Library Reader Issue 02: Source Of Clarification

Library resource awareness poster covering the difference between primary, secondary, and tertiary sources, along with UNE Library resources which carry each.https://dune.une.edu/libraryreader/1001/thumbnail.jp

Removal of five fluoroquinolone antibiotics during broiler manure composting

<p>Composting is a cost-effective approach for the removal of antibiotics from the environment; however, the consequence of this approach on fluoroquinolone antibiotics is limited. The fate of five representative fluoroquinolone antibiotics, namely ciprofloxacin, enrofloxacin, lomefloxacin, norfloxacin, and sarafloxacin, was investigated in a pilot-scale composting of broiler manure over 42 days. The effect of antibiotic concentrations (at a dose of 15, 30, or 60 mg/kg for each and a control without antibiotic addition) on the composting process was also assessed. The 42-day composting showed 45.3–75.4% of antibiotic removal with species-specific patterns. However, the observed variations in such removal among both antibiotics concentrations and composting times were not significant in most cases, possibly indicating a slight side-effect of the tested antibiotic concentrations on the composting process. To the best of our knowledge, this study is among few studies with a focus on the persistence of fluoroquinolone antibiotics during a pilot-scale composting, which warrants further study in regards to the mechanism underlying the removal of these compounds during composting.</p

Efficient CRISPR/Cas9-Mediated Gene Editing in <i>Arabidopsis thaliana</i> and Inheritance of Modified Genes in the T2 and T3 Generations

<div><p>The newly developed CRISPR/Cas9 system for targeted gene knockout or editing has recently been shown to function in plants in both transient expression systems as well as in primary T1 transgenic plants. However, stable transmission of genes modified by the Cas9/single guide RNA (sgRNA) system to the T2 generation and beyond has not been demonstrated. Here we provide extensive data demonstrating the efficiency of Cas9/sgRNA in causing modification of a chromosomally integrated target reporter gene during early development of transgenic Arabidopsis plants and inheritance of the modified gene in T2 and T3 progeny. Efficient conversion of a nonfunctional, out-of-frame <i>GFP</i> gene to a functional <i>GFP</i> gene was confirmed in T1 plants by the observation of green fluorescent signals in leaf tissues as well as the presence of mutagenized DNA sequences at the sgRNA target site within the <i>GFP</i> gene. All GFP-positive T1 transgenic plants and nearly all GFP-negative plants examined contained mutagenized <i>GFP</i> genes. Analyses of 42 individual T2 generation plants derived from 6 different T1 progenitor plants showed that 50% of T2 plants inherited a single T-DNA insert. The efficiency of the Cas9/sgRNA system and stable inheritance of edited genes point to the promise of this system for facile editing of plant genes.</p></div

Robust expression in guard cells of functional <i>GFP</i> genes created by Cas9/sgRNA-mediated mutagenesis.

<p>A) Detection of green fluorescence protein signals in guard cells of transgenic leaf stomata with lesser expression in surrounding leaf epidermal cells of T1 transgenic Arabidopsis plants. B) Merged image of red chlorophyll fluorescence and GFP fluorescence. Bar, 50 µm. Photographed twenty days after seed germination.</p

Cas9/sgRNA-mediated mutagenesis in Arabidopsis.

<p>Scheme for Cas9/sgRNA-mediated mutagenesis of a non-functional (out-of-frame) mutant <i>GFP</i> gene, detection of T1 Arabidopsis leaves with restored <i>GFP</i> gene function by confocal microscopy and documentation of target site DNA mutagenesis.</p

Efficiency of Cas9/sgRNA mutagenesis in Arabidopsis plants.

<p>PCR/Restriction Enzyme (PCR/RE) analysis of total DNA extracts from individual hygromycin resistant T1 Arabidopsis plants (lanes 1–12) showing the relative proportion of nonfunctional <i>GFP</i> genes mutagenized by Cas9/sgRNA activity. Bottom arrow indicates the expected ∼125 bp DNA fragments resulting from <i>Apa</i>LI cleavage of the ∼250 bp PCR product amplified from nonfunctional, out-of-frame, nonmutagenized <i>GFP</i> genes that contains an intact <i>Apa</i>LI cleavage site in the sgRNA target region. Top arrow indicates the expected ∼250 bp size of PCR products from <i>GFP</i> genes mutagenized by the Cas9/sgRNA system in such a manner that they are no longer are susceptible to cleavage by <i>Apa</i>LI. NG (<u>N</u>onfunctional <i>GFP</i><u>G</u>ene), the PCR products amplified from a sgRNA target site of a cloned nonfunctional, out-of-frame, <i>GFP</i> gene digested with <i>Apa</i>LI. % modified <i>GFP</i> Gene = (pixels in 250 bp band)/(pixels in 250 bp band+pixels in 125 bp band) ×100.</p

Confirmation by DNA sequencing of Cas9/sgRNA-mediated mutagenesis of the sgRNA target site within the nonfuctional, out-of-frame, <i>GFP</i> gene.

