Efficacy and Safety of Topical Corticosteroid Treatment under Occlusion for Severe Alopecia Areata in Children. A single-center retrospective analysis.

BACKGROUND: Alopecia areata (AA) has a poor clinical course in children. There are no reliable therapeutic options for children with severe AA, including alopecia totalis (AT) and alopecia universalis (AU). AIM: We evaluated the efficacy and adverse effects of a potent topical corticosteroid (TCS) under occlusion in pediatric patients with severe AA. METHODS: We reviewed records of 23 patients under the age of 10 years with AT or AU treated with a potent TCS (0.05% clobetasol propionate or 0.3% diflucortolone valerate) for 8 hours under occlusion with a plastic film. We used the Severity of Alopecia Tool (SALT) to measure clinical improvement. The primary endpoint was a Severity of Alopecia Tool (SALT) score of 20 or less at six months. We analyzed the change in cortisol levels to identify the long-term safety of TCS therapy on the hypothalamus-pituitary-adrenal axis. RESULTS: Nineteen patients reached SALT 20 or less at the 6-month treatment. Six patients relapsed over the 6-month follow-up period. Four patients were suspected of adrenal insufficiency. However, the cortisol level of the patients recovered to normal at least 1-month after lowering TCS potency or changing to non-steroidal treatments. LIMITATIONS: Retrospective design and small sample size. CONCLUSION: This study shows that a potent TCS occlusion may be a safe treatment option in pediatric patients with severe AA. Further long-term studies are required to evaluate the safety and recurrence of TCS occlusion therapy for pediatric AA.

Pitavastatin Induces Apoptosis of Cutaneous Squamous Cell Carcinoma Cells through Geranylgeranyl Pyrophosphate-Dependent c-Jun N-Terminal Kinase Activation.

BACKGROUND: Pitavastatin is a cholesterol-lowering drug and is widely used clinically. In addition to this effect, pitavastatin has shown the potential to induce apoptosis in cutaneous squamous cell carcinoma (SCC) cells. OBJECTIVE: The purpose of this study is to investigate the effects and possible action mechanisms of pitavastatin. METHODS: SCC cells (SCC12 and SCC13 cells) were treated with pitavastatin, and induction of apoptosis was confirmed by Western blot. To examine whether pitavastatin-induced apoptosis is related to a decrease in the amount of intermediate mediators in the cholesterol synthesis pathway, the changes in pitavastatin-induced apoptosis after supplementation with mevalonate, squalene, geranylgeranyl pyrophosphate (GGPP) and dolichol were investigated. RESULTS: Pitavastatin dose-dependently induced apoptosis of cutaneous SCC cells, but the viability of normal keratinocytes was not affected by pitavastatin at the same concentrations. In supplementation experiments, pitavastatin-induced apoptosis was inhibited by the addition of mevalonate or downstream metabolite GGPP. As a result of examining the effect on intracellular signaling, pitavastatin decreased Yes1 associated transcriptional regulator and Ras homolog family member A and increased Rac family small GTPase 1 and c-Jun N-terminal kinase (JNK) activity. All these effects of pitavastatin on signaling molecules were restored when supplemented with either mevalonate or GGPP. Furthermore, pitavastatin-induced apoptosis of cutaneous SCC cells was inhibited by a JNK inhibitor. CONCLUSION: These results suggest that pitavastatin induces apoptosis of cutaneous SCC cells through GGPP-dependent JNK activation.

Sequence determinant of small RNA production by DICER.

RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs)1,2. However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3' overhang and a terminal loop3-11. Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis-acting element, termed the 'GYM motif' (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified 'ruler'-like counting mechanisms from the 5' and 3' ends of pre-miRNA3-6. Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics.

Structure of the human DICER-pre-miRNA complex in a dicing state.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.

The potential role of fibroblast-derived multi-peptide factors in activation of growth factors and ß-Catenin in hair follicle cells.

BACKGROUND: Dermal fibroblasts play a pivotal role in hair follicle regeneration during wound repair. Recently, dermal fibroblast-conditioned medium (DFCM), which contains multi-peptide factors (MPFs), has been used to promote wound repair. AIM: This study aimed to investigate the stimulatory effects of MPF-containing DFCM on hair growth. METHODS: MPF-containing DFCM was prepared using human neonatal dermal fibroblasts. Outer root sheath (ORS) and dermal papilla (DP) cells were cultured in MPF-containing DFCM. We examined the expression and secretion of growth factors and cytokines using quantitative polymerase chain reaction and a growth factor array. In addition, the effect of MPFs on ß-catenin activity was determined using the TOPflash assay. All experiments were repeated at least three times with separate batches of cells. RESULTS: MPF-containing DFCM increased keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) mRNA expression in ORS cells and KGF and VEGF mRNA expression in DP cells. When ORS cells were treated with MPF-containing DFCM, the secretion of several growth factors, including EGF, VEGF, insulin-like growth factor-binding protein (IGFBP)-4, IGFBP-6, and fibroblast growth factor-7, was increased in the cell-cultured medium compared with that in control. Additionally, MPF-containing DFCM increased the transcriptional activation of ß-catenin in DP cells. CONCLUSIONS: These results suggest that MPF-containing DFCM might stimulate hair growth by inducing growth factors in ORS and DP cells and regulating ß-catenin in DP cells.

