silico and in vitro. Owing to their moderate sensitivity/coverage, the novelnirB-targeted primers are suitable for pure culture studies only. ThenirA-targeted primers showed sufficient sensitivity and phylogenetic specificity, but poor gene specificity. The primers designed for amplification of archaealnosZperformed well in all 3 criteria; their discrimination against bacterial homologs appears to be weakened when Archaea are strongly outnumbered by bacteria in a mixed community. The novelnifH-targeted primers showed high sensitivity and gene specificity, but failed to discriminate against bacterial homologs. Despite limitations, 4 of the new primer pairs are suitable tools in several molecular methods applied in archaeal ecology.

scholarly journals Differential Involvement of Histidine Kinase Receptors in Pseudohyphal Development, Stress Adaptation, and Drug Sensitivity of the Opportunistic Yeast Candida lusitaniae

2007 ◽  
Vol 6 (10) ◽  
pp. 1782-1794 ◽  
Author(s):  
Florence Chapeland-Leclerc ◽  
Paméla Paccallet ◽  
Gwenaël Ruprich-Robert ◽  
David Reboutier ◽  
Christiane Chastin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Histidine Kinase ◽  
High Sensitivity ◽  
Triple Mutant ◽  
Mating Ability ◽  
Reverse Transcription Pcr ◽  
Moderate Sensitivity ◽  
Candida Lusitaniae ◽  
High Level

ABSTRACT Fungal histidine kinase receptors (HKRs) sense and transduce many extracellular signals. We investigated the role of HKRs in morphogenetic transition, osmotolerance, oxidative stress response, and mating ability in the opportunistic yeast Candida lusitaniae. We isolated three genes, SLN1, NIK1, and CHK1, potentially encoding HKRs of classes VI, III, and X, respectively. These genes were disrupted by a transformation system based upon the “URA3 blaster” strategy. Functional analysis of disruptants was undertaken, except for the sln1 nik1 double mutant and the sln1 nik1 chk1 triple mutant, which are not viable in C. lusitaniae. The sln1 mutant revealed a high sensitivity to oxidative stress, whereas both the nik1 and chk1 mutants exhibited a more moderate sensitivity to peroxide. We also showed that the NIK1 gene was implicated in phenylpyrrole and dicarboximide compound susceptibility while HKRs seem not to be involved in resistance toward antifungals of clinical relevance. Concerning mating ability, all disruptants were still able to reproduce sexually in vitro in unilateral or bilateral crosses. The most important result of this study was that the sln1 mutant displayed a global defect of pseudohyphal differentiation, especially in high-osmolarity and oxidative-stress conditions. Thus, the SLN1 gene could be crucial for the C. lusitaniae yeast-to-pseudohypha morphogenetic transition. This implication is strengthened by a high level of SLN1 mRNAs revealed by semiquantitative reverse transcription-PCR when the yeast develops pseudohyphae. Our findings highlight a differential contribution of the three HKRs in osmotic and oxidant adaptation during the morphological transition in C. lusitaniae.

scholarly journals Excystation of Eimeria tenella Sporozoites Impaired by Antibody Recognizing Gametocyte/Oocyst Antigens GAM22 and GAM56

2007 ◽  
Vol 7 (2) ◽  
pp. 202-211 ◽  
Author(s):  
Jürgen Krücken ◽  
Ralf J. Hosse ◽  
Aimdip N. Mouafo ◽  
Rolf Entzeroth ◽  
Stefan Bierbaum ◽  
...  
Keyword(s):  
Phage Display Library ◽  
Northern Blotting ◽  
Eimeria Tenella ◽  
Genome Project ◽  
The Novel ◽  
Protein Sequence Analysis ◽  
Oocyst Wall ◽  
Body Type ◽  
Genes Encoding

ABSTRACT Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.

scholarly journals Evolution of resistance in vitro reveals mechanisms of artemisinin activity inToxoplasma gondii

