Studies of acute gastroenteritis (AGE) are hampered by the lack of routine diagnostic methods with good sensitivity and specificity. Molecular methods are increasingly used for clinical purposes, but ...the clinical significance of a positive result remains a challenge. In this study we aimed to compare results of routine diagnostic methods and molecular methods in symptomatic children and asymptomatic controls.
Patients presenting to the pediatric emergency departments of two university hospitals in Brussels with AGE were recruited prospectively from May 2015 to October 2016; asymptomatic controls were recruited from the same hospitals. Stool analyses were performed for all participants for common pathogenic bacteria (culture), virus (immunochromatography) and parasites (microscopy). Stools were also analysed with the Luminex Gastrointestinal Pathogen Panel, a multiplex-PCR for common enteropathogens.
Stools from 178 patients and 165 controls were analysed. An enteropathogen was detected in 62.4% (111/178) of cases when combining the two methods (56.2% (100/178) by Luminex, 42.7% (76/178) with routine methods) and 29.1% (48/165) of controls (24.2% (40/165) by Luminex and 10.3% (17/165) by routine methods). Some pathogens were detected more often with Luminex than with routine methods, such as Salmonella (16.3% (29/178) with Luminex and 3.9% (7/178) with routine method, p < 0.05), whereas others identified by culture methods, such as Campylobacter, Shigella, Yersinia, were missed by Luminex.
Molecular tools seem attractive methods, providing high positivity and a rapid turn-around time for the diagnosis of AGE. However, high rates of positivity in both cases and controls highlight the difficulty in interpreting results. Pathogens missed by Luminex but detected by culture methods raise more questions about the true clinical interest of the technique for our patients.
•SiSERK1 gene belonging to crop which is relatively less studied.•The construction process of SiSERK1 gene overexpression vector was detailed and completed.•Novel construction method of SiSERK1 gene ...overexpression vector.•The possible signal pathways involved in SiSERK1 gene were discussed.
Plant somatic embryogenesis receptor-like kinases (SERK), members of leucine-rich repeat receptor-like kinases (LRR-RLKs) subfamily, are widely involved in plant growth, development and innate immunity. In this study, the setaria italica somatic embryogenesis receptor-like kinase1 gene (SiSERK1) was cloned by gateway technology, and transferred into a brasssinosteroid (BR) receptor mutant of Arabidopsis thaliana WS2 (bri1-5). After BL treatment, the transgenic plants could partially restore the phenotype of bri1-5. After Pst DC3000 treatment, the CFU value of SiSERK1 overexpression plant pathogen was between WS2 and bri1-5. Stomatal opening and plant height were also between them. Therefore, it is speculated that SiSERK1 gene is involved in BR signaling pathway and can improve the resistance of bri1-5 to Pst DC3000 through SA and NHP mediated systemic acquired resistance (SAR).
Molecular methods can enable rapid identification of Bartonella spp. infections, which are difficult to diagnose by using culture or serology. We analyzed clinical test results of PCR that targeted ...bacterial 16S rRNA hypervariable V1-V2 regions only or in parallel with PCR of Bartonella-specific ribC gene. We identified 430 clinical specimens infected with Bartonella spp. from 420 patients in the United States. Median patient age was 37 (range 1-79) years; 62% were male. We identified B. henselae in 77%, B. quintana in 13%, B. clarridgeiae in 1%, B. vinsonii in 1%, and B. washoensis in 1% of specimens. B. quintana was detected in 83% of cardiac specimens; B. henselae was detected in 34% of lymph node specimens. We detected novel or uncommon Bartonella spp. in 9 patients. Molecular diagnostic testing can identify Bartonella spp. infections, including uncommon and undescribed species, and might be particularly useful for patients who have culture-negative endocarditis or lymphadenitis.
Environmental exposures pose a significant threat to human health. However, it is often difficult to study toxicological mechanisms in human subjects due to ethical concerns. Plant-derived ...aristolochic acids are among the most potent nephrotoxins and carcinogens discovered to date, yet the mechanism of bioactivation in humans remains poorly understood. Microphysiological systems (organs-on-chips) provide an approach to examining the complex, species-specific toxicological effects of pharmaceutical and environmental chemicals using human cells. We microfluidically linked a kidney-on-a-chip with a liver-on-a-chip to determine the mechanisms of bioactivation and transport of aristolochic acid I (AA-I), an established nephrotoxin and human carcinogen. We demonstrate that human hepatocyte-specific metabolism of AA-I substantially increases its cytotoxicity toward human kidney proximal tubular epithelial cells, including formation of aristolactam adducts and release of kidney injury biomarkers. Hepatic biotransformation of AA-I to a nephrotoxic metabolite involves nitroreduction, followed by sulfate conjugation. Here, we identify, in a human tissue-based system, that the sulfate conjugate of the hepatic NQO1-generated aristolactam product of AA-I (AL-I-NOSO3) is the nephrotoxic form of AA-I. This conjugate can be transported out of liver via MRP membrane transporters and then actively transported into kidney tissue via one or more organic anionic membrane transporters. This integrated microphysiological system provides an ex vivo approach for investigating organ-organ interactions, whereby the metabolism of a drug or other xenobiotic by one tissue may influence its toxicity toward another, and represents an experimental approach for studying chemical toxicity related to environmental and other toxic exposures.
