Polo-like kinase 1 (Plk1) in non-melanoma skin cancers

Polo-like kinase 1 (Plk1) is becoming an increasingly attractive target for cancer management. Plk1 has been shown to be over-expressed in a variety of cancers; however its role in skin cancers is not well-understood. We recently demonstrated that Plk1 is over-expressed in human melanoma and gene-knockdown as well as chemical-inhibition of Plk1 resulted in a significant decrease in melanoma cell viability and growth without affecting the growth of the normal human epidermal melanocytes (NHEMs). Further, the observed anti-proliferative response of Plk1 was found to be accompanied with a significant G2/M cell cycle arrest, mitotic catastrophe and induction of apoptosis in melanoma cells. In this study, we determined the expression profile of Plk1 in non-melanoma skin cancers viz. basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Our data demonstrated that like melanoma, Plk1 is significantly over-expressed in BCC and SCC samples. Further, we also found that compared to normal human epidermal keratinocytes (NHEKs), Plk1 was over-expressed at both the protein and mRNA levels in squamous A253 and A431 cells. In addition, a similar protein expression pattern was found for the downstream targets of Plk1, viz. Cdk1, Cyclin B1 and Cdc25C. We believe that the expression pattern of Plk1 in the various skin cancers, the insusceptibility of normal keratinocytes, to Plk1 inhibition and the easy accessibility for topical applications lends the skin as an attractive tissue for Plk1 based cancer chemoprevention and chemotherapeutic applications.     

Growth and progression of melanoma and non-melanoma skin cancers regulated by ubiquitination

Basal cell carcinomas (BCC), squamous cell carcinoma (SCC), and melanomas are the major types of skin tumors. Despite being skin cancers, the characteristics of each cancer are widely varied. BCCs often do not proliferate rapidly, and rarely metastasize. Squamous cell carcinomas are more malignant and a certain subtype of SCC is highly metastatic. Melanomas are highly proliferative and invasive, and are most frequently metastatic. Ubiquitin and ubiquitin-related proteins post-translationally modify proteins and thereby alter the functions of their target proteins. The ubiquitination process is involved in various physiological responses, including cell growth, cell death, and DNA damage repair. Accumulating evidence suggests that ubiquitin pathways are involved in different types of cancers, including skin cancers. This review describes the major ubiquitin pathways in BCC, SCC, and melanoma. The ubiquitin pathways that are activated among the skin cancers are highly diverse, which might reflect the various characteristics of these three cancer types. Meanwhile, there are also common pathways between BCC, SCC, and melanoma. Therefore, examining the ubiquitin pathways will reveal the mechanisms of these three major skin cancer types and will suggest treatment options.     

实时荧光定量PCR快速鉴别新型冠状病毒主要变异株北大核心CSCD

为建立一种快速鉴别严重急性呼吸综合征冠状病毒2 (severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)的5种主要变异株的Taq Man探针实时荧光定量PCR(real-time quantitative PCR, RT-qPCR)体系,基于SARS-CoV-2野生型及变异株alpha (N501Y、HV69-70del)、beta (E484K、K417N)、gamma (K417T、V1176F)、delta (L452R、T478K)和omicron (H655Y、N679K、P681H)序列设计特异性引物、探针,建立和优化一种鉴别新型冠状病毒(SARS-CoV-2) 5种主要变异株的Taq Man探针RT-qPCR方法,并进行该方法的特异性、敏感性、鉴别能力评价。该方法可准确区分出SARS-CoV-2野生型和突变型,与其他呼吸道病原体(n=21)无交叉,显示高特异性。该方法最低检测限为2×10;拷贝/mL,操作简单、快速、成本廉价,可用于监测SARS-CoV-2毒株的变异,精准指导疫情识别与防控。     

Rapid quantitative serological assay of staphylococcal enterotoxin B

A simple, rapid method, based on the Oudin single diffusion technique, is described for the quantitative assay of staphylococcal enterotoxin B. The method yields reproducible results without close control of such assay variables as temperature, antiserum dilution, and assay time, provided that the ionic strength is maintained above 0.2 n sodium chloride equivalent.     