2019 ◽  
Vol 116 (52) ◽  
pp. 26881-26891 ◽  
Author(s):  
Alex Rosenberg ◽  
Madeline R. Luth ◽  
Elizabeth A. Winzeler ◽  
Michael Behnke ◽  
L. David Sibley
Keyword(s):  
Point Mutations ◽  
Artemisinin Resistance ◽  
Wild Type ◽  
Resistant Lines ◽  
Genes Encoding ◽  
Evolution Of Resistance ◽  
Moderate Sensitivity ◽  
High Concentrations ◽  
Related Parasite

Artemisinins are effective against a variety of parasites and provide the first line of treatment for malaria. Laboratory studies have identified several mechanisms for artemisinin resistance inPlasmodium falciparum, including mutations in Kelch13 that are associated with delayed clearance in some clinical isolates, although other mechanisms are likely involved. To explore other potential mechanisms of resistance in parasites, we took advantage of the genetic tractability ofToxoplasma gondii, a related parasite that shows moderate sensitivity to artemisinin. Resistant populations ofT. gondiiwere selected by culture in increasing concentrations and whole-genome sequencing identified several nonconservative point mutations that emerged in the population and were fixed over time. Genome editing using CRISPR/Cas9 was used to introduce point mutations conferring amino acid changes in a serine protease homologous to DegP and a serine/threonine protein kinase of unknown function. Single and double mutations conferred a competitive advantage over wild-type parasites in the presence of drug, despite not changing EC50values. Additionally, the evolved resistant lines showed dramatic amplification of the mitochondria genome, including genes encoding cytochromeband cytochromecoxidase I. Prior studies in yeast and mammalian tumor cells implicate the mitochondrion as a target of artemisinins, and treatment of wild-type parasites with high concentrations of drug decreased mitochondrial membrane potential, a phenotype that was stably altered in the resistant parasites. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition of resistance inT. gondii.

scholarly journals Distinct Features of Cyanophage-encoded T-type Phycobiliprotein Lyase ΦCpeT

2017 ◽  
Vol 292 (8) ◽  
pp. 3089-3098 ◽  
Author(s):  
Raphael Gasper ◽  
Julia Schwach ◽  
Jana Hartmann ◽  
Andrea Holtkamp ◽  
Jessica Wiethaus ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Fluorescence Quantum Yield ◽  
Β Subunit ◽  
Metabolic Genes ◽  
Genes Encoding ◽  
Tetrapyrrole Chromophores ◽  
Auxiliary Metabolic Genes ◽  
Distinct Features ◽  
Host Genes

Auxiliary metabolic genes (AMG) are commonly found in the genomes of phages that infect cyanobacteria and increase the fitness of the cyanophage. AMGs are often homologs of host genes, and also typically related to photosynthesis. For example, the ΦcpeT gene in the cyanophage P-HM1 encodes a putative phycobiliprotein lyase related to cyanobacterial T-type lyases, which facilitate attachment of linear tetrapyrrole chromophores to Cys-155 of phycobiliprotein β-subunits, suggesting that ΦCpeT may also help assemble light-harvesting phycobiliproteins during infection. To investigate this possibility, we structurally and biochemically characterized recombinant ΦCpeT. The solved crystal structure of ΦCpeT at 1.8-Å resolution revealed that the protein adopts a similar fold as the cyanobacterial T-type lyase CpcT from Nostoc sp. PCC7120 but overall is more compact and smaller. ΦCpeT specifically binds phycoerythrobilin (PEB) in vitro leading to a tight complex that can also be formed in Escherichia coli when it is co-expressed with genes encoding PEB biosynthesis (i.e. ho1 and pebS). The formed ΦCpeT·PEB complex was very stable as the chromophore was not lost during chromatography and displayed a strong red fluorescence with a fluorescence quantum yield of ΦF = 0.3. This complex was not directly able to transfer PEB to the host phycobiliprotein β-subunit. However, it could assist the host lyase CpeS in its function by providing a pool of readily available PEB, a feature that might be important for fast phycobiliprotein assembly during phage infection.

