Early-life infections in association with the development of atopic dermatitis in infancy and early childhood: a nationwide nested case-control study.

BACKGROUND: Microbial dysbiosis has been implicated in the development of atopic dermatitis (AD). The risk of development of AD following early-life infections remains unclear. OBJECTIVE: To investigate the impact of early-life infections on AD development. METHODS: This population-based nested case-control study was conducted using the Taiwan's National Health Insurance Research Database. A total of 5454 AD patients and 16 362 control subjects without AD were identified, for the period 1997 to 2013. Demographic characteristics, comorbidities and maternal factors were compared. Adjusted odds ratio (aOR) was calculated to examine the associations between early-life infections and subsequent AD by conditional stepwise logistic regression analysis. RESULTS: Mean age was 2.6 ± 2.9 years in both groups. Overall infections (41.8% vs. 28.9%) before the diagnosis of AD were more common in AD patients than in control subjects (P < 0.001). Infectious diseases [aOR, 1.40; 95% confidence interval (CI), 1.29-1.51], skin infections (aOR, 1.55; 95% CI, 1.40-1.71) and systemic antibiotic exposure (aOR 1.67, 95% CI 1.55-1.79) before AD diagnosis were independently associated with AD development on multivariate analyses. These results were consistent across observation periods (0-1, 1-2 and >2 years after birth) and sensitivity analyses after redefining the index date as 3 or 6 months before the date of AD diagnosis. Other independent risk factors included asthma, allergic rhinitis, intussusception and neonatal hyperbilirubinemia. No association with subsequent AD was found for maternal age at delivery, Caesarean delivery or prenatal antibiotic exposure. CONCLUSION: Infections in early life are associated with AD development in infancy and early childhood.

Enhanced O-succinyl-l-homoserine production by recombinant Escherichia coli ΔIJBB*TrcmetL/pTrc-metAfbr -Trc-thrAfbr -yjeH via multilevel fermentation optimization.

AIMS: Constructing a strain with high yield of O-succinyl-l-homoserine (OSH) and improving the titre through multilevel fermentation optimization. METHODS AND RESULTS: OSH high-yielding strain was first constructed by deleting the thrB gene to block the threonine biosynthesis. Single-factor experiment was carried out, where a Plackett-Burman design was used to screen out three factors (glucose, yeast and threonine) from the original 11 factors that affected the titre of OSH. The Box-Behnken response surface method was used to optimize the fermentation conditions. Through gene editing and medium optimization, the titre of OSH increased from 7·20 to 8·70 g l-1 in 500 ml flask. Furthermore, the fermentation process and fed-batch fermentation conditions including pH, temperature, feeding strategy and feeding medium were investigated and optimized. Under the optimal conditions, the titre of OSH reached 102·5 g l-1 , which is 5·6 times higher than before (15·6 g l-1 ). CONCLUSIONS: O-succinyl-l-homoserine fermentation process was established and the combination of response surface methodology and metabolic pathway analysis effectively improved the titre of OSH. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the titre of OSH reached the needs for industrial production and the metabolic pathway of OSH was demonstrated for further optimization.

T-cell factor-4 and MHC upregulation in pigs receiving a live attenuated classical swine fever virus (CSFV) vaccine strain with interferon-gamma adjuvant.

The effect of co-administration of interferon (IFN)-γ in pigs undergoing vaccination with an attenuated strain (LPC) of classical swine fever virus (CSFV) was investigated. Unvaccinated pigs demonstrated pyrexia and died 7-9 days after challenge with virulent CSFV. Pigs receiving the attenuated vaccine remained healthy after virus challenge, except for mild, transient pyrexia, whereas pigs receiving IFN-γ simultaneously with the vaccine demonstrated normal body temperatures after virus challenge. Examination by nested RT-PCR revealed greater viral load in the spleens of the pigs vaccinated with the attenuated CSFV, compared with those that had additionally received IFN-γ. Expression of major histocompatibility complex (MHC) class I and MHC class II molecules was upregulated in the spleens of the IFN-γ treated vaccinated pigs, demonstrated by immunohistochemistry. Based on Western blot analysis, anti-CSFV IgG2 antibodies were elevated in vaccinated pigs by co-administration of IFN-γ (IFN-γ(Hi): P < 0.01; IFN-γ(Lo): P <0.05). By employing the suppression subtractive hybridization technique, RT-PCR, in situ hybridization, and immunohistochemistry, T-cell factor-4 (Tcf-4) mRNA and protein expression were found to be upregulated in the spleens of vaccinated pigs that had received IFN-γ. This study suggests involvement of Tcf-4 in IFN-γ-mediated immune regulation following CSFV vaccination.

Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells.

Hepatoma exhibits a series of heterogeneous subpopulations in its cell surface markers, tumorigenicity, invasion and metastatic capability. We previously demonstrated that the CD133(-)/EpCAM(-) hepatoma subpopulation was more metastatic than its counterpart; however, the controlling mechanisms are unexplored. The present study aimed to delineate the significance of aberrant hedgehog (Hh) signaling in the mediation of metastases. Fluorescence-activated cell sorting-enriched CD133(-)/EpCAM(-) (double negative, DN), Huh-7 cells underwent a transwell selection for metastatic cells (transwell-selected, TS). The TS cells displayed much greater metastatic activity as evidenced by an increased invasion rate, extremely upregulated expression of matrix metalloproteinase (MMP)-1/2/9 genes compared with DN and double-positive (DP) subpopulations. In contrast to DP cells, TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry. There was a transitional increase in Gli-1/2 gene expression levels from DP, DN to TS subpopulations, which was consistent with elevated Gli-1/2 or Twist-1 protein levels in the nuclear fraction. Furthermore, truncated Gli-1 (tGli-1), which transactivates molecules involved in metastasis, was detected in the highly invasive Huh-7 cell subpopulation, but not in less metastatic hepatoma cells or normal hepatocytes. The enhanced metastatic features with increased expression of MMPs as well as the presence of twist and snail genes in TS Huh-7 cells were reversed by LDE225, a potent Smoothened antagonist. In conclusion, the highly metastatic capability of a unique TS subpopulation was highly attributed to significant epithelial-mesenchymal transition, enhanced Hh activity and aberrant occurrence of a tGli-1 variant, which appears to be responsible for the highly invasive behavior.

Overexpression of miR-98 inhibits cell invasion in glioma cell lines via downregulation of IKKε.

OBJECTIVE: MicroRNAs (miRNAs) function as negative regulators for the expression of genes involved in cancer metastasis. The aim of this study was to investigate the potential role of miR-98 in gliomas and validate its regulatory mechanism. PATIENTS AND METHODS: Cell viability assays are used to measure proliferation of cell. mRNA expression is measured by qRT-PCR. Western blot analysis is used to measure protein expression. RESULTS: Functional studies showed that miR-98 overexpression inhibited glioma migration and invasion, but had no effect on the cell viability. An enhanced green fluorescent protein reporter assay, quantitative RT-PCR, and a western blot analysis confirmed that miR-98 suppressed the expression of IκB kinase (IKKε) by directly targeting its 3'-untranslated region, also, the NF-κB p65 nuclear translocation and matrix metalloproteinase (MMP)-9 expression were significantly arrested in glioma cells treated with miR-98 mimics. Accordingly, the overexpression of IKKε or NF-κB p65 can restore cell migration and invasion after being inhibited by miR-98, and can restore NF-κB p65 nuclear translocation as well as increase MMP-9 expression. CONCLUSIONS: These findings demonstrated that miR-98 functions as a tumor suppressor in gliomas. Furthermore, miR-98 may act as a potential therapeutic biomarker for glioma patients.

A small-molecule inhibitor of UBE2N induces neuroblastoma cell death via activation of p53 and JNK pathways.

Neuroblastoma (NB) is the most common extracranial neoplasm in children. In NB, loss of p53 function is largely due to cytoplasmic sequestration rather than mutation. Ubiquitin-conjugating enzyme E2 N (UBE2N), also known as Ubc13, is an E2 ubiquitin-conjugating enzyme that promotes formation of monomeric p53 that results in its cytoplasmic translocation and subsequent loss of function. Therefore, inhibition of UBE2N may reactivate p53 by promoting its nuclear accumulation. Here, we show that NSC697923, a novel UBE2N inhibitor, exhibits potent cytotoxicity in a panel of NB cell lines evidenced by its ability to induce apoptosis. In p53 wild-type NB cells, NSC697923 induced nuclear accumulation of p53, which led to its increased transcriptional activity and tumor suppressor function. Interestingly, in p53 mutant NB cells, NSC697923 induced cell death by activating JNK pathway. This effect was reversible by blocking JNK activity with its selective inhibitor, SP600125. More importantly, NSC697923 impeded cell growth of chemoresistant LA-N-6 NB cell line in a manner greater than conventional chemotherapy drugs doxorubicin and etoposide. NSC697923 also revealed in vivo antitumor efficacy in NB orthotopic xenografts. Taken together, our results suggest that UBE2N is a potential therapeutic target in NB and provide a basis for the rational use of UBE2N inhibitors like NSC697923 as a novel treatment option for NB patients.

