MCM can initiate duplex DNA unwinding from a short 3′ single-stranded overhang

<p><b>Copyright information:</b></p><p>Taken from "Use of a restriction enzyme-digested PCR product as substrate for helicase assays"</p><p>Nucleic Acids Research 2005;33(1):e8-e8.</p><p>Published online 13 Jan 2005</p><p>PMCID:PMC546180.</p><p>© 2005, the authors © </p> Two oligonucleotides were used to generate a helicase substrate with a 4-base ssDNA overhang. Helicase assays were performed as described in Material and Methods in 15 μl reactions with the indicated concentrations of protein and 10 fmol substrate. The P-labeled oligonucleotide is marked with an asterisk. Lane 1, substrate only; lane 2, boiled substrate; lanes 3–5 contain 0.13, 0.40 and 1.2 pmol of proteins (as monomer), respectively. The percent displacement of the labeled oligonucleotide is indicated as %

Instrument-Free Synthesizable Fabrication of Label-Free Optical Biosensing Paper Strips for the Early Detection of Infectious Keratoconjunctivitides

We introduce a surface-enhanced Raman scattering (SERS)-functionalized, gold nanoparticle (GNP)-deposited paper strip capable of label-free biofluid sensing for the early detection of infectious eye diseases. The GNP biosensing paper strip was fabricated by the direct synthesis and deposition of GNPs on wax-divided hydrophilic areas of a permeable porous substrate through a facile, power-free synthesizable, and highly reproducible successive ionic layer absorption and reaction (SILAR) technique. To maximize localized surface plasmon resonance-generated SERS activity, the concentration of the reactive solution and number of SILAR cycles were optimized by controlling the size and gap distance of GNPs and verified by computational modeling with geometrical hypotheses of Gaussian-estimated metallic nanoparticles. The responses of our SERS-functionalized GNP paper strip to Raman intensities exhibited an enhancement factor of 7.8 × 10⁸, high reproducibility (relative standard deviation of 7.5%), and 1 pM 2-naphthalenethiol highly sensitive detection limit with a correlation coefficient of 0.99, achieved by optimized SILAR conditions including a 10/10 mM/mM HAuCl₄/NaBH₄ concentration and six SILAR cycles. The SERS-functionalized GNP paper is supported by a multivariate statistics-preprocessed machine learning-judged bioclassification system to provide excellent label-free chemical structure sensitivity for identifying infectious keratoconjunctivitis. The power-free synthesizable fabrication, label-free, rapid analysis, and high sensitivity feature of the SILAR-fabricated SERS-functionalized GNP biosensing paper strip makes it an excellent alternative in point-of-care applications for the early detection of various infectious diseases

Label-Free Biochemical Analytic Method for the Early Detection of Adenoviral Conjunctivitis Using Human Tear Biofluids

Cell culture and polymerase chain reaction are currently regarded as the gold standard for adenoviral conjunctivitis diagnosis. They maximize sensitivity and specificity but require several days to 3 weeks to get the results. The aim of this study is to determine the potential of Raman spectroscopy as a stand-alone analytical tool for clinical diagnosis of adenoviral conjunctivitis using human tear fluids. A drop-coating deposition surface enhanced Raman scattering (DCD-SERS) method was identified as the most effective method of proteomic analysis in tear biofluids. The proposed DCD-SERS method (using a 2-μL sample) led to Raman spectra with high reproducibility, noise-independence, and uniformity. Additionally, the spectra were independent of the volume of biofluids used and detection zones, including the ring, middle, and central zone, with the exception of the outer layer of the ring zone. Assessments with an intensity ratio of 1242–1342 cm^–1 achieved 100% sensitivity and 100% specificity in the central zone. Principal component analysis assessments achieved 0.9453 in the area under the receiver operating characteristic curve (AUC) as well as 93.3% sensitivity and 94.5% specificity in the central zone. Multi-Gaussian peak assessments showed that the differences between these two groups resulted from the reduction of the amide III α-helix structures of the proteins. The presence of adenovirus in tear fluids could be detected more accurately in the center of the sample than in the periphery. The DCD-SERS technique allowed for high chemical structure sensitivity without additional tagging or chemical modification, making it a good alternative for early clinical diagnosis of adenoviral conjunctivitis. Therefore, we are hopeful that the DCD-SERS method will be approved for use in ophthalmological clinics in the near future

Scanning electron micrograph of <i>S</i>. <i>marcescens</i> RSC-14.

