Mutant Nrf2E79Q enhances the promotion and progression of a subset of oncogenic Ras keratinocytes and skin tumors.

Squamous cell carcinomas (SCCs), including lung, head & neck, bladder, and skin SCCs often display constitutive activation of the KEAP1-NRF2 pathway. Constitutive activation is achieved through multiple mechanisms, including activating mutations in NFE2L2 (NRF2). To determine the functional consequences of Nrf2 activation on skin SCC development, we assessed the effects of mutant Nrf2E79Q expression, one of the most common activating mutations in human SCCs, on tumor promotion and progression in the mouse skin multistage carcinogenesis model using a DMBA-initiation/TPA-promotion protocol where the Hras A->T mutation (Q61L) is the canonical driver mutation. Nrf2E79Q expression was temporally and conditionally activated in the epidermis at two stages of tumor development: 1) after DMBA initiation in the epidermis but before cutaneous tumor development and 2) in pre-existing DMBA-initiated/TPA-promoted squamous papillomas. Expression of Nrf2E79Q in the epidermis after DMBA initiation but before tumor occurrence inhibited the development/promotion of 70% of squamous papillomas. However, the remaining papillomas often displayed non-canonical Hras and Kras mutations and enhanced progression to SCCs compared to control mice expressing wildtype Nrf2. Nrf2E79Q expression in pre-existing tumors caused rapid regression of 60% of papillomas. The remaining papillomas displayed the expected canonical Hras A->T mutation (Q61L) and enhanced progression to SCCs. These results demonstrate that mutant Nrf2E79Q enhances the promotion and progression of a subset of skin tumors and alters the frequency and diversity of oncogenic Ras mutations when expressed early after initiation.

Efficacy of Short Novel Antimicrobial Peptides in a Mouse Model of Staphylococcus pseudintermedius Skin Infection.

As the clinical application of antibiotics for bacterial skin infections in companion animals becomes increasingly prevalent, the issue of bacterial resistance has become more pronounced. Antimicrobial peptides, as a novel alternative to traditional antibiotics, have garnered widespread attention. In our study, synthetic peptides ADD-A and CBD3-ABU were tested against Staphylococcus pseudintermedius skin infections in KM mice. ADD-A was applied topically and through intraperitoneal injection, compared with control groups and treatments including CBD3-ABU, ampicillin sodium, and saline. Wound contraction, bacterial counts and histology were assessed on days 3 and 11 post-infection. ADD-A and ampicillin treatments significantly outperformed saline in wound healing (p < 0.0001 and p < 0.001, respectively). ADD-A also showed a markedly lower bacterial count than ampicillin (p < 0.0001). Histologically, ADD-A-applied wounds had better epidermal continuity and a thicker epidermis than normal, with restored follicles and sebaceous glands. ADD-A's effectiveness suggests it as a potential alternative to antibiotics for treating skin infections in animals.

Molecular heterogeneity of quiescent melanocyte stem cells revealed by single-cell RNA-sequencing.

Melanocyte stem cells (McSCs) of the hair follicle are a rare cell population within the skin and are notably underrepresented in whole-skin, single-cell RNA sequencing (scRNA-seq) datasets. Using a cell enrichment strategy to isolate KIT+/CD45- cells from the telogen skin of adult female C57BL/6J mice, we evaluated the transcriptional landscape of quiescent McSCs (qMcSCs) at high resolution. Through this evaluation, we confirmed existing molecular signatures for qMcCS subpopulations (e.g., Kit+, Cd34+/-, Plp1+, Cd274+/-, Thy1+, Cdh3+/-) and identified novel qMcSC subpopulations, including two that differentially regulate their immune privilege status. Within qMcSC subpopulations, we also predicted melanocyte differentiation potential, neural crest potential, and quiescence depth. Taken together, the results demonstrate that the qMcSC population is heterogeneous and future studies focused on investigating changes in qMcSCs should consider changes in subpopulation composition.

Molecular and spatial landmarks of early mouse skin development.