<p>Five cloned DNA fragments of 250 bp containing DNA from PCR amplified sgRNA target regions of (previously) nonfunctional <i>GFP</i> genes from three different GFP-positive T1 Arabidopsis plants (Plants 21, 22 and 25) and two different GFP-negative plants (Plants 23 and 24) were subjected to DNA sequencing. DNA sequences of a segment of 48 nucleotides surrounding the sgRNA target site are shown for each clone with the sequence of the nonmutagenized DNA region shown as the top line of each group. The 20 nucleotide target sequence for the Cas9/sgRNA complex is depicted in blue, the PAM site in red and the <i>Apa</i>LI recognition site is underlined in blue. Brackets ([]) denote nondisplayed DNA sequences. For the Cas9/sgRNA-mutagenized DNA sequences, deleted nucleotides are depicted as red dots and inserted nucleotides are shown in green. The net length of insertions and/or deletions (In/Del) are presented in the column to the right.</p

The information of two data sets.

As a result of climate change and rapid urbanization, urban waterlogging commonly caused by rainstorm, is becoming more frequent and more severe in developing countries. Urban waterlogging sometimes results in significant financial losses as well as human casualties. Accurate waterlogging depth prediction is critical for early warning system and emergency response. However, the existing hydrological models need to obtain more abundant hydrological data, and the model construction is complicated. The waterlogging depth prediction technology based on object detection model are highly dependent on image data. To solve the above problem, we propose a novel approach based on Temporal Convolutional Networks and Long Short-Term Memory networks to predicting urban waterlogging depth with Waterlogging Monitoring Station. The difficulty of data acquisition is small though Waterlogging Monitoring Station and TCN-LSTM model can be used to predict timely waterlogging depth. Waterlogging Monitoring Station is developed which integrates an automatic rain gauge and a water gauge. The rainfall and waterlogging depth can be obtained by periodic sampling at some areas with Waterlogging Monitoring Station. Precise hydrological data such as waterlogging depth and rainfall collected by Waterlogging Monitoring Station are used as training samples. Then training samples are used to train TCN-LSTM model, and finally a model with good prediction effect is obtained. The experimental results show that the difficulty of data acquisition is small, the complexity is low and the proposed TCN-LSTM hybrid model can properly predict the waterlogging depth of the current regional. There is no need for high dependence on image data. Meanwhile, compared with machine learning model and RNN model, TCN-LSTM model has higher prediction accuracy for time series data. Overall, the low-cost method proposed in this study can be used to obtain timely waterlogging warning information, and enhance the possibility of using existing social networks and traffic surveillance video systems to perform opportunistic waterlogging sensing.</div

Confirmation of inheritance of a single modified or nonmodified <i>GFP</i> gene in each of 7 T2 progeny from 6 individual T1 generation plants – and evidence of Cas9 gene and/or sgRNA gene silencing in T2 progeny.

<p>DNA was isolated from each of 7 T2 progeny from each of 6 different progenitor T1 plants (T1 Plants #3 to #8). PCR was used to amplify a 250 bp DNA fragment containing the sgRNA target region of the nonfunctional, out-of-frame, <i>GFP</i> gene. DNA sequencing of the fragment provided the sequence of the 31 bp region displayed for each of the 42 T2 plants. The DNA sequence of the original <i>GFP</i> gene is provide as the top line in each column along with the sequence of one mutagenized <i>GFP</i> gene found in a leaf of the original progenitor T1 plant. A) DNA sequences of three groups of T2 plants in which there has been a Cas9/sgRNA-mediated gene modification including insertion of an A nucleotide (Plant #4 progeny), a T nucleotide (Plant #5 progeny), or deletion of an A nucleotide (Plant #6 progeny) that restored a proper reading frame and resulted in T2 progeny displaying a green fluorescence phenotype, B) DNA sequences of three groups of T2 plants (progeny of T1 Plants #3, #7 and #8) in which there was no inherited Cas9/sgRNA-mediated gene modification. (GF−), No green fluorescence phenotype; (GF+), Green fluorescence phenotype; (Mut+), Inherited mutagenized <i>GFP</i> gene; (Mut-), No inherited mutagenized <i>GFP</i> gene. C, D, E, and F) DNA sequencing traces from sequencing of PCR amplified Cas9/sgRNA target sites from a single leaf of an individual T2 progeny from T1 progenitor Plants #3, #7 and #8, respectively. G) A DNA sequencing trace from sequencing of the PCR amplified Cas9/sgRNA target sites isolated from a single leaf of T1 Plant #1 showing multiple overlapping DNA peaks caused by the presence of multiple different DNA sequences in the separate mutagenized <i>GFP</i> genes present in different patches of cells scattered throughout the leaf.</p

Design of a Cas9/sgRNA system for mutagenesis and restoration of activity of a non-functional (out-of-frame) mutant <i>GFP</i> gene in Arabidopsis.

<p>Design of a Cas9/sgRNA system for mutagenesis and restoration of activity of a non-functional (out-of-frame) mutant <i>GFP</i> gene in Arabidopsis.</p