Probiotic candidates for controlling Paenibacillus larvae, a causative agent of American foulbrood disease in honey bee.

BACKGROUND: American foulbrood (AFB) disease caused by Paenibacillus larvae is dangerous, and threatens beekeeping. The eco-friendly treatment method using probiotics is expected to be the prospective method for controlling this pathogen in honey bees. Therefore, this study investigated the bacterial species that have antimicrobial activity against P. larvae. RESULTS: Overall, 67 strains of the gut microbiome were isolated and identified in three phyla; the isolates had the following prevalence rates: Firmicutes 41/67 (61.19%), Actinobacteria 24/67 (35.82%), and Proteobacteria 2/67 (2.99%). Antimicrobial properties against P. larvae on agar plates were seen in 20 isolates of the genus Lactobacillus, Firmicutes phylum. Six representative strains from each species (L. apis HSY8_B25, L. panisapium PKH2_L3, L. melliventris HSY3_B5, L. kimbladii AHS3_B36, L. kullabergensis OMG2_B25, and L. mellis OMG2_B33) with the largest inhibition zones on agar plates were selected for in vitro larvae rearing challenges. The results showed that three isolates (L. apis HSY8_B25, L. panisapium PKH2_L3, and L. melliventris HSY3_B5) had the potential to be probiotic candidates with the properties of safety to larvae, inhibition against P. larvae in infected larvae, and high adhesion ability. CONCLUSIONS: Overall, 20 strains of the genus Lactobacillus with antimicrobial properties against P. larvae were identified in this study. Three representative strains from different species (L. apis HSY8_B25, L. panisapium PKH2_L3, and L. melliventris HSY3_B5) were evaluated to be potential probiotic candidates and were selected for probiotic development for the prevention of AFB. Importantly, the species L. panisapium isolated from larvae was identified with antimicrobial activity for the first time in this study.

Actinidia arguta Sprout as a Natural Antioxidant: Ameliorating Effect on Lipopolysaccharide-Induced Cognitive Impairment.

Here, we investigated the prebiotic and antioxidant effects of Actinidia arguta sprout water extract (AASWE) on lipopolysaccharide (LPS)-induced cognitive deficit mice. AASWE increased viable cell count, titratable acidity, and acetic acid production in Lactobacillus reuteri strain and showed a cytoprotective effect on LPS-induced inflammation in HT-29 cells. We assessed the behavior of LPSinduced cognitive deficit mice using Y-maze, passive avoidance and Morris water maze tests and found that administration of AASWE significantly improved learning and memory function. The AASWE group showed antioxidant activity through downregulation of malondialdehyde levels and upregulation of superoxide dismutase levels in brain tissue. In addition, the AASWE group exhibited activation of the cholinergic system with decreased acetylcholinesterase activity in brain tissue. Furthermore, AASWE effectively downregulated inflammatory mediators such as phosphorylated- JNK, phosphorylated-NF-κB, TNF-α and interleukin-6. The major bioactive compounds of AASWE were identified as quercetin-3-O-arabinopyranosyl(1→2)-rhamnopyranosyl(1→6)-glucopyranose, quercetin-3-O-apiosyl(1→2)-galactoside, rutin, and 3-caffeoylquinic acid. Based on these results, we suggest that AASWE not only increases the growth of beneficial bacteria in the intestines, but also shows an ameliorating effect on LPS-induced cognitive impairment.

Pentacyclic triterpenoid-rich fraction of the Hardy kiwi (Actinidia arguta) improves brain dysfunction in high fat diet-induced obese mice.

This study was performed to investigate the effect of the chloroform fraction from Actinidia arguta (CFAA) on cognitive dysfunction in a C57BL/6 mouse model fed a high-fat diet (HFD) for 12 weeks. The CFAA has the protective effect on high glucose-induced neurotoxicity in MC-IXC cell (neuroblastoma cell line). In a C57BL/6 mouse model fed a HFD for 12 weeks, the improved glucose tolerance and cognitive dysfunction were observed in a group ingesting CFAA. In the brain tissue analysis, the impaired cholinergic, antioxidant system and mitochondria functions were improved in the CFAA group. In addition, in a molecular biology study, it was observed that CFAA improves HFD-induced abnormal insulin signaling such as increase of IRS phosphorylation at serine residues and reduction of Akt phosphorylation caused by the increase of JNK phosphorylation and then inhibited apoptosis. In the UPLC Q-TOF/MS analysis, pentacyclic triterpenoids such as asiatic acid (AA), madecassic acid (MA) were identified in CFAA as main compounds. Therefore, these results propose that Actinidia arguta rich in pentacyclic triterpenoids may be effective as preventive matter a therapeutic strategy to improve neurodegenerative disease caused by HFD.

