Tenebrio molitor Spätzle 1b Is Required to Confer Antibacterial Defense Against Gram-Negative Bacteria by Regulation of Antimicrobial Peptides.

Innate immunity is the ultimate line of defense against invading pathogens in insects. Unlike in the mammalian model, in the insect model, invading pathogens are recognized by extracellular receptors, which activate the Toll signaling pathway through an extracellular serine protease cascade. In the Toll-NF-κB pathway, the extracellular spätzle protein acts as a downstream ligand for Toll receptors in insects. In this study, we identified a novel Spätzle isoform (TmSpz1b) from RNA sequencing database of Tenebrio molitor. TmSpz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of TmSpz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in TmSpz1b was involved in three disulfide bridges and the formation of a spätzle dimer. TmSpz1b was mostly expressed in the hemocytes of T. molitor late instar larvae. The mRNA expression of TmSpz1b was highly induced in the hemocytes after Escherichia coli, Staphylococcus aureus, and Candida albicans stimulation of T. molitor larvae. TmSpz1b silenced larvae were significantly more susceptible to E. coli infection. In addition, RNAi-based functional assay characterized TmSpz1b to be involved in the positive regulation of antimicrobial peptide genes in hemocytes and fat bodies. Further, the TmDorX2 transcripts were downregulated in TmSpz1b silenced individuals upon E. coli challenge suggesting the relationship to Toll signaling pathway. These results indicate that TmSpz1b is involved in the T. molitor innate immunity, causes the sequestration of Gram-negative bacteria by the regulatory action of antimicrobial peptides, and enhances the survival of T. molitor larvae.

TmSpz-like Plays a Fundamental Role in Response to E. coli but Not S. aureus or C. albican Infection in Tenebrio molitor via Regulation of Antimicrobial Peptide Production.

The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor (TmSpz-like) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor. Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post-E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli-infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli-infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes (TmDorX1, TmDorX2, and TmRelish) were downregulated in the E. coli-infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.

Molecular Cloning and Expression Analysis of Three Suppressors of Cytokine Signaling Genes (SOCS5, SOCS6, SOCS7) in the Mealworm Beetle Tenebrio molitor.

Suppressors of cytokine signaling (SOCS) influence cytokine and growth factor signaling by negatively regulating the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway to maintain homeostasis during immune responses. However, functional characterization of SOCS family members in invertebrates is limited. Here, we identified and evaluated three SOCS genes (type I sub-family) in the mealworm beetle Tenebrio molitor. The full-length open reading frames (ORFs) of TmSOCS5, TmSOCS6, and TmSOCS7 comprised of 1389, 897, and 1458 nucleotides, encoding polypeptides of 462, 297, and 485 amino acids, respectively. The SH2 and SOCS box domains of the TmSOCS C-terminal region were highly conserved. Phylogenetic analysis revealed that these SOCS genes were clustered within the type I subfamily that exhibits the highest amino acid identity with Tribolium castaneum SOCS genes. Contrary to TmSOCS7 expression, the expression levels of TmSOCS5 and TmSOCS6 were lower in the larval, pupal, and adult stages. In larvae and adults, the expression levels of TmSOCS5 and TmSOCS6 were highest in the hemocytes and ovaries, respectively. SOCS transcripts were also highly upregulated in the hemocytes of T. molitor larvae within 3⁻6 h post-infection with the fungus Candida albicans. Collectively, these results provide valuable information regarding the involvement of TmSOCS type-I subfamily in the host immune response of insects.

Short-term application of dexamethasone on stem cells derived from human gingiva reduces the expression of RUNX2 and ß-catenin.