A wealth of specialized cell populations within the skin facilitates its hair-producing, protective, sensory, and thermoregulatory functions. How the vast cell-type diversity and tissue architecture develops is largely unexplored. Here, with single-cell transcriptomics, spatial cell-type assignment, and cell-lineage tracing, we deconstruct early embryonic mouse skin during the key transitions from seemingly uniform developmental precursor states to a multilayered, multilineage epithelium, and complex dermal identity. We identify the spatiotemporal emergence of hair-follicle-inducing, muscle-supportive, and fascia-forming fibroblasts. We also demonstrate the formation of the panniculus carnosus muscle (PCM), sprouting blood vessels without pericyte coverage, and the earliest residence of mast and dendritic immune cells in skin. Finally, we identify an unexpected epithelial heterogeneity within the early single-layered epidermis and a signaling-rich periderm layer. Overall, this cellular and molecular blueprint of early skin development-which can be explored at https://kasperlab.org/tools-establishes histological landmarks and highlights unprecedented dynamic interactions among skin cells.

mleS in Staphylococcus aureus Contributes to Microaerobic Metabolic Activity, Abscess Formation, and Survival in Macrophages.

Staphylococcus aureus is subdivided into lineages termed sequence types (STs), infections of which necessitate the expression of virulence factors and metabolic adaptation to the host niche. Given that mechanisms underlying the dynamic replacement of sequence types in S. aureus populations have yet to be sufficiently determined, we investigated the role of metabolic determinants in epidemic clones. mleS, encoding the NAD+-dependent malolactic enzyme, was found to be carried by the epidemic clones ST59 and ST398, although not by ST239 and ST5. The genomic location of mleS in the metabolism-associated region flanked by the thiol-specific redox system and glycolysis operon implies that it plays significant roles in metabolism and pathogenesis. Mouse skin abscess caused by the BS19-mleS mutant strain (isogenic mleS mutant in an ST59 isolate) was significantly attenuated and associated with reductions in interleukin-6 (IL-6) and lactic acid production. mleS deletion also impaired S. aureus biofilm formation and survival in RAW264.7 cells. The BS19-mleS-mutant was also characterized by reduced ATP and lactic acid production under microaerobic conditions; however, NAD+/NADH levels remained unaffected. mleS is thus identified as an epidemiological marker that plays an important role in the microaerobic metabolism and pathogenesis of epidemic S. aureus clones. IMPORTANCE Given the importance of metabolic adaptation during infection, new insights are required regarding the pathogenesis of S. aureus, particularly for epidemic clones. We accordingly investigated the role of metabolic determinants that are unique to the epidemic clones ST59 and ST398. Our results provide evidence that the NAD+-dependent malolactic enzyme-coding gene mleS is an epidemiological marker that plays an important role in the microaerobic metabolism and pathogenesis of epidemic S. aureus clones.

Topical Application of Butyl Flufenamate Ointment Promotes Cranial Defect Healing in Mice by Inducing BMP2 Secretion in Skin Mesenchymal Stem Cells.

Bone defects and fractures heal slowly compared with injuries to other tissues, creating a heavy burden for patients, their families, and society. Alongside conventional treatment methods for fractures and bone defects, adjuvant therapies play an important but underappreciated role. In a previous study, we found that systemic administration of flufenamic acid promoted osteogenesis in vivo, but its side effects limited the application of our findings. In the present study, we assess the effects of external butyl flufenamate ointment on the healing of cranial defects in mice. We found that application of butyl flufenamate ointment on the surface of the skin accelerated the healing of cranial defects in mice by promoting BMP2 secretion from mouse-skin mesenchymal stem-cells. These findings indicate that butyl flufenamate ointment has potential therapeutic value for treating superficial fractures or bone defects while avoiding the toxicity and side effects of systemic medication, representing a safe and convenient adjuvant therapy to promote healing of superficial bone defects and fractures.

Overactive Wnt5a signaling disrupts hair follicle polarity during mouse skin development.