Chronic Alcohol Exposure Induced Neuroapoptosis: Diminishing Effect of Ethyl Acetate Fraction from Aralia elata.

An ethyl acetate fraction from Aralia elata (AEEF) was investigated to confirm its neuronal cell protective effect on ethanol-induced cytotoxicity in MC-IXC cells and its ameliorating effect on neurodegeneration in chronic alcohol-induced mice. The neuroprotective effect was examined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) assays. As a result, AEEF reduced alcohol-induced cytotoxicity and oxidative stress. To evaluate the improvement of learning, memory ability, and spatial cognition, Y-maze, passive avoidance, and Morris water maze tests were conducted. The AEEF groups showed an alleviation of the decrease in cognitive function in alcohol-treated mice. Then, malondialdehyde (MDA) levels and the superoxide dismutase (SOD) content were measured to evaluate the antioxidant effect of AEEF in the brain tissue. Treatment with AEEF showed a considerable ameliorating effect on biomarkers such as SOD and MDA content in alcohol-induced mice. To assess the cerebral cholinergic system involved in neuronal signaling, acetylcholinesterase (AChE) activity and acetylcholine (ACh) content were measured. The AEEF groups showed increased ACh levels and decreased AChE activities. In addition, AEEF prevented alcohol-induced neuronal apoptosis via improvement of mitochondrial activity, including reactive oxygen species levels, mitochondrial membrane potential, and adenosine triphosphate content. AEEF inhibited apoptotic signals by regulating phosphorylated c-Jun N-terminal kinases (p-JNK), phosphorylated protein kinase B (p-Akt), Bcl-2-associated X protein (BAX), and phosphorylated Tau (p-Tau). Finally, the bioactive compounds of AEEF were identified as caffeoylquinic acid (CQA), 3,5-dicaffeoylquinic acid (3,5-diCQA), and chikusetsusaponin IVa using the UPLC-Q-TOF-MS system.

Ethyl acetate fraction from Hibiscus sabdariffa L. attenuates diabetes-associated cognitive impairment in mice.

The ameliorating effects of the ethyl acetate fraction from Hibiscus sabdariffa L. (EFHS)2 against diabetes mellitus (DM)3 and DM-induced cognitive impairment were investigated on streptozotocin (STZ)4-induced DM mice. The EFHS groups showed improved hyperglycemia and glucose tolerance compared to the STZ group. Furthermore, their liver and kidney function and lipid metabolic imbalance in the blood serum were effectively recovered. The EFHS groups significantly ameliorated STZ-induced cognitive impairment in Y-maze, passive avoidance, and Morris water maze (MWM)5 tests. The EFHS groups showed significant improvement in the antioxidant and cholinergic systems of the brain tissue. In addition, EFHS had an excellent ameliorating effect on protein expression levels from the tau hyperphosphorylation pathways, such as phospho-c-Jun N-terminal kinases (p-JNK),6 phospho-tau (p-tau),7 and cleaved poly (ADP-ribose) polymerase (c-PARP).8 The main compounds of EFHS were identified as various phenolic compounds, including hibiscus acid, caffeoylquinic acid (CQA)9 isomers, and quercetin derivates. Therefore, EFHS containing various physiologically active materials can potentially be used for improving DM-induced cognitive impairment via its antioxidant activity, improvement of the cholinergic system, and hyperphosphorylation tau signaling.

Incidences and Prognostic Impact of c-KIT, WT1, CEBPA, and CBL Mutations, and Mutations Associated With Epigenetic Modification in Core Binding Factor Acute Myeloid Leukemia: A Multicenter Study in a Korean Population.