Enhancement of seed germination and microbial disinfection on ginseng by cold plasma treatment.

BACKGROUND: This study aimed to investigate the effect of cold plasma treatment on the improvement of seed germination and surface sterilization of ginseng seeds. METHODS: Dehisced ginseng (Panax ginseng) seeds were exposed to dielectric barrier discharge (DBD) plasma operated in argon (Ar) or an argon/oxygen mixture (Ar/O2), and the resulting germination and surface sterilization were compared with those of an untreated control group. Bacterial and fungal detection assays were performed for plasma-treated ginseng seeds after serial dilution of surface-washed suspensions. The microbial colonies (fungi and bacteria) were classified according to their phenotypical morphologies and identified by molecular analysis. Furthermore, the effect of cold plasma treatment on the in vitro antifungal activity and suppression of Cylindrocarpon destructans in 4-year-old ginseng root discs was investigated. RESULTS: Seeds treated with plasma in Ar or Ar/O2 exhibited a higher germination rate (%) compared with the untreated controls. Furthermore, the plasma treatment exhibited bactericidal and fungicidal effects on the seed surface, and the latter effect was stronger than the former. In addition, plasma treatment exhibited in vitro antifungal activity against C. destructans and reduced the disease severity (%) of root rot in 4-year-old ginseng root discs. The results demonstrate the stimulatory effect of plasma treatment on seed germination, surface sterilization, and root rot disease suppression in ginseng. CONCLUSION: The results of this study indicate that the cold plasma treatment can suppress the microbial community on the seed surface root rot in ginseng.

A quantitative map of human primary microRNA processing sites.

Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). More than 1,800 miRNA loci are annotated in humans, but it remains largely unknown whether and at which sites pri-miRNAs are cleaved by DROSHA. Here, we performed in vitro processing on a full set of human pri-miRNAs (miRBase version 21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs on the basis of DROSHA dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing and unproductive cleavage events such as "nick" or "inverse" processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.

A Mechanism for microRNA Arm Switching Regulated by Uridylation.

Strand selection is a critical step in microRNA (miRNA) biogenesis. Although the dominant strand may change depending on cellular contexts, the molecular mechanism and physiological significance of such alternative strand selection (or "arm switching") remain elusive. Here we find miR-324 to be one of the strongly regulated miRNAs by arm switching and identify the terminal uridylyl transferases TUT4 and TUT7 to be the key regulators. Uridylation of pre-miR-324 by TUT4/7 re-positions DICER on the pre-miRNA and shifts the cleavage site. This alternative processing produces a duplex with a different terminus from which the 3' strand (3p) is selected instead of the 5' strand (5p). In glioblastoma, the TUT4/7 and 3p levels are upregulated, whereas the 5p level is reduced. Manipulation of the strand ratio is sufficient to impair glioblastoma cell proliferation. This study uncovers a role of uridylation as a molecular switch in alternative strand selection and implicates its therapeutic potential.

Viral hijacking of the TENT4-ZCCHC14 complex protects viral RNAs via mixed tailing.

TENT4 enzymes generate 'mixed tails' of diverse nucleotides at 3' ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4-ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for mixed tailing and protection of viral poly(A) tails. We find that the HBV post-transcriptional regulatory element (PRE), specifically the CNGGN-type pentaloop, is critical for TENT4-dependent regulation. HCMV uses a similar pentaloop, an interesting example of convergent evolution. This pentaloop is recognized by the sterile alpha motif domain-containing ZCCHC14 protein, which in turn recruits TENT4. Overall, our study reveals the mechanism of action of PRE, which has been widely used to enhance gene expression, and identifies the TENT4-ZCCHC14 complex as a potential target for antiviral therapeutics.

Power-constrained contrast enhancement for emissive displays based on histogram equalization.

A power-constrained contrast-enhancement algorithm for emissive displays based on histogram equalization (HE) is proposed in this paper. We first propose a log-based histogram modification scheme to reduce overstretching artifacts of the conventional HE technique. Then, we develop a power-consumption model for emissive displays and formulate an objective function that consists of the histogram-equalizing term and the power term. By minimizing the objective function based on the convex optimization theory, the proposed algorithm achieves contrast enhancement and power saving simultaneously. Moreover, we extend the proposed algorithm to enhance video sequences, as well as still images. Simulation results demonstrate that the proposed algorithm can reduce power consumption significantly while improving image contrast and perceptual quality.

Coding order decision of B frames for rate-distortion performance improvement in single-view video and multiview video coding.

The coding gain that can be achieved by improving the coding order of B frames in the H.264/AVC standard is investigated in this work. We first represent the coding order of B frames and their reference frames with a binary tree. We then formulate a recursive equation to find out the binary tree that provides a suboptimal, but very efficient, coding order. The recursive equation is efficiently solved using a dynamic programming method. Furthermore, we extend the coding order improvement technique to the case of multiview video sequences, in which the quadtree representation is used instead of the binary tree representation. Simulation results demonstrate that the proposed algorithm provides significantly better R-D performance than conventional prediction structures.