The polarity of mouse hair follicles is controlled by the Frizzled (Fzd) receptors and other membrane planar cell polarity (PCP) proteins. Whether Wnt proteins can act as PCP ligands in the skin remains unknown. Here, we show that overexpression of Wnt5a in the posterior part of mouse embryos causes a local disruption of hair follicle orientation. The misoriented hair follicle phenotype in Wnt5a overexpressing mice can be rescued by a heterozygous loss of Fzd6, suggesting Wnt5a is likely to signal through Fzd6. Although the membrane distribution of PCP proteins seems unaffected by Wnt5a overexpression, transcriptional profiling analyses identify a set of genes as potential targets of the skin polarization program controlled by Wnt5a/Fzd6 signaling. Surprisingly, deletion of Wnt5a globally or in the posterior part of the mouse embryos does not affect hair follicle orientation. We show that many other Wnts are highly expressed in the developing skin. They can activate the Fzd6 signaling pathway in vitro and may act together with Wnt5a to regulate the Fzd6-mediated skin polarization. Our experiments demonstrate for the first time that Wnt5a can function as an orienting cue for mouse skin PCP.

Elucidating the Ability of CGRP to Modulate Microvascular Events in Mouse Skin.

Oedema formation and polymorphonuclear leukocyte (neutrophil) accumulation are involved in both acute and chronic inflammation. Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide that is released from stimulated sensory nerves. CGRP is a potent vasodilator neuropeptide, especially when administered to the cutaneous microvasculature, with a long duration of action. Here, we have investigated the ability of vasodilator amounts of CGRP to modulate oedema formation and neutrophil accumulation induced in the cutaneous microvasculature of the mouse. To learn more about the mechanism of action of endogenous CGRP, we have investigated the response to the inflammatory stimulants tumour necrosis factor alpha (TNFα) and carrageenan in three different murine models: a model where sensory nerves were depleted by resiniferatoxin (RTX); a pharmacological method to investigate the effect of a selective CGRP receptor antagonist; and a genetic approach using wildtype (WT) and αCGRP knockout (KO) mice. Our results show that exogenous CGRP potentiates oedema formation induced by substance P (SP) and TNFα. This is further supported by our findings from sensory nerve-depleted mice (in the absence of all neuropeptides), which indicated that sensory nerves are involved in mediating the oedema formation and neutrophil accumulation induced by TNFα, and also carrageenan in cutaneous microvasculature. Furthermore, endogenous CGRP was shown to contribute to this inflammatory response as carrageenan-induced oedema formation is attenuated in WT mice treated with the CGRP receptor antagonist, and in αCGRPKO mice. It is therefore concluded that CGRP can contribute to inflammation by promoting oedema formation in skin, but this response is dependent on the pro-inflammatory stimulus and circumstance.

Mouse dendritic cells and other myeloid subtypes in healthy lymph nodes and skin: 26-Color flow cytometry panel for immune phenotyping.

This novel 26-color flow cytometry panel allows the detailed immune phenotyping of the complex network of myeloid cells in murine lymph nodes and skin. With the optimized panel the different murine DC-subsets and other myeloid cell types can be identified and further characterized for co-stimulatory and inhibitory surface molecules.

Uptake of nanoparticles from sunscreen physical filters into cells arising from increased environmental microwave radiation: increased potential risk of the use of sunscreens to human health.

This study examines the microwave chemical risks posed by photocatalysts present in sunscreens (physical filters) against the increasing use of microwaves (radio waves) in the environment, sometimes referred to as electronic smog. Specifically, the study assesses the damage caused by silica-coated physical filters (photocatalysts, TiO2⋅ and/or ZnO) contained in commercially available sunscreens and fresh silica-coated ZnO for sunscreens to mouse skin fibroblasts cells (NIH/3T3) evaluated in vitro by the life/death of cells using two types of electromagnetic waves: UV light and microwave radiation, and under simultaneous irradiation with both UV light and microwaves. Conditions of the electromagnetic waves were such as to be of lower light irradiance than that of UVA/UVB radiation from incident sunlight, and with microwaves near the threshold power levels that affect human health. The photocatalytic activity of the physical filters was investigated by examining the degradation of the rhodamine B (RhB) dye in aqueous media and by the damage caused to DNA plasmids from E. coli. Compared to the photocatalytic activity of ZnO and TiO2 when irradiated with UV light alone, a clear enhanced photocatalytic activity was confirmed upon irradiating these physical filters concurrently with UV and microwaves. Moreover, the uptake of these metal oxides into the NIH/3T3 cells led to the death of these cells as a result of the enhanced photocatalytic activity of the metal oxides on exposure to microwave radiation.