Objective Next-generation sequencing was performed to evaluate the effects of short-term application of dexamethasone on human gingiva-derived mesenchymal stem cells. Methods Human gingiva-derived stem cells were treated with a final concentration of 10-7 M dexamethasone and the same concentration of vehicle control. This was followed by mRNA sequencing and data analysis, gene ontology and pathway analysis, quantitative real-time polymerase chain reaction of mRNA, and western blot analysis of RUNX2 and ß-catenin. Results In total, 26,364 mRNAs were differentially expressed. Comparison of the results of dexamethasone versus control at 2 hours revealed that 7 mRNAs were upregulated and 25 mRNAs were downregulated. The application of dexamethasone reduced the expression of RUNX2 and ß-catenin in human gingiva-derived mesenchymal stem cells. Conclusion The effects of dexamethasone on stem cells were evaluated with mRNA sequencing, and validation of the expression was performed with qualitative real-time polymerase chain reaction and western blot analysis. The results of this study can provide new insights into the role of mRNA sequencing in maxillofacial areas.

Effects of Cimicifugae Rhizoma on the osteogenic and adipogenic differentiation of stem cells.

Cimicifugae Rhizoma, a herb with a long history of use in traditional Oriental medicine is reported to have anti-inflammatory, antioxidant, anti-complement and anticancer effects. The aim of the present study was to evaluate the effects of Cimicifugae Rhizoma extracts on the osteogenic and adipogenic differentiation of human stem cells derived from gingiva. Stem cells derived from gingiva were grown in the presence of Cimicifugae Rhizoma at final concentrations of 0.1, 1 and 10 µg/ml. Cell proliferation analyses were performed at day 15. For osteogenic differentiation experiments, the stem cells were cultured in osteogenic media containing ß-glycerophosphate, ascorbic acid-2-phosphate and dexamethasone, and osteogenic differentiation was evaluated by analysis of osteocalcin expression at 21 days. For adipogenic differentiation experiments, the stem cells were grown in adipogenic induction medium, and the adipogenic differentiation was evaluated by analysis of adipocyte fatty acid-binding protein at day 14. The cultures grown in the presence of 0.1 µg/ml Cimicifugae Rhizoma showed a significant increase in cellular proliferation at day 15 compared with the control group. The relative osteogenic differentiation in the presence of Cimicifugae Rhizoma for the 0.1, 1 and 10 µg/ml groups was 171.5±13.7, 125.6±28.7 and 150.5±9.0, respectively, when that of the untreated control group on day 21 was considered to be 100%. The relative adipogenic differentiation at day 14 of the 0.1, 1 and 10 µg/ml groups in the presence of Cimicifugae Rhizoma was 97.5±15.0, 102.9±12.8 and 87.0±6.8%, respectively when that of the untreated control group on day 14 was considered to be 100%. Within the limits of this study, Cimicifugae Rhizoma increased the proliferation of stem cells derived from the gingiva, and low concentrations of Cimicifugae Rhizoma may increase the osteogenic differentiation of stem cells.

Decreased Cystatin C-Estimated Glomerular Filtration Rate Is Correlated with Prolonged Hospital Stay in Transient Tachypnea of Newborn Infants.

BACKGROUND: Transient tachypnea of the newborn (TTN) is a benign disorder with a variable clinical course that often leads to hospitalization. The aim of this study was to assess and validate the relationship between the serum cystatin C level and symptom duration in infants with TTN. METHODS: Forty newborns presenting with TTN and who had undergone serum cystatin C (Cys C) tests on the first day of admission to the Kyung Hee University Hospital (Seoul, Korea) from 2009 to 2013 were included. The serum Cys C level, creatinine (Cr) level, estimated glomerular filtration rate (eGFR), and tachypnea duration were correlated retrospectively. RESULTS: The median gestation period was 37.8 ± 3.8 weeks and the mean birth weight was 3.2 ± 0.4 kg. Tachypnea duration was 3.3 ± 2.0 days. Serum Cys C and Cr levels were 1.7 ± 0.2 mg/L and 0.8 ± 1.2 mg/dL, respectively. Tachypnea duration was significantly positively correlated with the serum levels of Cys C and significantly negatively correlated with Cys C-based eGFR (p = 0.016), but was not significantly correlated with the serum Cr level or Cr-based eGFR. When tachypnea duration was compared between infants with Cys C level <1.6 mg/L (n = 15; Group A) and infants with Cys C level ≥ 1.6 mg/L (n = 25; Group B), the symptom duration was significantly shorter in Group A infants (p = 0.011). CONCLUSION: Tachypnea duration was shorter with higher Cys C-based eGFR in infants with TTN.