BACKGROUND: To identify potential molecular prognostic markers in core binding factor (CBF) AML, we analyzed incidences and prognostic impacts of mutations in c-KIT, WT1, CEBPA, CBL, and a number of epigenetic genes in CBF AML. METHODS: Seventy one and 21 AML patients with t(8;21) and inv(16) were enrolled in this study, respectively. NPM1, CEBPA, c-KIT, IDH1/2, DNMT3A, EZH2, WT1, and CBL mutations were analyzed by direct sequencing. Patients were categorized with respect to c-KIT and WT1 mutation status, and both clinical features and prognoses were compared. RESULTS: The incidences of FLT3 internal tandem duplication (ITD), NPM1, CEBPA, IDH1/2, DNMT3A, EZH2, and CBL mutations were low (≤5%) in CBF AML patients. However, c-KIT and WT1 mutations occurred frequently (10.9% and 13.8%, respectively). t(8;21) patients with c-KIT mutations showed significantly shorter overall survival (OS) and disease free survival (DFS) periods than those without mutations (P<0.001, for both); however, although the limited number of t(8;21) patients were analyzed, WT1 mutation status did not affect prognosis significantly. Relapse or death during follow-up occurred more frequently in t(8;21) patients carrying c-KIT mutations than in those without the mutation, although the difference was significant only in a specific patient subgroup with no WT1 mutations (P=0.014). CONCLUSIONS: The incidences of mutations in epigenetic genes are very low in CBF AML; however, c-KIT and WT1 mutations occur more frequently than others. The poor prognostic impact of c-KIT mutation in t(8;21) AML patients only applies in a specific patient subgroup without WT1 mutations. The prognostic impact of WT1 mutation in CBF AML is not evident and further investigation is required.

Risk-Reducing Genetic Variant of Wilms Tumor 1 Gene rs16754 in Korean Patients With BCR-ABL1-Negative Myeloproliferative Neoplasm.

The genetic variant rs16754 of Wilms tumor gene 1 (WT1) has recently been described as an independent prognostic factor in AML patients. It is of great interest to test whether WT1 single nucleotide polymorphism can be used as a molecular marker in other types of cancer, to improve risk and treatment stratification. We performed sequencing analysis of exons 7 and 9 of WT1, which are known mutational hotspots, in a total of 73 patients with BCR-ABL1-negative myeloproliferative neoplasm (MPN) and 93 healthy controls. No previously reported WT1 mutations were identified in the present study. In Korean patients with BCR-ABL1-negative MPN, WT1 genetic variant rs16754 had no significant impact on clinical outcomes. We observed a significant difference in the allelic frequencies of WT1 rs16754 in Koreans between BCR-ABL1-negative MPN cases and healthy controls. Individuals carrying variant G alleles of WT1 rs16754 showed a relatively low prevalence of BCR-ABL1-negative MPN, compared with those carrying wild A alleles of WT1 rs16754 (Hazard ratio 0.10-0.65, P<0.05). Therefore, possession of the variant G allele of WT1 rs16754 may reduce the risk of developing BCR-ABL1-negative MPN.

Comparison of methylation profiling in cancerous and their corresponding normal tissues from korean patients with breast cancer.

BACKGROUND: Aberrant DNA hypermethylation plays a pivotal role in carcinogenesis and disease progression; therefore, accurate measurement of differential gene methylation patterns among many genes is likely to reveal biomarkers for improved risk assessment. We evaluated the gene hypermethylation profiles of primary breast tumors and their corresponding normal tissues and investigated the association between major clinicopathological features and gene hypermethylation. METHODS: A single reaction using methylation-specific multiplex ligation-dependent probe amplification was used to analyze the DNA methylation status of 24 tumor suppressor genes in 60 cancerous tissues and their corresponding normal tissues from patients with primary breast cancer. RESULTS: In cancerous breast tissues, 21 of 24 genes displayed promoter methylation in one or more samples. The most frequently methylated genes included RASSF1 (43.3%), APC (31.7%), CDKN2B (25.0%), CDH13 (23.3%), GSTP1 (16.7%), and BRCA1 (10%). APC was associated with lymph node metastasis, and BRCA1 was associated with negative estrogen receptor and negative progesterone receptor expression. In normal breast tissues, 8 of 24 tumor suppressor genes displayed promoter hypermethylation; CDKN2B (28.3%) and RASSF1 (8.3%) hypermethylation were most frequently observed. CONCLUSIONS: RASSF1 and CDKN2B hypermethylation in Korean breast cancer patients were the most frequent in cancerous tissue and corresponding normal tissue, respectively. Our data indicates that methylation of specific genes is a frequent event in morphologically normal breast tissues adjacent to breast tumors as well as the corresponding breast cancers. This study also suggests that gene methylation is linked to various pathological features of breast cancer; however, this requires confirmation in a larger study.