Dual nature of mouse periderm structure, function and fate.

The periderm is the outer layer of embryonic skin, which is essential for the development of the epidermis and the establishment of its barrier function. In humans, the periderm is a monolayer and is shed prenatally. The structure and fate of the mouse periderm remain puzzling. Using electron microscopy of mouse skin between the embryonic day 14.5 and the postnatal Day 2, we have assessed the periderm structure and its shedding patterns. In contrast to human periderm, the mouse periderm appears to be bilayered, comprising a presumably absorptive outer periderm with numerous microvilli and an inner periderm packed with specific (glycogen-containing?) granules. The desmosomes between the inner periderm and the underlying epidermis are scarce, and they are totally absent between the inner and the outer periderm. The inner and outer periderms in mice are shed at different stages of development: the outer periderm is shed in utero in conjunction with barrier acquisition (E16-18), while the inner periderm is retained postnatally. We assume that the bilayered structure of the mouse periderm and the postnatal retention of its inner layer represent the evolutionary adaptations in ancestral rodents which helps altricial newborns of their extant descendants to cope with acute dehydration right after birth and/or provides a substrate for proper bacterial colonization of newborn skin.

Evaluation of Short-Chain Antimicrobial Peptides With Combined Antimicrobial and Anti-inflammatory Bioactivities for the Treatment of Zoonotic Skin Pathogens From Canines.

The incidence of zoonotic Staphylococcus pseudintermedius and Microsporum canis infections is rapidly growing worldwide in the context of an increasing frequency of close contact between animals and humans, presenting challenges in both human and veterinary medicine. Moreover, the development of microbial resistance and emergence of recalcitrant biofilms, accompanied by the insufficiency of new antimicrobial agents, have become major obstacles in treating superficial skin infections caused by various microbes including S. pseudintermedius and M. canis. Over recent years, the prospects of antimicrobial peptides as emerging antimicrobials to combat microbial infections have been demonstrated. In our study, two novel short-chain peptides, namely, allomyrinasin and andricin B, produced by Allomyrina dichotoma and Andrias davidianus, were revealed to exhibit potent antimicrobial efficacy against clinical isolates of S. pseudintermedius and M. canis with remarkable and rapid fungicidal and bactericidal effects, while allomyrinasin exhibited inhibition of biofilm formation and eradication of mature biofilm. These peptides displayed synergistic activity when combined with amoxicillin and terbinafine against S. pseudintermedius and M. canis. Cytoplasmic leakage via cytomembrane permeabilization serves as a mechanism of action. Extremely low hemolytic activity and serum stability in vitro, as well as superior anti-infective efficacy in reducing bacterial counts and relieving the inflammatory response in vivo, were detected. The potent antibacterial, antifungal, and anti-inflammatory activities of allomyrinasin and andricin B might indicate promising anti-infective alternatives for the treatment of S. pseudintermedius and M. canis infections in the context of human and veterinary medicine.

Histological and microbiological evaluation of surgical wound closure in mouse skin with cyanoacrylate (Histoacryl®) in comparison to poliglecaprone (Monocryl®) traditional suture.

