BCL2A1 is a lineage-specific antiapoptotic melanoma oncogene that confers resistance to BRAF inhibition.

Although targeting oncogenic mutations in the BRAF serine/threonine kinase with small molecule inhibitors can lead to significant clinical responses in melanoma, it fails to eradicate tumors in nearly all patients. Successful therapy will be aided by identification of intrinsic mechanisms that protect tumor cells from death. Here, we used a bioinformatics approach to identify drug-able, "driver" oncogenes restricted to tumor versus normal tissues. Applying this method to 88 short-term melanoma cell cultures, we show that the antiapoptotic BCL2 family member BCL2A1 is recurrently amplified in ∼30% of melanomas and is necessary for melanoma growth. BCL2A1 overexpression also promotes melanomagenesis of BRAF-immortalized melanocytes. We find that high-level expression of BCL2A1 is restricted to melanoma due to direct transcriptional control by the melanoma oncogene MITF. Although BRAF inhibitors lead to cell cycle arrest and modest apoptosis, we find that apoptosis is significantly enhanced by suppression of BCL2A1 in melanomas with BCL2A1 or MITF amplification. Moreover, we find that BCL2A1 expression is associated with poorer clinical responses to BRAF pathway inhibitors in melanoma patients. Cotreatment of melanomas with BRAF inhibitors and obatoclax, an inhibitor of BCL2A1 and other BCL2 family members, overcomes intrinsic resistance to BRAF inhibitors in BCL2A1-amplified cells in vitro and in vivo. These studies identify MITF-BCL2A1 as a lineage-specific oncogenic pathway in melanoma and underscore its role for improved response to BRAF-directed therapy.

The brain circuitry mediating antipruritic effects of acupuncture.

Itch is an aversive sensory experience and while systemic therapies, such as acupuncture, have shown promise in alleviating itch in patients suffering from chronic itch, their antipruritic mechanisms are unknown. As several lines of evidence implicate brain-focused mechanisms, we applied functional magnetic resonance imaging and our validated temperature-modulation itch model to evaluate the underlying brain circuitry supporting allergen-induced itch reduction in atopic dermatitis patients by acupuncture, antihistamine, and respective placebo treatments. Brain response to allergen itch demonstrated phase dependency. During an increasing itch phase, activation was localized in anterior insula and striatum, regions associated with salience/interoception and motivation processing. Once itch reached peak plateau, robust activation was noted in prefrontal cognitive and premotor areas. Acupuncture reduced itch and itch-evoked activation in the insula, putamen, and premotor and prefrontal cortical areas. Neither itch sensation nor itch-evoked brain response was altered following antihistamine or placebo acupuncture. Greater itch reduction following acupuncture was associated with greater reduction in putamen response, a region implicated in motivation and habitual behavior underlying the urge to scratch, specifically implicating this region in acupuncture's antipruritic effects. Understanding brain circuitry underlying itch reduction following acupuncture and related neuromodulatory therapies will significantly impact the development and applicability of novel therapies to reduce an itch.

YY1 regulates melanocyte development and function by cooperating with MITF.

Studies of coat color mutants have greatly contributed to the discovery of genes that regulate melanocyte development and function. Here, we generated Yy1 conditional knockout mice in the melanocyte-lineage and observed profound melanocyte deficiency and premature gray hair, similar to the loss of melanocytes in human piebaldism and Waardenburg syndrome. Although YY1 is a ubiquitous transcription factor, YY1 interacts with M-MITF, the Waardenburg Syndrome IIA gene and a master transcriptional regulator of melanocytes. YY1 cooperates with M-MITF in regulating the expression of piebaldism gene KIT and multiple additional pigmentation genes. Moreover, ChIP-seq identified genome-wide YY1 targets in the melanocyte lineage. These studies mechanistically link genes implicated in human conditions of melanocyte deficiency and reveal how a ubiquitous factor (YY1) gains lineage-specific functions by co-regulating gene expression with a lineage-restricted factor (M-MITF)-a general mechanism which may confer tissue-specific gene expression in multiple lineages.

Fractional epidermal grafting in combination with laser therapy as a novel approach in treating radiation dermatitis.

Radiation injury to the skin is a major source of dysfunction, disfigurement, and complications for thousands of patients undergoing adjunctive treatment for internal cancers. Despite the great potential for affecting quality of life, radiation injury has received little attention from dermatologists and is primarily being managed by radiation oncologists. During our volunteer work in Vietnam, we encountered numerous children with significant scarring and depigmentation of skin from the outdated use of radioactive phosphorus P32 in the treatment of hemangiomas. This dangerous practice has left thousands of children with significant fibrosis and disfigurement. Currently, there is no treatment for radiation dermatitis. Here, we report a case series using the combination of laser treatment, including pulsed-dye laser, fractional CO2 laser, and epidermal grafting to improve the appearance and function of the radiation scars in these young patients. We hope that by improving the appearance and function of these scars, we can improve the quality of life for these young patients and potentially open up a new avenue of treatment for cancer patients affected with chronic radiation dermatitis, potentially improving their range of motion, cosmesis, and reducing their risk of secondary skin malignancies.