scholarly journals Molecular Characterization of Carbapenem-Nonsusceptible Enterobacterial Isolates Collected during a Prospective Interregional Survey in France and Susceptibility to the Novel Ceftazidime-Avibactam and Aztreonam-Avibactam Combinations

2015 ◽  
Vol 60 (1) ◽  
pp. 215-221 ◽  
Author(s):  
Hervé Dupont ◽  
Olivier Gaillot ◽  
Anne-Sophie Goetgheluck ◽  
Claire Plassart ◽  
Jean-Philippe Emond ◽  
...  
Keyword(s):  
Outer Membrane Proteins ◽  
Surveillance Program ◽  
Northwestern Part ◽  
The Novel ◽  
Content Type ◽  
Active Efflux ◽  
Fixed Concentration ◽  
Sds Page ◽  
Genes Encoding

ABSTRACTAn interregional surveillance program was conducted in the northwestern part of France to determine the prevalence of carbapenem-nonsusceptibleEnterobacteriaceae(CNSE) isolates and their susceptibility to ceftazidime-avibactam and aztreonam-avibactam combinations. Nonduplicate CNSE clinical isolates were prospectively collected from six hospitals between June 2012 and November 2013. MICs of ceftazidime and aztreonam, alone or combined with a fixed concentration of avibactam (4 μg/ml), and those of carbapenems (comparator agents) were determined. MICs of ertapenem in combination with phenylalanine arginine-naphthylamide dihydrochloride (PAβN) were also determined to assess active efflux. Genes encoding carbapenemases, plasmid-mediated AmpC enzymes, extended-spectrum β-lactamases (ESBLs), and major outer membrane proteins (OMPs) were amplified and sequenced. OMPs were also extracted for SDS-PAGE analysis. Among the 139 CNSE isolates, mainlyEnterobacterspp. andKlebsiella pneumoniae, 123 (88.4%) were ertapenem nonsusceptible, 12 (8.6%) exhibited reduced susceptibility to all carbapenems, and 4Proteeaeisolates (2.9%) were resistant to imipenem. Carbapenemase production was detected in only two isolates (producing OXA-48 and IMI-3). In contrast, OMP deficiency, in association with AmpCs and/or ESBLs (mainly CTX-M-9, SHV-12, and CTX-M-15), was largely identified among CNSE isolates. The ceftazidime-avibactam and aztreonam-avibactam combinations exhibited potent activity against CNSE isolates (MIC50/MIC90, 1/1 μg/ml and 0.5/0.5 μg/ml, respectively) compared to that of ceftazidime and aztreonam alone (MIC50/MIC90, 512/512 μg/ml and 128/512 μg/ml, respectively). This study reveals thein vitroactivity of ceftazidime-avibactam and aztreonam-avibactam combinations against a large collection of porin-deficient enterobacterial isolates that are representative of the CNSE recovered in the northern part of France.

scholarly journals Advances in Microtechnology for Improved Cytotoxicity Assessment

2020 ◽  
Vol 7 ◽  
Author(s):  
Maite Garcia-Hernando ◽  
Fernando Benito-Lopez ◽  
Lourdes Basabe-Desmonts
Keyword(s):  
Surface Engineering ◽  
Current Trend ◽  
High Sensitivity ◽  
The Novel ◽  
Skilled Personnel ◽  
Cytotoxic Compounds ◽  
Novel Approaches ◽  
Cytotoxicity Assessment ◽  
User Intervention

In vitro cytotoxicity testing is essential in the pharmaceutical and environmental industry to study the effects of potential harmful compounds for human health. Classical assays present several disadvantages: they are commonly based on live-death labelling, are highly time consuming and/or require skilled personnel to be performed. The current trend is to reduce the number of required cells and the time during the analysis, while increasing the screening capability and the accuracy and sensitivity of the assays, aiming single cell resolution. Microfabrication and surface engineering are enabling novel approaches for cytotoxicity assessment, offering high sensitivity and the possibility of automation in order to minimize user intervention. This review aims to overview the different microtechnology approaches available in this field, focusing on the novel developments for high-throughput, dynamic and real time screening of cytotoxic compounds.