Incisional wound closure is a key surgical step to facilitate tissue healing, reduce the risk of infection and obtain esthetic and functional recovery. Cyanoacrylates such as Histoacryl® have become a popular choice in surgical veterinary practice. However, how Histoacryl® is affecting tissue regeneration and bacterial load in the wound in comparison to poliglecaprone (Monocryl®) traditional suture methods remains to be determined. This work aimed to evaluate how wounded tissue responds to traditional suture with Monocryl® (poliglecaprone 25/4-0) and Histoacryl®, as well as provide evidence of their effects on wound healing in mice. Fortyeight hours after the incisional procedure, wound tissue biopsies were prepared for histological and microbiological analysis. Biopsies were fixed and colored with Mallory's trichrome and hematoxylin-eosin stains. For microbiological assays, biopsies were suspended in tryptic soy broth (TSB) and 1/10 diluted to evaluate the number of CFU in nutrient agar plates. Our results show no differences between Histoacryl® and Monocryl® traditional suture suggesting that both methods could be used to treat wounds in small animals such as rodents.

Topical Vitamin D Receptor Antagonist/Partial-Agonist Treatment Induces Epidermal Hyperproliferation via RARγ Signaling Pathways.

Vitamin D and A derivatives are well-known endogenous substances responsible for skin homeostasis. In this study we topically treated shaved mouse skin with a vitamin D agonist (MC903) or vitamin D antagonist/partial agonist (ZK159222) and compared the changes with acetone (control treatment) treatment for 14 days. Topical treatment with ZK159222 resulted in increased expression of genes involved in retinoic acid synthesis, increased retinoic acid concentrations and increased expression of retinoid target genes. Clustering the altered genes revealed that heparin-binding epidermal growth factor-like growth factor, the main driver of epidermal hyperproliferation, was increased via RARγ-mediated pathways, while other clusters of genes were mainly decreased which were comparable to the changes seen upon activation of the RARα-mediated pathways. In summary, we conclude that epidermal hyperproliferation of mouse skin in response to a topically administered vitamin D receptor antagonist/partial agonist (ZK159222) is induced via increased retinoic acid synthesis, retinoic acid levels and increased RARγ-mediated pathways.

Magnetic resonance contrast agents in optical clearing: Prospects for multimodal tissue imaging.

Skin optical clearing effect ex vivo and in vivo was achieved by topical application of low molecular weight paramagnetic magnetic resonance contrast agents. This novel feature has not been explored before. By using collimated transmittance the diffusion coefficients of three clinically used magnetic resonance contrast agents, that is Gadovist, Magnevist and Dotarem as well as X-ray contrast agent Visipaque in mouse skin were determined ex vivo as (4.29 ± 0.39) × 10-7 cm2 /s, (5.00 ± 0.72) × 10-7 cm2 /s, (3.72 ± 0.67) × 10-7 cm2 /s and (1.64 ± 0.18) × 10-7 cm2 /s, respectively. The application of gadobutrol (Gadovist) resulted in efficient optical clearing that in general, was superior to other contrast agents tested and allowed to achieve: (a) more than 12-fold increase of transmittance over 10 minutes after application ex vivo; (b) markedly improved images of skin architecture obtained with optical coherence tomography; (c) an increase of the fluorescence intensity/background ratio in TagRFP-red fluorescent marker protein expressing tumor by five times after 15 minutes application into the skin in vivo. The obtained results have immediate implications for multimodality imaging because many contrast agents are capable of simultaneously enhancing the contrast of multiple imaging modalities.

Coordinated hedgehog signaling induces new hair follicles in adult skin.

Hair follicle (HF) development is orchestrated by coordinated signals from adjacent epithelial and mesenchymal cells. In humans this process only occurs during embryogenesis and viable strategies to induce new HFs in adult skin are lacking. Here, we reveal that activation of Hedgehog (Hh) signaling in adjacent epithelial and stromal cells induces new HFs in adult, unwounded dorsal mouse skin. Formation of de novo HFs recapitulated embryonic HF development, and mature follicles produced hair co-occurring with epithelial tumors. In contrast, Hh-pathway activation in epithelial or stromal cells alone resulted in tumor formation or stromal cell condensation respectively, without induction of new HFs. Provocatively, adjacent epithelial-stromal Hh-pathway activation induced de novo HFs also in hairless paw skin, divorced from confounding effects of pre-existing niche signals in haired skin. Altogether, cell-type-specific modulation of a single pathway is sufficient to reactivate embryonic programs in adult tissues, thereby inducing complex epithelial structures even without wounding.