Osteopontin expression in normal skin and non-melanoma skin tumors.

Osteopontin (OPN) is an adhesive, matricellular glycoprotein, whose expression is elevated in many types of cancer and has been shown to facilitate tumorigenesis in vivo. To understand the role of OPN in human skin cancer, this study is designed to determine whether OPN is expressed in premalignant [solar/actinic keratosis (AK)] and in malignant skin lesions such as squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), as well as in normal skin exposed or not exposed to sunlight. Immunohistochemical analyses showed that OPN is expressed in SCC (20/20 cases) and in AK (16/16 cases), which are precursors to SCC, but is absent or minimally expressed in solid BCC (17 cases). However, positive staining for OPN was observed in those BCC that manifest differentiation toward epidermal appendages such as keratotic BCC. In sunlight-exposed normal skin, OPN is minimally expressed in the basal cell layer, but in contrast to those not exposed to sunlight, OPN is more prominent in the spinous cell layer with increasing intensity toward the granular cell layer. Additionally, OPN is expressed in the hair follicles, sebaceous glands, and sweat glands of normal skin. In conclusion, these data suggest that OPN is associated with keratinocyte differentiation and that it is expressed in AK and SCC, which have metastatic potential, but minimally expressed in solid BCC.     

Genetic determinants of UV-susceptibility in non-melanoma skin cancer

A milieu of cytokines and signaling molecules are involved in the induction of UV-induced immune suppression and thus the etiology of non-melanoma skin cancer (NMSC). Targeting the UV-induced immunosuppression pathway, and using a large population based study of NMSC, we have investigated the risk associated with functional variants in 10 genes (IL10, IL4, IL4R, TNF, TNFR2, HTR2A, HRH2, IL12B, PTGS2, and HAL). The most prominent single genetic effect was observed for IL10. There was increasing risk for both basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) with increasing number of variant IL10 haplotypes (BCC: p_trend = 0.0048; SCC: p_trend = 0.031). Having two IL10 GC haplotypes was associated with increased odds ratios of BCC and SCC (OR_BCC = 1.5, 95% CI 1.1–1.9; OR_SCC = 1.4, 95% CI 1.0–1.9), and these associations were largely confined to women (OR_BCC = 2.2, 95% CI 1.4–3.4; SCC: OR_SCC = 1.8, 95% CI 1.1–3.0). To examine how combinations of these variants contribute to risk of BCC and SCC, we used multifactor dimensionality reduction (MDR) and classification and regression trees (CART). Results from both of these methods found that in men, a combination of skin type, burns, IL10, IL4R, and possibly TNFR2 were important in both BCC and SCC. In women, skin type, burns, and IL10 were the most critical risk factors in SCC, with risk of BCC involving these same factors plus genetic variants in HTR2A, IL12B and IL4R. These data suggest differential genetic susceptibility to UV-induced immune suppression and skin cancer risk by gender.     

Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide, associated with declines in amphibian populations. Diagnosis of chytridiomycosis to date has largely relied upon histological and immunohistochemical examination of toe clips. This technique is invasive and insensitive particularly at early stages of infection when treatment may be possible. We have developed a real-time PCR Taqman assay that can accurately detect and quantify one zoospore in a diagnostic sample. This assay will assist the early detection of B. dendrobatidis in both captive and wild populations, with a high degree of sensitivity and specificity, thus facilitating treatment and protection of endangered populations, monitoring of pristine environments and preventing further global spread via amphibian trade.     

A quantitative cytochemical assay of beta-galactosidase in single cultured human skin fibroblasts