Self-assembly of biogenic gold nanoparticles and their use to enhance drug delivery into cells.

Integration of the principles of green chemistry into nanotechnology is one of the key issues in nanobio-science research. There is a growing need for development of a synthesis method for producing environmentally harmless nanoparticles in order to avoid adverse effects in medical applications. Here, we report the use of a simple and rapid in vivo biosynthesis method for the preparation of gold nanoparticles (AuNPs) using heavy metal binding proteins (HMBPs) in recombinant Escherichia coli. The HMBPs were found to act as reducing, stabilizing, and capping agents to form the spherical nanoparticles with 5-20 nm in diameter. The size and the shape of AuNPs were modulated by varying the concentration ratio of recombinant proteins in the medium. Only 20 min was required to form AuNPs at room temperature, suggesting that the reaction rate of the proposed method is faster than that of the chemical methods commonly used for nanoparticle synthesis. The AuNPs could be applied as drug carriers in therapeutic applications to improve drug delivery, since they exhibit higher biocompatibility and less toxic effects than chemically synthesized materials. To achieve high cytotoxicity for cancer chemotherapy, doxorubicin (Dox) was released from AuNPs, which can be a more efficient anti-cancer agent than free Dox.

Evaluation of the effects of Angelicae dahuricae radix on the morphology and viability of mesenchymal stem cells.

Angelicae dahuricae radix is a traditional herbal medicine used to treat various diseases in China and Korea, such as colds, headaches, rhinitis and psoriasis. Angelicae dahuricae radix has been used as an anti-inflammatory, analgesic, antipyretic and antioxidant remedy. This study was performed in order to evaluate the effects of the extracts of Angelicae dahuricae radix on the morphology and viability of mesenchymal stem cells derived from the gingiva. Mesenchymal stem cells derived from the gingiva were grown in the presence of Angelicae dahuricae radix at final concentrations that ranged from 0.001 to 100 µg/ml. The morphology of the cells was viewed under an inverted microscope, and the analysis of cell proliferation was performed with cell counting kit-8 (CCK-8) on days 1, 3 and 7. The cells in the control group had spindle-shaped, fibroblast-like morphology at days 1, 3 and 7 under optical microscopy. The shapes of the cells in 0.001, 0.01, 0.1, 1, 10 and 100 µg/ml Angelicae dahuricae radix were similar to the shapes of the cells in the control group. The relative values of the CCK-8 assays of 0.001, 0.01, 0.1, 1, 10, and 100 µg/ml Angelicae dahuricae radix were 102.5 ± 0.6, 133.3 ± 9.6, 148.4 ± 20.5, 147.7 ± 12.6, 132.3 ± 27.7 and 101.1 ± 4.6%, respectively, when the CCK-8 result of the control group on day 1 was considered to be 100%. There was a marginal increase in cell proliferation at 0.1 and 1 µg/ml groups at day 1; however, this did not achieve statistical significance (P=0.052). The relative values of the CCK-8 assays of 0.001, 0.01, 0.1, 1, 10 and 100 µg/ml Angelicae dahuricae radix were 96.5 ± 1.3, 89.3 ± 0.9, 90.3 ± 3.0, 84.8 ± 12.2, 92.3 ± 4.5 and 86.8 ± 11.7%, respectively, when the CCK-8 result of the control group on day 3 was considered to be 100% (P>0.05). The relative values of the CCK-8 assays of 0.001, 0.01, 0.1, 1, 10 and 100 µg/ml Angelicae dahuricae radix day 7 were 94.9 ± 22.3, 102.8 ± 22.1, 127.4 ± 7.4, 130.4 ± 1.3, 129.2 ± 10.8 and 124.8 ± 9.1%, respectively, when the CCK-8 result of the control group on day 7 was considered to be 100%, but there were no statistically significant differences among the groups (P>0.05). Within the limits of this study, Angelicae dahuricae radix at the tested concentrations did not produce statistically significant differences in the viability of stem cells derived from the gingiva.