Direct Read and Quantify Damage Nucleotide from an Oligonucleotide Using a Single Molecule Interface

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Meng-Yin Li ◽  
Jie Yang ◽  
Ya-Qian Wang ◽  
Xue-Yuan Wu ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Genetic Diseases ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Lesion Detection ◽  
The Novel ◽  
Structural Variations ◽  
Dna Lesion ◽  
Base Lesion

DNA lesion such as metholcytosine(<sup>m</sup>C), 8-OXO-guanine(<sup>O</sup>G), inosine(I) <i>etc</i> could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (<sup>m</sup>C, <sup>O</sup>G, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

The Antiviral and Antimalarial Drug Repurposing in Quest of Chemotherapeutics to Combat COVID-19 Utilizing Structure-Based Molecular Docking

2020 ◽  
Vol 23 ◽  
Author(s):  
Sisir Nandi ◽  
Mohit Kumar ◽  
Mridula Saxena ◽  
Anil Kumar Saxena
Keyword(s):  
Molecular Docking ◽  
In Silico ◽  
Drug Repurposing ◽  
Clinical Settings ◽  
The Novel ◽  
In Silico Docking ◽  
Biochemical Mechanisms ◽  
Novel Coronavirus ◽  
In Silico Molecular Docking

Background: The novel coronavirus disease (COVID-19) is caused by a new strain (SARS-CoV-2) erupted in 2019. Nowadays, it is a great threat that claims uncountable lives worldwide. There is no specific chemotherapeutics developed yet to combat COVID-19. Therefore, scientists have been devoted in the quest of the medicine that can cure COVID- 19. Objective: Existing antivirals such as ASC09/ritonavir, lopinavir/ritonavir with or without umifenovir in combination with antimalarial chloroquine or hydroxychloroquine have been repurposed to fight the current coronavirus epidemic. But exact biochemical mechanisms of these drugs towards COVID-19 have not been discovered to date. Method: In-silico molecular docking can predict the mode of binding to sort out the existing chemotherapeutics having a potential affinity towards inhibition of the COVID-19 target. An attempt has been made in the present work to carry out docking analyses of 34 drugs including antivirals and antimalarials to explain explicitly the mode of interactions of these ligands towards the COVID-19protease target. Results: 13 compounds having good binding affinity have been predicted towards protease binding inhibition of COVID-19. Conclusion: Our in silico docking results have been confirmed by current reports from clinical settings through the citation of suitable experimental in vitro data available in the published literature.

Synthetic and semi-synthetic drugs as promising therapeutic option for the treatment of COVID-19

2020 ◽  
Vol 20 ◽  
Author(s):  
Ekta Shirbhate ◽  
Preeti Patel ◽  
Vijay K Patel ◽  
Ravichandran Veerasamy ◽  
Prabodh C Sharma ◽  
...  
Keyword(s):  
Therapeutic Option ◽  
Antiviral Treatment ◽  
The Novel ◽  
Clinical Experiences ◽  
Synthetic Compounds ◽  
Synthetic Drugs ◽  
Global Pandemic ◽  
The World ◽  
Novel Coronavirus

: The novel coronavirus disease-19 (COVID-19), a global pandemic that emerged from Wuhan, China has today travelled all around the world, so far 216 countries or territories with 21,732,472 people infected and 770,866 deaths globally (as per WHO COVID-19 update dated August 18, 2020). Continuous efforts are being made to repurpose the existing drugs and develop vaccines for combating this infection. Despite, to date, no certified antiviral treatment or vaccine prevails. Although, few candidates have displayed their efficacy in in vitro studies and are being repurposed for COVID-19 treatment. This article summarizes synthetic and semi-synthetic compounds displaying potent activity in their clinical experiences or studies against COVID-19 and also focuses on mode of action of drugs being repositioned against COVID-19.

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