A quantitative cytochemical method for the measurement of beta-galactosidase activity in cultured human skin fibroblasts has been developed using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside as the indigogenic substrate. The method relies upon the oxidation of the primary reaction product by ferro/ferricyanide during which an insoluble indigo dye is generated as the final reaction product. The reaction was linear with time up to 60 min using the final cytochemical standard procedure. The enzyme showed maximum activity at pH 4.0 to 4.1. The concentration optima of indigogenic substrate and potassium ferro/ferricyanide were 3.67 mM and 3.13 mM respectively. The presence of sodium chloride activated beta-galactosidase up to 100 mM, but was inhibitory above that concentration. The enzyme was inhibited by N-ethylmaleimide, N-acetyl-D-galactosamine and heparin. The enzyme molecules were shown to diffuse out of the cells using media without a suitable inert colloid stabilizer. However, diffusion was completely prevented by using polyvinyl alcohol (PVA) grade G18/140. Air-drying of cells was essential to make the cell membrane permeabel to the substrate and, thereby, to avoid a pronounced lag phase. However, in a biochemical analysis, air-drying itself caused a decrease in enzyme activity to 43% of the control. Even after air-drying lysosomal latency could still be demonstrated by using PVA grade G04/140. Control persons, one carrier of and two patients with beta-galactosidase deficiency were easily identified as belonging to three separate groups by using the cytochemical assay. It is proposed that the quantitative cytochemical approach may also be applied to cultured human amniotic fluid cells or chorion biopsies giving a rapid prenatal diagnosis of beta-galactosidase deficiency due to the small number of cells needed in the analysis.     

Rapid chemosensitivity assay with human normal and tumor cells in vitro

Summary Neutral red assay, as an index of cytotoxicity, has been applied to predictive screening of chemotherapeutic agents. Human hepatoma and melanoma tumor cells and normal melanocytes, keratinocytes and fibroblasts were incubated for 2, 24, and 48 h with graded concentrations of cis-platinum (0.1 to 80 μM), doxorubicin (0.01 to 100 μM), and 5-fluorouracil (1 to 1000 μM). Cells were most sensitive after 48 h. Tumor cells, based on 50% toxicity values, were 2–4 times more sensitive than the normal cells, except for cis-platinum, where only melanoma cells, as compared to normal melanocytes, showed a marked difference in cytotoxic response. Methotrexate (1 to 10 μM) toxicity could be reversed in the presence of 100 μM of leucovorin. This sensitive, rapid, and economical assay is suitable for preclinical screening and drug development. This work has been supported, in part, by funds from Schering Corporation, New Jersey, and Chevron Environmental Health Center, Inc., California.     

Comparing quantitative measures of erythema,pigmentation and skin response using reflectometry

We measured a number of pigmentation and skin response phenotypes in a sample of volunteers (n=397) living in State College, PA. The majority of this sample was composed of four groups based on stated ancestry: African-American, European-American, Hispanic and East Asian. Several measures of melanin concentration (L*, melanin index and adjusted melanin index) were estimated by diffuse reflectance spectroscopy and compared. The efficacy of these measures for assessing constitutive pigmentation and melanogenic dose-response was evaluated. Similarly, several measures of erythema (a*, erythema index and adjusted erythema index) were compared and evaluated in their efficacy in measuring erythema and erythemal dose-response. We show a high correspondence among all of the measures for the assessment of constitutive pigmentation and baseline erythema. However, our results demonstrate that evaluating melanogenic dose-response is highly dependent on the summary statistic used: while L* is a valid measure of constitutive pigmentation it is not an effective measure of melanogenic dose-response. Our results also confirm the use of a*, as it is shown to be highly correlated with the adjusted erythema index, a more advanced measure of erythema based on the apparent absorbance. Diffuse reflectance spectroscopy can be used to quantify the constitutive pigmentation, melanogenic dose-response at 7 d and erythemal dose-response at both 24 h and 7 d postexposure.     

Rapid protein-folding assay using green fluorescent protein.

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.     

Modification of the quantitative DNA assay using adriamycin.

A modification of the adriamycin quantitative assay for DNA is presented which is sensitive to 1–20 μg DNA. The assay is simple, rapid, and highly reproducible but, contrary to previous claims, not specific for DNA, RNA interferes with the assay while protein does not. The mechanism and sensitivity of the assay is interpreted with reference to published data on adriamycin DNA binding.     

Rapid protein kinase assay using phosphocellulose-paper absorption.

Protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins. Histone is widely used as an acceptor substrate in measuring the activity of this enzyme isolated from a variety of sources. We have devised a rapid procedure for resolving phosphohistone from ATP and its metabolites based on the specific absorption of phosphorylated histone onto phosphocellulose paper. Using [γ-³²P]ATP as the phosphoryl donor, aliquots of the protein kinase assay mixture are applied to phosphocellulose-paper disks that are then immersed in water which elutes [γ-³²P]ATP and metabolites. After brief organic solvent extraction and drying, bound radioactivity is measured by liquid scintillation spectrometry.     

Rapid spectrophotometric assay of dodecyl sulfate using acridine orange

A rapid assay that is useful for quantitating as little as 1 μg sodium dodecyl sulfate in a 100-μl sample is described. Except for trichloroacetic acid, a variety of other substances tested caused little or no interference with the assay. The method is based on the extraction of a dodecyl sulfate-acridine orange complex from aqueous solution into toluene. Both extraction and spectrophotometric measurement were performed in the same tube, resulting in high precision in replicate determinations and added safety in handling toluene solutions.     

Hedgehog signaling in skin cancers

An increasing progress on the role of Hedgehog (Hh) signaling for carcinogenesis has been achieved since the link of Hh pathway to human cancer was firstly established. In particular, the critical role of Hh signaling in the development of Basal cell carcinoma (BCC) has been convincingly demonstrated by genetic mutation analyses, mouse models of BCCs, and successful clinical trials of BCCs using Hh signaling inhibitors. In addition, the Hh pathway activity is also reported to be involved in the pathogenesis of Squamous Cell Carcinoma (SCC), melanoma and Merkel Cell Carcinoma. These findings have significant new paradigm on Hh signaling transduction, its mechanisms in skin cancer and even therapeutic approaches for BCC. In this review, we will summarize the major advances in the understanding of Hh signaling transduction, the roles of Hh signaling in skin cancer development, and the current implications of “mechanism-based” therapeutic strategies.     

Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture

Acyl glucuronides of nonsteroidal anti-inflammatory drugs having a chiral center are known to be chemically very active and form covalently bound adducts with proteins, such as human serum albumin, which may be the cause of hypersensitive reactions. Hepatic acyl glucuronosyltransferase catalyzes the transformation of alpha-aryl propionates into these diastereoisomeric acyl glucuronides, and, hence, its activity needs to be characterized. From this point of view, we developed a rapid, accurate and reproducible analytical method for the separation and determination of diastereoisomeric glucuronides of flurbiprofen, one of the nonsteroidal anti-inflammatory drugs, in the incubation mixture of the hepatic microsomal preparation by high-performance liquid chromatography with a simple column-switching technique for deproteinization. The glucuronides were separated on a TSKgel ODS-80Ts column with 20 mM ammonium acetate buffer (pH 5.6)-ethanol-acetonitrile as the mobile phase and monitored with a UV detector at 246 nm. The detection limit of the proposed method was 600 fmol/injection at a signal-to-noise ratio of 10. The validation results indicated that this method would be very useful for the determination of diastereomeric acyl glucuronides formed from flurbiprofen in an incubation mixture.     

Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay

Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.     

The prediction of human skin responses by using the combined in vitro fluorescein leakage/Alamar Blue (resazurin) assay

A range of cosmetics formulations with human patch-test data were supplied in a coded form, for the examination of the use of a combined in vitro permeability barrier assay and cell viability assay to generate, and then test, a prediction model for assessing potential human skin patch-test results. The target cells employed were of the Madin Darby canine kidney cell line, which establish tight junctions and adherens junctions able to restrict the permeability of sodium fluorescein across the barrier of the confluent cell layer. The prediction model for interpretation of the in vitro assay results included initial effects and the recovery profile over 72 hours. A set of the hand-wash, surfactant-based formulations were tested to generate the prediction model, and then six others were evaluated. The model system was then also evaluated with powder laundry detergents and hand moisturisers: their effects were predicted by the in vitro test system. The model was under-predictive for two of the ten hand-wash products. It was over-predictive for the moisturisers, (two out of six) and eight out of ten laundry powders. However, the in vivo human patch test data were variable, and 19 of the 26 predictions were correct or within 0.5 on the 0-4.0 scale used for the in vivo scores, i.e. within the same variable range reported for the repeat-test hand-wash in vivo data.