miR-1207-5p and miR-1266 suppress gastric cancer growth and invasion by targeting telomerase reverse transcriptase.

hTERT is the catalytic subunit of the telomerase complex. Elevated expression of hTERT is associated with the expansion and metastasis of gastric tumor. In this study, we aimed to identify novel tumor suppressor miRNAs that restrain hTERT expression. We began our screen for hTERT-targeting miRNAs with a miRNA microarray. miRNA candidates were further filtered by bioinformatic analysis, general expression pattern in different cell lines, gain-of-function effects on hTERT protein and the potential of these effects to suppress hTERT 3' untranslated region (3'UTR) luciferase activity. The clinical relevance of two miRNAs (miR-1207-5p and miR-1266) was evaluated by real-time RT-PCR. The effects of these miRNAs on cell growth, cell cycle and invasion of gastric cancer cells were measured with CCK-8, flow cytometry and transwell assays. Finally, the ability of these miRNAs to suppress the transplanted tumors was also investigated. Fourteen miRNAs were identified using a combination of bioinformatics and miRNA microarray analysis. Of these fourteen miRNAs, nine were expressed at significantly lower levels in hTERT-positive cell lines compared with hTERT-negative cell lines and five could downregulate hTERT protein expression. Only miR-1207-5p and miR-1266 interacted with the 3' UTR of hTERT and the expression levels of these two miRNAs were significantly decreased in gastric cancer tissues. These two miRNAs also inhibited gastric tumor growth in vitro and in vivo. Altogether, miR-1207-5p and miR-1266 were determined to be hTERT suppressors in gastric cancer, and the delivery of these two miRNAs represents a novel therapeutic strategy for gastric cancer treatment.

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis.

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.

Notch signaling may negatively regulate neonatal rat cardiac fibroblast-myofibroblast transformation.

Cardiac fibroblast-myofibroblast transformation (CMT) is a critical event in the initiation of myocardial fibrosis. Notch signaling has been shown to regulate myofibroblast transformation from other kinds of cells. However, whether Notch signaling is also involved in CMT remains unclear. In the present study, expressions of Notch receptors in cardiac fibroblasts (CFs) were examined, effects of Notch signaling inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) and transforming growth factor-beta1 (TGF-beta1) on CMT were determined by increasing alpha-smooth muscle actin (alpha-SMA) expression and collagen synthesis, and Notch signaling was examined by analyzing expressions of Notch receptors. The results showed that: (1) Notch receptor 1, 2, 3 and 4 were all expressed in CFs; (2) DAPT promoted CMT in a time-dependent manner; (3) During the period of CMT induced by TGF-beta1, expressions of Notch receptor 1, 3 and 4 in CFs were down-regulated, whereas there was no change for Notch receptor 2. Moreover, the downtrends of Notch 1, 3 and 4 were corresponding to the trend growth of alpha-SMA expression and collagen synthesis. These results suggested that inhibiting of Notch signaling might promote CMT. The down-regulations of Notch receptor 1, 3 and 4 induced by TGF-beta1 may facilitate CMT. In conclusion, inhibition of Notch signaling might be a novel mechanism of CMT in myocardial fibrosis.

USP4 targets TAK1 to downregulate TNFα-induced NF-κB activation.

Lys63-linked polyubiquitination of transforming growth factor-ß-activated kinase 1 (TAK1) has an important role in tumor necrosis factor-α (TNFα)-induced NF-κB activation. Using a functional genomic approach, we have identified ubiquitin-specific peptidase 4 (USP4) as a deubiquitinase for TAK1. USP4 deubiquitinates TAK1 in vitro and in vivo. TNFα induces association of USP4 with TAK1 to deubiquitinate TAK1 and downregulate TAK1-mediated NF-κB activation. Overexpression of USP4 wild type, but not deuibiquitinase-deficient C311A mutant, inhibits both TNFα- and TAK1/TAB1 co-overexpression-induced TAK1 polyubiquitination and NF-κB activation. Notably, knockdown of USP4 in HeLa cells enhances TNFα-induced TAK1 polyubiquitination, IκB kinase phosphorylation, IκBα phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Moreover, USP4 negatively regulates IL-1ß-, LPS- and TGFß-induced NF-κB activation. Together, our results demonstrate that USP4 serves as a critical control to downregulate TNFα-induced NF-κB activation through deubiquitinating TAK1.

Transcripts of enriched germ cells responding to heat shock as potential markers for porcine semen quality.

