DNA提取过程中混入的实验室空气微生物DNA定量

元基因组测序方法为微生物研究提供了有力的工具。但其中的DNA提取过程,会不可避免地混入实验室中的空气微生物。这些微生物DNA,是否会对一些极微量的元基因组检测(如皮肤样本等)结果造成影响,有多大影响,仍没有明确结论。本研究首先收集了实验室空气样品,用16S rRNA引物建立了基于qPCR的标准曲线,并检测了在开放环境下提取DNA过程中可掺杂的环境微生物DNA量。然后在开放环境下提取纯水DNA样品并进行元基因组分析,以确定掺杂环境微生物的种类。最后分别在生物安全柜和实验室开放环境下提取皮肤样本,并用鸟枪测序方法对样本的微生物组成进行分析,以评估掺杂环境微生物对元基因组检测结果的影响。结果显示,在实验室开放环境的DNA提取过程中,环境微生物的DNA残留可达28.9 pg,可达某些极微量样本DNA总量的30%。元基因组分析显示,样品中掺杂的环境微生物主要是痤疮杆菌Cutibacteriumacnes、大肠杆菌Escherichia coli等皮肤常见细菌。与洁净皮肤样本的信息相比,开放环境下提取掺杂了数十种环境微生物,并导致主要菌种的丰度大幅降低,从而影响结果的真实性。因此,微量样品的DNA...     

Psychophysical Studies of the Itch Sensation and Itchy Skin (“Alloknesis”) Produced by Intracutaneous Injection of Histamine

Psychophysical measurements of itch and itchy skin (“alloknesis”—itch produced by innocuous mechanical stimulation) were obtained in human volunteers following intracutaneous or subcutaneous injections of histamine or papain into the volar forearm. Histamine and papain were given in doses of 0.1, 1, or 10 μg in 10 μl of saline. The effects of the depth of injection and of skin temperature on the latency, magnitude, and duration of itch were examined. Also, dose-response functions were obtained for the area of alloknesis produced by intracutaneous injections of histamine. Finally, the neural mechanisms underlying the spread of alloknesis were investigated via local anesthesia of the skin.

Intracutaneous and subcutaneous injections of histamine, but not papain, produced a sensation of itch without pain. The latency of itch was shorter after an intracutanous than after a subcutaneous injection of histamine. The mean latencies of itch produced by a 1-μg dose were 9.5 and 23.0 sec for intracutaneous and subcutaneous injections, respectively. No differences were observed in the magnitude or duration of itch. Similarly, the latency of itch was increased when the skin temperature at injection site was lowered to 15°C, whereas the magnitude and duration of itch were unaffected.

Intracutaneous and subcutaneous injections of histamine produced similar areas of alloknesis. However, the magnitude and duration of alloknesis were dependent on dose. The mean maximum areas of alloknesis produced by intracutaneous injections of 0.1, 1, and 10 μg of histamine were 28.3, 47.2, and 43.8 cm², respectively. Alloknesis was present at 2 min after injection, increased to a maximum area without 10 min, and then gradually decreased during the next 25-40 min. Once developed, the area was typically abolished when the injection site was cooled to 1-4°C. Rewarming the injection site to 38°C returned the area to its original size. Also, when histamine was injected into a small area of skin anesthetized with Xylocaine, alloknesis failed to develop until the anesthetic wore off. In addition, when histamine was injected 5 mm distal to a thin mediolateral anesthetic barrier, alloknesis did not develop on the proximal side of the barrier.

These results demonstrate that histamine is effective in producing itch and alloknesis, and should be useful in correlative neurophysiological studies of the underlying mechanisms. It is suggested that both peripheral and central neural mechanisms are involved in the development of alloknesis.     