7,3',4'-Trihydroxyisoflavone Ameliorates the Development of Dermatophagoides farinae-Induced Atopic Dermatitis in NC/Nga Mice.

Atopic dermatitis is an inflammatory and chronically relapsing skin disorder that commonly occurs in children; the number of atopic dermatitis patients is increasing. The cause and mechanism of atopic dermatitis have not been defined clearly, although many studies are ongoing. Epidemiological studies suggest that soybean and its isoflavones have immunoregulatory activities. Here, we report that 7,3',4'-trihydroxyisoflavone (7,3',4'-THIF), a major metabolite of daidzin, effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO), tumor necrosis factor (TNF)- α , and interleukin (IL)-6 production in RAW 264.7 cells, and also reduced ß -hexosaminidase secretion in RBL-2H3 cells. Moreover, 7,3',4'-THIF significantly reduced scratching time, transepidermal water loss, and mast cell infiltration. It also decreased protease-activated receptor (PAR)-2 and IL-4 expression and increased filaggrin expression in skin lesions of NC/Nga mice. These results suggest that 7,3',4'-THIF improves Dermatophagoides farina body extract-induced atopic dermatitis in NC/Nga mice.

Current status of registry of vaccine clinical trials conducted by Korean investigators in ClinicalTrials.gov, database of US National Institutes of Health.

PURPOSE: PubMed is not only includes international medical journals but also has a registration site for the ongoing clinical trials, such as ClinicalTrials.gov, under the supervision of US National Institutes of Health. We analyzed current status of vaccine clinical trials conducted by Korean investigators in database of ClinicalTrial.gov. MATERIALS AND METHODS: As of October 2012, there are total of 72 trials found on registry of vaccine clinical trials conducted by Korean investigators in database of ClinicalTrial.gov. These trials were analyzed and classified by conditions of vaccine clinical trials, biologicals or drugs used in vaccine clinical trials, status of proceeding research, and list of sponsor and collaborators. RESULTS: Total 72 trials of vaccine clinical trials conducted by Korean investigators are classified by groups of infection (64 trials), cancer (4 trials), and others (4 trials). Infections group shown are as follows: poliomyelitis, pertussis, diphtheria, tetanus, and Haemophilus influenzae type b (10), influenza (9), human papillomavirus infection (8), pneumococcal vaccine (6), herpes zoster (4), smallpox (4), hepatitis B (4), etc. One trial of each in lung cancer, breast cancer, prostate cancer, and colorectal cancer are shown in cancer group. One trial of each in Crohn's disease, ulcerative colitis, renal failure, and rheumatoid arthritis are shown in other group. CONCLUSION: Vaccine clinical trials conducted by Korean investigators in ClinicalTrial.gov reflects the current status of Korean research on vaccine clinical trials at the international level and can indicate research progress. It is hoped that this aids the development of future vaccine clinical trials in Korea.

Enzymatic production of 3,6-anhydro-L-galactose from agarose and its purification and in vitro skin whitening and anti-inflammatory activities.

3,6-Anhydro-L-galactose (L-AHG) constitutes 50% of agarose, which is the main component of red macroalgae. No information is currently available on the mass production, metabolic fate, or physiological effects of L-AHG. Here, agarose was converted to L-AHG in the following three steps: pre-hydrolysis of agarose into agaro-oligosaccharides by using acetic acid, hydrolysis of the agaro-oligosaccharides into neoagarobiose by an exo-agarase, and hydrolysis of neoagarobiose into L-AHG and galactose by a neoagarobiose hydrolase. After these three steps, L-AHG was purified by adsorption and gel permeation chromatographies. The final product obtained was 95.6% pure L-AHG at a final yield of 4.0% based on the initial agarose. In a cell proliferation assay, L-AHG at a concentration of 100 or 200 µg/ mL did not exhibit any significant cytotoxicity. In a skin whitening assay, 100 µg/ mL of L-AHG showed significantly lower melanin production compared to arbutin. L-AHG at 100 and 200 µg/ mL showed strong anti-inflammatory activity, indicating the significant suppression of nitrite production. This is the first report on the production of high-purity L-AHG and its physiological activities.