Cutaneous drug delivery: an update.

Cutaneous delivery of therapeutics represents a proven and attractive option for treating a variety of dermatologic conditions with minimal systemic side effects. Although there have been many innovations in drug delivery systems, the number of effective cutaneous drugs remains small, primarily because of the stratum corneum permeability barrier. Overcoming this barrier safely and reversibly to deliver large hydrophilic drugs cutaneously is one of the major challenges in the field of dermatologic therapy.

The Basic Mechanism of Hair Growth Stimulation by Adipose-derived Stem Cells and Their Secretory Factors.

BACKGROUND: Adipose-derived stem cells (ADSCs) are mesenchymal stem cells (MSCs) within the stromal vascular fraction of subcutaneous adipose tissue. ADSCs secrete growth factors and other proteins, and have been used to regenerate skin with satisfactory results. OBJECTIVE: This review focuses on the effect of ADSCs and their secretory factors on the stimulation of hair growth in vitro, ex vivo and in vivo. RESULTS: The conditioned media of ADSCs (ADSC-CM) increases the proliferation rate of human follicular cells. ADSCs-derived proteins improve hair growth and protect human dermal papilla cells against cytotoxic injury caused by androgen and reactive oxygen species. Moreover, ADSC-CM induces the anagen phase and promotes hair growth in mice, and enhances the elongation of hair shafts in ex vivo human hair organ cultures. CONCLUSION: ADSC-CM promotes hair growth in vitro, ex vivo, and in vivo. Given that ADSCs are one of the most accessible sources of MSCs, ADSC-derived proteins may be feasible clinical therapeutic agents for the treatment of hair loss.

Enzyme-linked immunoassay for dengue virus IgM and IgG antibodies in serum and filter paper blood.

BACKGROUND: The reproducibilty of dengue IgM and IgG ELISA was studied in serum and filter paper blood spots from Vietnamese febrile patients. METHODS: 781 pairs of acute (t0) and convalescent sera, obtained after three weeks (t3) and 161 corresponding pairs of filter paper blood spots were tested with ELISA for dengue IgG and IgM. 74 serum pairs were tested again in another laboratory with similar methods, after a mean of 252 days. RESULTS: Cases were classified as no dengue (10 %), past dengue (55%) acute primary (7%) or secondary (28%) dengue. Significant differences between the two laboratories' results were found leading to different diagnostic classification (kappa 0.46, p < 0.001). Filter paper results correlated poorly to serum values, being more variable and lower with a mean (95% CI) difference of 0.82 (0.36 to 1.28) for IgMt3, 0.94 (0.51 to 1.37) for IgGt0 and 0.26 (-0.20 to 0.71) for IgGt3. This also led to differences in diagnostic classification (kappa value 0.44, p < 0.001) The duration of storage of frozen serum and dried filter papers, sealed in nylon bags in an air-conditioned room, had no significant effect on the ELISA results. CONCLUSION: Dengue virus IgG antibodies in serum and filter papers was not affected by duration of storage, but was subject to inter-laboratory variability. Dengue virus IgM antibodies measured in serum reconstituted from blood spots on filter papers were lower than in serum, in particular in the acute phase of disease. Therefore this method limits its value for diagnostic confirmation of individual patients with dengue virus infections. However the detection of dengue virus IgG antibodies eluted from filter paper can be used for sero-prevalence cross sectional studies.

Nanoparticle-aptamer bioconjugates: a new approach for targeting prostate cancer cells.

Nucleic acid ligands (aptamers) are potentially well suited for the therapeutic targeting of drug encapsulated controlled release polymer particles in a cell- or tissue-specific manner. We synthesized a bioconjugate composed of controlled release polymer nanoparticles and aptamers and examined its efficacy for targeted delivery to prostate cancer cells. Specifically, we synthesized poly(lactic acid)-block-polyethylene glycol (PEG) copolymer with a terminal carboxylic acid functional group (PLA-PEG-COOH), and encapsulated rhodamine-labeled dextran (as a model drug) within PLA-PEG-COOH nanoparticles. These nanoparticles have the following desirable characteristics: (a) negative surface charge (-50 +/- 3 mV, mean +/- SD, n = 3), which may minimize nonspecific interaction with the negatively charged nucleic acid aptamers; (b) carboxylic acid groups on the particle surface for potential modification and covalent conjugation to amine-modified aptamers; and (c) presence of PEG on particle surface, which enhances circulating half-life while contributing to decreased uptake in nontargeted cells. Next, we generated nanoparticle-aptamer bioconjugates with RNA aptamers that bind to the prostate-specific membrane antigen, a well-known prostate cancer tumor marker that is overexpressed on prostate acinar epithelial cells. We demonstrated that these bioconjugates can efficiently target and get taken up by the prostate LNCaP epithelial cells, which express the prostate-specific membrane antigen protein (77-fold increase in binding versus control, n = 150 cells per group). In contrast to LNCaP cells, the uptake of these particles is not enhanced in cells that do not express the prostate-specific membrane antigen protein. To our knowledge, this represents the first report of targeted drug delivery with nanoparticle-aptamer bioconjugates.