Design of NK‐2‐derived peptides with improved activity against equine sarcoid cells

Equine sarcoid is a topically accessible model for the evaluation of anticancer peptides acting by physical membrane disruption avoiding the complexity of a systemic application. We aim at evaluating and improving natural peptides for host defence as lead structures, where we focus on the cationic and amphipathic peptide NK‐2. Cytotoxicity tests, fluorescence microscopy and a chip‐based biosensor, which enabled real‐time monitoring of cell metabolism, were applied. Cancer cell killing was dynamic with an initial phase of increased cellular respiration, followed by membrane destruction. NK‐2 was substantially improved and shortened. Novel peptides exhibited a fivefold improved activity against sarcoid cells, while haemolysis remained almost unaltered. Similar Zeta potential and similar amount of surface phosphatidylserine of sarcoid and normal skin cells are responsible for a lack of selectivity between these two cell types. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Review of the results of the in vivo dosimetry during total skin electron beam therapy

This work reviews results of in vivo dosimetry (IVD) for total skin electron beam (TSEB) therapy, focusing on new methods, data emerged within 2012. All quoted data are based on a careful review of the literature reporting IVD results for patients treated by means of TSEB therapy. Many of the reviewed papers refer mainly to now old studies and/or old guidelines and recommendations (by IAEA, AAPM and EORTC), because (due to intrinsic rareness of TSEB-treated pathologies) only a limited number of works and reports with a large set of numerical data and proper statistical analysis is up-to-day available in scientific literature. Nonetheless, a general summary of the results obtained by the now numerous IVD techniques available is reported; innovative devices and methods, together with areas of possible further and possibly multicenter investigations for TSEB therapies are highlighted.     

Confocal Raman microspectroscopy on excised human skin: uncertainties in depth profiling and mathematical correction applied to dermatological drug permeation

Confocal Raman microspectroscopy represents the advantage of giving structural and conformational information on samples without any destructive treatment. Recently, several studies were achieved to study the skin hydration, endogenous and exogenous molecules repartition in the skin using the confocal feature of this technique. Meanwhile, when working through a material boundary with a different refractive index, the main limitation remains the spatial precision, especially the distortion in the depth and the depth resolution. Recently, several authors described mathematical models to correct the depth and the resolution values. In this study, we combined theoretical approaches, proposed by different authors with experimental measurements to try to find out the most appropriate approach for correction. We then applied the corrections on in‐depth profiles tracking the penetration of Metronidazole, a drug produced by Galderma for rosacea treatment, through excised human skin. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Angiogenesis QTL on Mouse Chromosome 8 Colocalizes with Differential β-Defensin Expression

Identification of genetic factors that modify complex traits is often complicated by gene-environment interactions that contribute to the observed phenotype. In model systems, the phenotypic outcomes quantified are typically traits that maximize observed variance, which in turn, should maximize the detection of quantitative trait loci (QTL) in subsequent mapping studies. However, when the observed trait is dependent on multiple interacting factors, it can complicate genetic analysis, reducing the likelihood that the modifying mutation will ultimately be found. Alternatively, by focusing on intermediate phenotypes of a larger condition, we can reduce a model’s complexity, which will, in turn, limit the number of QTL that contribute to variance. We used a novel method to follow angiogenesis in mice that reduces environmental variance by measuring endothelial cell growth from culture of isolated skin biopsies that varies depending on the genetic source of the tissue. This method, in combination with a backcross breeding strategy, is intended to reduce genetic complexity and limit the phenotypic effects to fewer modifier loci. We determined that our approach was an efficient means to generate recombinant progeny and used this cohort to map a novel s.c. angiogenesis QTL to proximal mouse chromosome (Chr.) 8 with suggestive QTL on Chr. 2 and 7. Global mRNA expression analysis of samples from parental reference strains revealed β-defensins as potential candidate genes for future study.     

Two loss-of-function alleles of the glutathione S-transferase (GST) gene cause anthocyanin deficiency in flower and fruit skin of peach (Prunus persica)

Flower and fruit colors are important agronomic traits. To date, there is no forward genetic evidence that the glutathione S-transferase (GST) gene is responsible for the white flower color in peach (Prunus persica). In this study, genetic analysis indicated that the white-flower trait is monogenetic, is recessive to the non-white allele, and shows pleiotropic effects with non-white-flowered types. The genetic locus underpinning this trait was mapped onto chromosome 3 between 0.421951 and 3.227115 Mb by using bulked segregant analysis in conjunction with whole-genome sequencing, and was further mapped between 0 and 1.178149 Mb by using the backcross 1 (BC₁) population. Finally, the locus was fine-mapped within 535.974- and 552.027-kb intervals by using 151 F₂ individuals and 75 individuals from a BC₁ self-pollinated (BC₁S₁) population, respectively. Pp3G013600, encoding a GST that is known to transport anthocyanin, was identified within the mapping interval. The analysis of genome sequence data showed Pp3G013600 in white flowers has a 2-bp insertion or a 5-bp deletion in the third exon. These variants likely render the GST non-functional because of early stop codons that reduce the protein length from 215 amino acids to 167 and 175 amino acids, respectively. Genetic markers based on these variants validated a complete correlation between the GST loss-of-function alleles and white flower in 128 peach accessions. This correlation was further confirmed by silencing of Pp3G013600 using virus-induced gene silencing technology, which reduced anthocyanin accumulation in peach fruit. The new knowledge from this study is useful for designing peach breeding programs to generate cultivars with white flower and fruit skin.     