<p>Fatty acid analysis indicated that the sum of C:14 3OH (3.8%), C16:0 (29.8%), C16:1 (8.2%) and C18:1 (5%) in RSC-14 is about 50%, which is consistent with the ratio of 50 to 80% observed in other <i>S</i>. <i>marcescens</i> strains [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171534#pone.0171534.ref033" target="_blank">33</a>]</p

Complete genome analysis of <i>Serratia marcescens</i> RSC-14: A plant growth-promoting bacterium that alleviates cadmium stress in host plants

<div><p><i>Serratia marcescens</i> RSC-14 is a Gram-negative bacterium that was previously isolated from the surface-sterilized roots of the Cd-hyperaccumulator <i>Solanum nigrum</i>. The strain stimulates plant growth and alleviates Cd stress in host plants. To investigate the genetic basis for these traits, the complete genome of RSC-14 was obtained by single-molecule real-time sequencing. The genome of <i>S</i>. <i>marcescens</i> RSC-14 comprised a 5.12-Mbp-long circular chromosome containing 4,593 predicted protein-coding genes, 22 rRNA genes, 88 tRNA genes, and 41 pseudogenes. It contained genes with potential functions in plant growth promotion, including genes involved in indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis, and phosphate solubilization. Moreover, annotation using NCBI and Rapid Annotation using Subsystem Technology identified several genes that encode antioxidant enzymes as well as genes involved in antioxidant production, supporting the observed resistance towards heavy metals, such as Cd. The presence of IAA pathway-related genes and oxidative stress-responsive enzyme genes may explain the plant growth-promoting potential and Cd tolerance, respectively. This is the first report of a complete genome sequence of Cd-tolerant <i>S</i>. <i>marcescens</i> and its plant growth promotion pathway. The whole-genome analysis of this strain clarified the genetic basis underlying its phenotypic and biochemical characteristics, underpinning the beneficial interactions between RSC-14 and plants.</p></div

Gibberellins synthesized by the entomopathogenic bacterium, <i>Photorhabdus temperata</i> M1021 as one of the factors of rice plant growth promotion

<div><p>In the present study, different types of gibberellins (GAs) in the culture filtrate (CF) of <i>Photorhabdus temperata</i> M1021 were quantified. The analysis of CF helped in profiling various bioactive GAs: GA₁, GA₃, GA₄, and GA₇. Several physiologically inactive GAs: GA₉, GA₁₂, and GA₂₀ were detected as well. Siderophore production was also investigated by growing <i>P. temperata</i> M1021 on chrome azurol-S blue agar plates. Furthermore, the strain was inoculated into ‘<i>Waito</i>-C’ (<i>Oryza sativa</i> L.) rice plants, which significantly (<i>P</i> < 0.05) increased plant growth attributes such as plant length, chlorophyll content, and fresh and dry biomass compared with those in controls. In a separate experiment, canola (<i>Brassica napus</i> L.) seeds treated with CF of M1021 were significantly (<i>P</i> < 0.05) accelerated germination rate as well as biomass production. Findings of the present study suggest that the strain M1021 contributes an important role in the plant growth by synthesizing a wide array of bioactive metabolites.</p></div

Bacterial metabolic pathways involved in indole-3-acetic acid, tryptophan, and acetoin biosynthesis in <i>S</i>. <i>marcescens</i> RSC-14.

<p><b>(a)</b> Structural gene cluster of the tryptophan biosynthesis pathway. <b>(b)</b> The biosynthetic pathways were constructed based on the KEGG database, NCBI genome annotation, and the RAST server. Black shaded arrows indicate known pathway steps for which related genes were not detected in the RSC-14 genome. Red arrows indicate putative enzymes that are encoded by the genome. The enzymes that were identified in the RSC-14 genome are shown with their NCBI locus tag.</p

Phylogenetic tree highlighting the position of <i>S</i>. <i>marcescens</i> RSC-14 with other closely related species within the genus of <i>Serratia</i>.

<p>The phylogenetic tree was constructed based on concatenated sequences of 16S rRNA, <i>gyr</i>B, and <i>rpo</i>B genes aligned in ClustalW2 using the neighbor-joining algorithm in CLC Main Workbench and rooted with <i>Photorhabdus luminescens subsp</i>. <i>laumondii</i> TTO1. All <i>Serratia</i> species clustered together and were distinct from other Enterobacteriaceae. The tree also highlights the close relationship of <i>S</i>. <i>marcescens</i> RSC-14 strain with the <i>S</i>. <i>marcescens</i> type strain WW4.</p

Venn diagram of the CDSs shared by the genomes of five closely related <i>Serratia</i> spp.; <i>S</i>. <i>marcescens</i> FGI94 (CP003942), <i>S</i>. <i>marcescens</i> WW4 (CP003959), <i>S</i>. <i>liquefaciens</i> ATCC27592 (CP006252) and <i>S</i>. <i>plymuthica</i> S13 (CP006566).

<p>The number outside the overlapping regions represents the number of CDSs present in each genome without homologs in other <i>Serratia</i> strains. While the overlapping regions indicates the CDSs shared by the respective strains.</p

Genes (and their locus tags) involved in phosphate solubilization.

<p>Genes (and their locus tags) involved in phosphate solubilization.</p