Characterization of chemisorbed hyaluronic acid directly immobilized on solid substrates.

Hyaluronic acid (HA) has a number of potential biomedical applications in drug delivery and tissue engineering. For these applications, a prerequisite is to understand the characteristic of HA films directly immobilized to solid substrates. Here, we demonstrate that high molecular weight HA can be directly immobilized onto hydrophilic substrates without any chemical manipulation, allowing for the formation of an ultrathin chemisorbed layer. Hyaluronic acid is stabilized on these surfaces through hydrogen bonding between the hydrophilic moieties in HA [such as carboxylic acid (-COOH) or hydroxyl (-OH) groups] with silanol (-SiOH), carboxylic acid or hydroxyl groups on the hydrophilic substrates. Despite the water solubility, the chemisorbed HA layer remained stable on glass or silicon oxide substrates for at least 7 days in phosphate-buffered saline. Furthermore, HA immobilized on silicon and other dioxide surfaces in much higher quantities than other polysaccharides including dextran sulfate, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, and alginic acid. This behavior is related to the molecular entanglement and intrinsic stiffness of HA as a result of strong internal and external hydrogen bonding as well as high molecular weight. These results demonstrate that HA can be used to coat surfaces through direct immobilization.

Hsp70 Knockdown by siRNA Decreased Collagen Production in Keloid Fibroblasts.

PURPOSE: There are currently no consistently effective treatments for the excessive collagen produced by keloid fibroblasts. Previously, we reported that heat shock protein 70 (Hsp70) is up-regulated in keloid fibroblasts and keloid tissue. We, therefore, investigated whether Hsp70 is related to excessive collagen production in keloid fibroblasts. MATERIALS AND METHODS: We inhibited Hsp70 in keloid fibroblasts by RNA interference and examined the resulting collagen expression. Thus, we selected small interfering RNAs (siRNAs) specific for human Hsp70, transfected them into keloid fibroblasts, and evaluated the resulting phenotypes and protein production using real-time polymerase chain reaction (PCR), Western blot, and a collagen assay. RESULTS: The siRNAs dramatically suppressed Hsp70 mRNA expression, resulting in a decrease in collagen production in the keloid fibroblasts compared with controls. The siRNAs did not influence the viability of the keloid fibroblasts. CONCLUSION: Hsp70 overexpression likely plays an important role in the excessive collagen production by keloid fibroblasts. RNA interference has therapeutic potential for the treatment of keloids.

Design and fabrication of human skin by three-dimensional bioprinting.

Three-dimensional (3D) bioprinting, a flexible automated on-demand platform for the free-form fabrication of complex living architectures, is a novel approach for the design and engineering of human organs and tissues. Here, we demonstrate the potential of 3D bioprinting for tissue engineering using human skin as a prototypical example. Keratinocytes and fibroblasts were used as constituent cells to represent the epidermis and dermis, and collagen was used to represent the dermal matrix of the skin. Preliminary studies were conducted to optimize printing parameters for maximum cell viability as well as for the optimization of cell densities in the epidermis and dermis to mimic physiologically relevant attributes of human skin. Printed 3D constructs were cultured in submerged media conditions followed by exposure of the epidermal layer to the air-liquid interface to promote maturation and stratification. Histology and immunofluorescence characterization demonstrated that 3D printed skin tissue was morphologically and biologically representative of in vivo human skin tissue. In comparison with traditional methods for skin engineering, 3D bioprinting offers several advantages in terms of shape- and form retention, flexibility, reproducibility, and high culture throughput. It has a broad range of applications in transdermal and topical formulation discovery, dermal toxicity studies, and in designing autologous grafts for wound healing. The proof-of-concept studies presented here can be further extended for enhancing the complexity of the skin model via the incorporation of secondary and adnexal structures or the inclusion of diseased cells to serve as a model for studying the pathophysiology of skin diseases.

A reversibly switching surface.

We report the design of surfaces that exhibit dynamic changes in interfacial properties, such as wettability, in response to an electrical potential. The change in wetting behavior was caused by surface-confined, single-layered molecules undergoing conformational transitions between a hydrophilic and a moderately hydrophobic state. Reversible conformational transitions were confirmed at a molecular level with the use of sum-frequency generation spectroscopy and at a macroscopic level with the use of contact angle measurements. This type of surface design enables amplification of molecular-level conformational transitions to macroscopic changes in surface properties without altering the chemical identity of the surface. Such reversibly switching surfaces may open previously unknown opportunities in interfacial engineering.