Although melatonin biosynthetic genes from plants have been cloned, the melatonin catabolism mechanisms remain unclear. To clone the genes responsible for melatonin metabolism, we ectopically ...expressed 35 full‐length cDNAs of rice 2‐oxoglutarate‐dependent dioxygenase (2‐ODD) in Escherichia coli and purified the corresponding recombinant proteins. In vitro 2‐ODD assays showed four independent 2‐ODD proteins that were able to catalyze melatonin into 2‐hydroxymelatonin, exhibiting melatonin 2‐hydroxylase (M2H). These M2H proteins had peak activities at pH 8.0 and 30°C. The Km ranged from 121 μm to 371 μm with the Vmax ranging from 1.7 to 18.5 pkat/mg protein, respectively. The M2H enzyme activities were dependent on cofactors such as α‐ketoglutarate, ascorbate, and Fe2+, similar to the 2‐ODD enzymes. M2H activity was inhibited by prohexadione‐Ca, an inhibitor of 2‐ODD, in a dose‐dependent manner. M2H activity was high in the roots of rice seedlings, concurrent with high transcription levels of 2‐ODD 21, suggesting that 2‐ODD 21 was a major gene for M2H activity. Analogous to the high M2H activity in the roots, 2‐hydroxymelatonin was found in large quantities in roots treated with melatonin. These results suggest that melatonin was metabolized into 2‐hydroxymelatonin by the M2H genes in plants, but the physiological significance of 2‐hydroxymelatonin remains to be examined in the future.
Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences ...(promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing β-carotene and co-producing β-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.
Although environmental DNA surveys improve our understanding of biodiversity, interpretation of unidentified lineages is limited by the absence of associated morphological traits and living cultures. ...Unidentified lineages of marine stramenopiles are called “MAST clades”. Twenty‐five MAST clades have been recognized: MAST‐1 through MAST‐25; seven of these have been subsequently discarded because the sequences representing those clades were found to either (1) be chimeric or (2) affiliate within previously described taxonomic groups. Eighteen MAST clades remain without a cellular identity. Moreover, the discarded “MAST‐13” has been used in different studies to refer to two different environmental sequence clades. After establishing four cultures representing two different species of heterotrophic stramenopiles and then characterizing their morphology and molecular phylogenetic positions, we determined that the two different species represented the two different MAST‐13 clades: (1) a lorica‐bearing Bicosoeca kenaiensis and (2) a microaerophilic flagellate previously named “Cafeteria marsupialis”. Both species were previously described with only light microscopy; no cultures, ultrastructural data or DNA sequences were available from these species prior to this study. The molecular phylogenetic position of three different “C. marsupialis” isolates was not closely related to the type species of Cafeteria; therefore, we established a new genus for these isolates, Cantina gen. nov.
Highlights • The new frontier in biomarker analysis is single-molecule counting. • The review reports on techniques allowing digital detection of biomolecules. • The first part focuses on microbead ...confinement in microenvironments. • The second part describes detection of biological nanoparticles. • Discussion emphasizes clinical applicability.
Molecular manipulations, including DNA cloning and mutagenesis are basic tools used on a routine basis in all life-science disciplines. Over the last decade new methodologies have emerged that ...facilitated and expanded the applications for DNA cloning and mutagenesis. Ligation-Independent Cloning (LIC) techniques were developed and replaced the classical Ligation Dependent Cloning (LDC) platform. Restriction Free (RF) cloning was originally developed for introduction of foreign DNA into a plasmid at any predetermined position. RF cloning is based on PCR amplification of a DNA fragment, which serves as a mega-primer for the linear amplification of the vector and insert. Here we present several novel applications of the Restriction Free (RF) cloning platform for DNA cloning and mutagenesis. The new applications include simultaneous cloning of several DNA fragments into distinct positions within an expression vector, simultaneous multi-component assembly, and parallel cloning of the same PCR product into a series of different vectors. In addition, we have expanded the application of the RF cloning platform for multiple alterations of the target DNA, including simultaneous multiple-site mutagenesis and simultaneous introduction of deletions and insertions at different positions. We further demonstrate the robustness of the new applications for facilitating recombinant protein expression in the Escherichia coli system.
CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme ...properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5' handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2'-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2'-hydroxyl sensitivity. Modified 5' pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5' pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5' pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.
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