We are born with all the hair follicles that we will ever have in our life. These structures are maintained by different types of cells (such as keratinocytes and fibroblasts) that work together to create hair. Follicles form in the embryo thanks to complex molecular signals, which include a molecular cascade known as the Hedgehog signaling pathway. After birth however, these molecular signals are shut down to avoid conflicting messages ­ inappropriate activation of Hedgehog signaling in adult skin, for instance, leads to tumors. This means that our skin loses the ability to make new hair follicles, and if skin is severely damaged it cannot regrow hair or produce the associated sebaceous glands that keep skin moisturized. Being able to create new hair follicles in adult skin would be both functionally and aesthetically beneficial for patients in need, for example, burn victims. Overall, it would also help to understand if and how it is possible to reactivate developmental programs after birth. To investigate this question, Sun, Are et al. triggered Hedgehog signaling in the skin cells of genetically modified mice; this was done either in keratinocytes, in fibroblasts, or in both types of cells. The experiments showed that Hedgehog signaling could produce new hair follicles, but only when activated in keratinocytes and fibroblasts together. The process took several weeks, mirrored normal hair follicle development and resulted in new hair shafts. The follicles grew on both the back of mice, where hair normally occurs, and even in paw areas that are usually hairless. Not unexpectedly the new hair follicles were accompanied with skin tumors. But, promisingly, treatment with Hedgehog-pathway inhibitor Vismodegib restricted tumor growth while keeping the new follicles intact. This suggests that future work on improving "when and where" Hedgehog signaling is activated may allow the formation of new follicles in adult skin with fewer adverse effects.

The Molecular Anatomy of Mouse Skin during Hair Growth and Rest.

Skin homeostasis is orchestrated by dozens of cell types that together direct stem cell renewal, lineage commitment, and differentiation. Here, we use single-cell RNA sequencing and single-molecule RNA FISH to provide a systematic molecular atlas of full-thickness skin, determining gene expression profiles and spatial locations that define 56 cell types and states during hair growth and rest. These findings reveal how the outer root sheath (ORS) and inner hair follicle layers coordinate hair production. We found that the ORS is composed of two intermingling but transcriptionally distinct cell types with differing capacities for interactions with stromal cell types. Inner layer cells branch from transcriptionally uncommitted progenitors, and each lineage differentiation passes through an intermediate state. We also provide an online tool to explore this comprehensive skin cell atlas, including epithelial and stromal cells such as fibroblasts, vascular, and immune cells, to spur further discoveries in skin biology.

17-Oxo-docosahexaenoic acid induces Nrf2-mediated expression of heme oxygenase-1 in mouse skin in vivo and in cultured murine epidermal cells.

Recently, growing attention has been given to new classes of bioactive lipid mediators derived from ω-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), especially in the context of their role as endogenous signal modulators. One such molecule is 17-oxo-DHA, generated from DHA by the action of COX2 and a dehydrogenase. The redox-sensitive transcription factor, Nrf2 plays a key role in cellular stress responses. In the present study, the effects of 17-oxo-DHA on Nrf2-mediated expression of cytoprotective enzymes were examined in mouse skin in vivo and cultured murine epidermal JB6 cells. Topical application of 17-oxo-DHA markedly elevated the nuclear localization of Nrf2 and expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 in hairless mouse skin. In contrast to 17-oxo-DHA, the non-electrophilic metabolic precursor 17-hydroxy-DHA was a much weaker inducer of Nrf2 activation and its target protein expression. Likewise, 17-oxo-DHA significantly enhanced nuclear translocation and transcriptional activity of Nrf2 with concomitant upregulation of HO-1 expression in cultured JB6 cells. 17-Oxo-DHA was a much stronger inducer of Nrf2-mediated antioxidant response than its parent molecule, DHA. HO-1 expression was abolished in Nrf2 knockdown JB6 cells or embryo fibroblasts from Nrf2 knock out mice. 17-Oxo-DHA also markedly reduced the level of Keap1 protein by inducing ubiquitination. Mutation of Cys151 and Cys273 in Keap1 abrogated 17-oxo-DHA-induced ubiquitination and proteasome-mediated degradation of Keap1 as well as HO-1 expression, suggesting that these cysteine residues are putative sites for 17-oxo-DHA binding. Further, Keap1 degradation stimulated by 17-oxo-DHA coincided with accumulation of the autophagy substrate, p62/SQSTM1.