A cDNA microarray-assisted experiment was conducted to survey genes that respond early to heat shock in enriched immature porcine germ cells; the 5'-UTR flanking the highest upregulated gene, heat shock 105/110 kDa protein 1 (Hsph1 or Hsp105), in response to heat shock was also investigated. We established a porcine testis cDNA microarray with 9944 transcripts from two libraries constructed from the testes of mature boars, with or without heat shock. After a mild heat shock treatment (39 degrees C for 1h and recovered at 34 degrees C for 2h), 380 transcripts demonstrated significant gene expression in enriched immature germ cells; 326 were upregulated and 54 were downregulated. Ten transcripts of interest exhibiting significance analysis of microarrays (SAM) scores higher than the median were subjected to quantitative real-time PCR; three (Hsp105, Hspa4l and Thap4) were upregulated >1.5-fold. The sequence of the 5'-UTR of Hsp105, the highest upregulated transcript, was cloned and analyzed. A single nucleotide polymorphism (SNP) was found at position -762 (C or T) upstream of the translational start site (ATG codon). Only two genotypes (CC or TC) were found in the mature boars that were studied (n=31). A heterozygous genotype (TC) at this SNP site revealed an elevated percentage of morphologically normal sperm during hot and cold seasons; this SNP may be a useful marker for semen quality in boars. Furthermore, the cell-model established from enriched primitive germ cells has potential for the study of reproduction in mature animals.

Lidocaine suppresses subthreshold oscillations by inhibiting persistent Na(+) current in injured dorsal root ganglion neurons.

The aim of this study was to determine the effect and mechanism of low concentration of lidocaine on subthreshold membrane potential oscillations (SMPO) and burst discharges in chronically compressed dorsal root ganglion (DRG) neurons. DRG neurons were isolated by enzymatic dissociation method. SMPO, burst discharges and single spike were elicited by whole cell patch-clamp technique in current clamp mode. Persistent Na(+) current (I(NaP)) and transient Na(+) current (I(NaT)) were elicited in voltage clamp mode. The results showed that SMPO was suppressed and burst discharges were eliminated by tetrodotoxin (TTX, 0.2 micromol/l) in current clamp mode, I(NaP) was blocked by 0.2 micromol/l TTX in voltage clamp mode. SMPO, burst discharges and I(NaP) were also suppressed by low concentration of lidocaine (10 micromol/l) respectively. However, single spike and I(NaT) could only be blocked by high concentration of lidocaine (5 mmol/l). From these results, it is suggested that I(NaP) mediates the generation of SMPO in injured DRG neurons. Low concentration of lidocaine (10 micromol/l) suppresses SMPO by selectively inhibiting I(NaP), but not I(NaT), in chronically compressed DRG neurons.

Use of a recombinant protein containing major epitopes of hnRNP G to detect anti-hnRNP G antibodies in dogs with systemic lupus erythematosus.

The objective of this study was to express major epitopes of heterogeneous nuclear ribonucleoprotein G (hnRNP G) for detecting anti-hnRNP G antibodies in dogs with systemic lupus erythematosus (SLE). HnRNP G cDNA clone was isolated from HEp-2 cells, and a DNA fragment encoding immunodominant region (residues 189-272) of hnRNP G (hnRNP Gi) was subcloned into pET32 vector to construct a prokaryotic expression plasmid named pEThnRNPGi. After induction, Escherichia coli carrying pEThnRNPGi expressed a recombinant protein of 28 kDa, comprising recombinant hnRNP Gi and fusion tag. Purified recombinant hnRNP Gi protein was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and its identity was confirmed. Western blot analysis showed that recombinant hnRNP Gi was specifically recognized by anti-hnRNP G positive sera of SLE dogs, and not by negative control sera. In conclusion, recombinant hnRNP Gi protein expressed in this study may serve as a useful reagent to assist in the immunological diagnosis of canine SLE.

Dynamical studies of gratings formed in polymer-dispersed liquid crystal films doped with a guest-host dye

This study investigated the dynamic behavior of the first-order diffraction efficiency of gratings formed in polymer-dispersed liquid crystal (PDLC) films doped with a guest-host dye. PDLC films were fabricated using various LC-polymer mixing ratios, and written with various powers. Experimental results indicated that several peaks appeared in the curve of the first-order diffraction efficiency versus time. According to the light scattering study, we believe that the first peak was due to the superposition of density and absorption gratings. The density grating was associated with the spatially varied molecular weight of polymer molecules across the sample, and the absorption grating resulted from the spatially varied density of free electrons. The other peaks were caused by the superposition of the absorption and phase gratings. The phase grating was generated by the formation of a periodic structure of polymer-rich and LC-rich regions in the sample. This study also proposes a model to explain these experimental results. Moreover, the theory derived from this model correlates well with the experimental results, allowing us to determine the amplitude of the final grating.