<i>In vitro</i> method to quantify dermal absorption of pesticide residues

All pesticides must go through a rigorous risk assessment process in order to show that they are safe for use. With respect to dermal risk assessment for re-entry workers, the absorption value applied to predict systemic dose from this external exposure is obtained by testing liquid forms of the pesticide <i>in vivo</i> and/or <i>in vitro</i>. However, in a real exposure scenario, the worker would be exposed to a dried residue, for which little or no absorption data are available. This study has developed a novel methodology for assessing the dermal absorption of pesticides from dried residues and aims ultimately to use this methodology to obtain more realistic absorption values for the risk assessment

Sequence analysis and editing for bisulphite genomic sequencing projects

Bisulphite genomic sequencing is a widely used technique for detailed analysis of the methylation status of a region of DNA. It relies upon the selective deamination of unmethylated cytosine to uracil after treatment with sodium bisulphite, usually followed by PCR amplification of the chosen target region. Since this two-step procedure replaces all unmethylated cytosine bases with thymine, PCR products derived from unmethylated templates contain only three types of nucleotide, in unequal proportions. This can create a number of technical difficulties (e.g. for some base-calling methods) and impedes manual analysis of sequencing results (since the long runs of T or A residues are difficult to align visually with the parent sequence). To facilitate the detailed analysis of bisulphite PCR products (particularly using multiple cloned templates), we have developed a visually intuitive program that identifies the methylation status of CpG dinucleotides by analysis of raw sequence data files produced by MegaBace or ABI sequencers as well as Staden SCF trace files and plain text files. The program then also collates and presents data derived from independent templates (e.g. separate clones). This results in a considerable reduction in the time required for completion of a detailed genomic methylation project

Is the Skin Absorption of Hydrocortisone Modified by the Variability in Dosing Topical Products?

Fingertip units have been proposed as a tool to standardize topical therapy with semisolid formulations. However, no studies to date have characterized the variability in dosing by patients using this concept and whether this variability ultimately affects the topical absorption of drugs. This work aimed to answer these two questions. A first study determined the dose measured, the area of spread and the area-normalized dose for a 1% hydrocortisone cream and ointment applied by members of the public using this dosing approach before and after brief counselling. Then, in vivo tape-stripping and in vitro permeation studies investigated whether the variability in the area-normalized dose altered the skin absorption of hydrocortisone. Participants applied greater doses and spread them over larger areas after a short counselling intervention leading to smaller area-normalized doses. In vivo hydrocortisone uptake by the stratum corneum was significantly greater for the higher normalized dose and the differences were further supported by the in vitro permeation studies. However, these differences were relatively small and not proportional to the increase in normalized dose. This work shows that, following brief advice, patients and carers can apply consistent and sufficient doses of corticosteroids whilst minimizing risks and variability in hydrocortisone absorption

非線型Klein-Gordon方程式の大域解の存在に対する一注意(函数解析を用いた偏微分方程式の研究)

<p><b>Fig 1A: Comparison of <i>PLAGL1</i> transcription derived from three alternative promoters (P1; P2; P5) in a panel of human tissues</b>. For each tissue, three individual RT-PCR reactions were performed, using primers that specifically amplify transcripts from P1 (lane ‘1’), P2 (lane ‘2’) or P5 (lane ‘5’) (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185678#sec009" target="_blank">Materials and methods</a>). The top two panels show <i>PLAGL1</i> in cDNA samples: Ov, ovary; Ki, kidney; Th, thymus; Co, colon; He, heart; Li, liver; Br, brain; Pr, prostate; Sp, spleen; Pl, placenta; Ad, adipose tissue; Bl, bladder; Ce, cervix; Oe, oesophagus; Lu, lung; Sk, skeletal muscle; Sm, small intestine; Te, testes; Th, thyroid; Tr, trachea; Pa, pancreas. The molecular weight marker is GeneRuler 100bp+ ladder (Invitrogen). <i>PLAGL1</i> transcripts are subject to complex alternative splicing of the 5′-UTR, and the two major bands (arrowed) result from alternative splicing of the coding exons. The third panel down shows RT-PCR of the housekeeping gene <i>RPLP0</i> as a loading control for each reaction. All non-template (water) controls were negative. <b>Fig 1B: Comparison of <i>PLAGL1</i> transcription from the three alternative promoters in primary blood cells.</b> cDNA samples: leukocytes (peripheral blood leukocytes); CD14+ cells (monocytes); activated CD4+ cells (T helper/inducer cells); CD8+ cells (T suppressor/cytotoxic cells); activated CD8+ cells; CD19+ cells (B-lymphocytes); activated CD19+; NK cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185678#sec009" target="_blank">Materials and methods</a>). The panel below shows <i>RPLP0</i> as loading controls for each reaction. All non-template controls were negative. <b>Fig 1C: qPCR analysis of promoter P5 transcription in a range of blood cell RNAs, compared with pancreas and placenta tissue.</b> The expression levels of P5 transcripts were normalised to that of the endogenous control gene <i>RPLP0</i> in each sample, and are expressed in arbitrary units. <b>Fig 1D: qPCR analysis of promoter P2 transcription using the same cDNA samples as in Fig. 1C, for comparison.</b> Similar to the P5 qPCR, expression levels were normalised to RPLP0 expression, and are in arbitrary units.</p

Alternative splicing of <i>PLAGL1</i> P5 transcripts isolated from A) activated CD4+ cell cDNA and B) activated CD19+ cell cDNA.

<p>The locus is shown at the top with the location of the five <i>PLAGL1</i> promoters indicated, and exon ‘3a’ and ‘3b’ (not to scale).</p

Allelic expression of <i>PLAGL1</i> transcripts derived from promoter P5, isolated from peripheral blood leukocytes from a normal individual (sample H3), heterozygous for SNP rs2092894.

<p><b>A</b>: SNP alleles represented in cloned P5 transcripts indicate biallelic transcription as both alleles are represented. Data for P1 and P2 for sample H3 have been published previously, showing monoallelic and biallelic expression respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185678#pone.0185678.ref007" target="_blank">7</a>]. <b>B:</b> Representative electropherograms of two cloned P5 transcripts indicating the alternative alleles at the location of the SNP (arrows). The sequence shown is 5'-3' with respect to the <i>PLAGL1</i> sequence. A representative sequence from this amplicon (clone 11; lower panel) has been deposited in GenBank (MF361142).</p

Methylation analysis of CpG sites close to the P5 TSS in peripheral blood leukocytes and placenta tissue.

<p>A) DNA sequence of the region close to the promoter P5 exon, written in the 5′-3′ orientation with respect to <i>PLAGL1</i> (chr6: 144054338–144055350; GRCh38/hg38). The 70-bp P5 exon is shown in bold. The amplicons for methylation analysis are highlighted in grey, and the locations of PCR primers are underlined. (Note: actual primer sequences are for bisulphite-converted DNA, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185678#sec009" target="_blank">Materials and methods</a>). Amplicon 1: CpG sites 1–3, labelled from 5′ end with respect to P5, are highlighted in white. CpG site 4 was not analyzed. Amplicon 2: CpG sites 5–9 highlighted in white (note that CpGs 5 and 6 are located within a primer). A further CpG in the UCSC Genome browser reference sequence is a SNP (rs6901529). B) Methylation analysis by bisulphite sequencing for the two separate amplicons for peripheral blood leukocyte genomic DNA (individual H1 and H7, and amplicon 1 only for H2) and placenta genomic DNA. Squares represent CpG sites: black indicates a methylated CpG site; white, an unmethylated CpG site. Grey shading indicates a CpG site that was neither CG nor TG.</p