Modified Trizol Method for Extracting RNA from Mouse Skin / 中山大学学报(医学科学版)

@#【Objective】Due to the tough nature of skin tissue and a high presence of RNases，the isolation of skin RNA by the classical Trizol method presents a challenge. Therefore，we adapted and tested different sample treatment protocols to improve the Trizol method for high- quality extraction of skin RNA.【Methods】In this study，normal skin of mice processed by different treatments（Tri：submersion Trizol；Pro：RNA sample protector；Cry：cryopreservation in liquid nitrogen frozen and then - 80 ℃ refrigerator；LNG：liquid nitrogen grinding；Cut：scissor cutting）were used as the experimental groups. Spinal cord tissue（Sp）was used as the reference group，and skin tissue of mouse psoriasis model induced by imiquimod（IMQ）was used as the validation group. We compared skin RNA concentration，purity and integrity， as well as IL- 1β mRNA expression extracted by conventional Trizol methods（1-Tri，Nor）and modified Trizol methods（2-Tri，LNG-Tri，Tri-Cut，Pro），which were determined by UV spectrophotometry，agar gel electrophoresis and quantitative reverse transcription PCR（qRT- PCR）.【Results】① Compared with spinal cord（Sp），the total RNA of normal skin tissue extracted with the same classical Trizol method（1-Tri）was with lower yields，more obvious DNA contamination and 5S RNA bands，and higher IL-1β mRNA relative expression，suggesting that skin tissue was relatively special and the classical Trizol methods of skin RNA extraction should be improved. ② Among the different treatment methods of skin tissue，2-Tri and LNG-Tri methods resulted in higher RNA concentrations，lower RNA degradation and lower DNA contamination，and the expression of IL-1β mRNA was closer to normal levels. More importantly，the skin RNA samples extracted by the 2-Tri method can reflect more realistically the variation of IL-1β mRNA expression between normal and psoriasiform groups.【Conclusion】Improved 2-Tri or LNG-Tri method has the advantage of high quality of total RNA，and 2-Tri can more reliably reflect the mRNA expression pattern under physiological and pathological conditions.

Evaporative cooling provides a major metabolic energy sink.

OBJECTIVE: Elimination of food calories as heat could help redress the excess accumulation of metabolic energy exhibited as obesity. Prior studies have focused on the induction of thermogenesis in beige and brown adipose tissues as the application of this principle, particularly because the ß-adrenergic environment associated with thermogenic activation has been shown to have positive health implications. The counterpoint to this strategy is the regulation of heat loss; we propose that mammals with inefficient heat conservation will require more thermogenesis to maintain body temperature. METHODS: Surface temperature thermography and rates of trans-epidermal water loss were integrated to profile the total heat transfer of genetically-engineered and genetically variable mice. RESULTS: These data were incorporated with energy expenditure data to generate a biophysical profile to test the significance of increased rates of evaporative cooling. CONCLUSIONS: We show that mouse skins vary considerably in their heat retention properties, whether because of naturally occurring variation (SKH-1 mice), or genetic modification of the heat-retaining lipid lamellae (SCD1, DGAT1 or Agouti Ay obese mice). In particular, we turn attention to widely different rates of evaporative cooling as the result of trans-epidermal water loss; higher rates of heat loss by evaporative cooling leads to increased demand for thermogenesis. We speculate that this physiology could be harnessed to create an energy sink to assist with strategies aimed at treating metabolic diseases.