Effects of scalding and dehairing of pig carcasses at abattoirs on the visibility of welfare-related lesions

There is increasing interest in developing abattoir-based measures to assist in determining the welfare status of pigs. The primary aim of this study was to determine the most appropriate place on the slaughter line to conduct assessments of welfare-related lesions, namely apparent aggression-related skin lesions (hereafter referred to as ‘skin lesions’), loin bruising and apparent tail biting damage. The study also lent itself to an assessment of the prevalence of these lesions, and the extent to which they were linked with production variables. Finishing pigs processed at two abattoirs on the Island of Ireland (n=1950 in abattoir A, and n=1939 in abattoir B) were used. Data were collected over 6 days in each abattoir in July 2014. Lesion scoring took place at two points on the slaughter line: (1) at exsanguination (slaughter stage 1 (SS1)), and (2) following scalding and dehairing of carcasses (slaughter stage 2 (SS2)). At both points, each carcass was assigned a skin and tail lesion score ranging from 0 (lesion absent) to 3 or 4 (severe lesions), respectively. Loin bruising was recorded as present or absent. Differences in the percentage of pigs with observable lesions of each type were compared between SS1 and SS2 using McNemar/McNemar-Bowker tests. The associations between each lesion type, and both cold carcass weight and condemnations, were examined at batch level using Pearson’s correlations. Batch was defined as the group of animals with a particular farm identification code on a given day. The overall percentage of pigs with a visible skin lesion (i.e. score>0) decreased between SS1 and SS2 (P<0.001). However, the percentage of pigs with a severe skin lesion increased numerically from SS1 to SS2. The percentage of pigs with a visible tail lesion and with loin bruising also increased between SS1 and SS2 (P<0.001). There was a positive correlation between the percentage of carcasses that were partially condemned, and the percentage of pigs with skin lesions, tail lesions and loin bruising (P<0.05). In addition, as the batch-level frequency of each lesion type increased, average cold carcass weight decreased (P<0.001). These findings suggest that severe skin lesions, tail lesions and loin bruising are more visible on pig carcasses after they have been scalded and dehaired, and that this is when abattoir-based lesion scoring should take place. The high prevalence of all three lesion types, and the links with economically important production parameters, suggests that more research into identifying key risk factors is warranted.     

Mesoglycan induces keratinocyte activation by triggering syndecan-4 pathway and the formation of the annexin A1/S100A11 complex

Wound healing is a dynamic process comprising multiple events, such as inflammation, re-epithelialization, and tissue remodeling. Re-epithelialization phase is characterized by the engagement of several cell populations, mainly of keratinocytes that sequentially go through cycles of migration, proliferation, and differentiation to restore skin functions. Troubles can arise during the re-epithelialization phase of skin wound healing particularly in keratinocyte migration, resulting in chronic non-healing lesions, which represent a serious clinical problem. Over the last decades, the efforts aimed to find new pharmacological approaches for wound care were made, yet almost all current therapeutic strategies used remain inadequate or even ineffective. As such, it is crucial to identify new drugs that can enable a proper regeneration of the epithelium in wounded skin. Here, we have investigated the effects of the fibrinolytic drug mesoglycan, a glycosaminoglycans mixture derived from porcine intestinal mucosa on HaCaT human keratinocytes that were used as in vitro experimental model of skin re-epithelialization. We found that mesoglycan induces keratinocyte migration and early differentiation by triggering the syndecan-4/PKCα pathway and that these effects were at least in part, because of the formation of the annexin A1/S100A11 complex. Our data suggest that mesoglycan may be useful as a new pro-healing drug for skin wound care.