A case of partial trisomy 20p resulting from meiotic recombination of a maternal pericentric inversion.

Here we report the cytogenetic and clinical manifestations observed in a patient with a rec(20)dup(20p)inv(20)(p11.2q13.3)mat. The patient was a full-term newborn girl with asymmetric intrauterine growth restriction and multiple congenital malformations, including a ventricular septal defect, pulmonary atresia, ambiguous genitalia, clinodactyly, and sacral dimpling. To our knowledge, this is the 4th report in the world and the 1st one in Korea of a patient with rec(20)dup(20p).

Effects of RBC removal and TRIzol of peripheral blood samples on RNA stability.

BACKGROUND: Purification of mRNA from stored specimens is very important because results from RT-PCR and microarray analyses are largely affected by the quality of mRNA. Moreover, many preanalytical factors during collection, processing, and storage may affect mRNA quality and the expression of peripheral blood mononuclear cells (PBMC). In this study, we evaluate the effects of RBC removal techniques and TRIzol on RNA quality in blood samples. METHODS: We obtained EDTA-blood samples from 50 adult volunteers, and made 10 pools of buffy coats for comparison between protocols and also evaluated RNA quality of clinical samples in biobank. Use of TRIzol and RBC removal (RBC lysis or cell separation) were evaluated their effect on the quality of mRNA from the stored blood samples. RESULTS: RNA integrity with TRIzol was significantly better than that without TRIzol (RIN 4.5 vs. 9.2, respectively; P=0.002). The change in RIN of the PBMC separation method was equivalent to that of the RBC lysis method. After 12 months, IL6 mRNA expression from stored clinical samples in cell separation/TRIzol was stable. CONCLUSIONS: The blood samples frozen in TRIzol after RBC removal preserved RNA quality well. PBMC/TRIzol preservation for storage of blood samples could be a simple protocol for rapid, low-cost biobanking.

[Evaluation of ARCHITECT HCV core antigen assay].

BACKGROUND: Hepatitis C virus (HCV) core antigen (Ag) levels are known to be well correlating with HCV RNA levels, and may be used as an alternative marker of HCV replication for monitoring the response to HCV treatment. However, the low sensitivity of HCV core Ag assay has been an obstacle for clinical use. In this study, recently developed ARCHITECT HCV Ag assay (Abbott Laboratories, USA) was evaluated for analytical performance and clinical usefulness. METHODS: A total of 109 sera from HCV infected patients including various genotypes of HCV (1b, 2, 2a/2c, 2b, and 3a) and 20 sera from healthy donors were used for evaluating the sensitivity, precision, and linearity of the HCV core Ag assay. The cross reactivity with HIV, hepatitis B virus and myeloma proteins (N=5, each) and correlation with HCV RNA PCR assay were also evaluated. RESULTS: The sensitivity of the HCV core Ag assay was 97.2% (106/109) and there were no false positive results and cross reactivity. The within-run, between-run and between-day CVs were 3.0%, 2.5% and 3.0%, respectively. The levels of HCV core antigen showed a good correlation with those of HCV RNA quantification (r=0.940). The HCV Ag assay showed an excellent linearity in the range from 0.63 to 17,114 fmol/L (r=0.999). CONCLUSIONS: The ARCHITECT HCV Ag assay was good in sensitivity, precision, and linearity and its results well correlated with HCV RNA levels. This assay could be used as a good marker of viral replication for monitoring the therapy response in chronically HCV infected patients.

[Antimicrobial susceptibility patterns of Legionella isolates in the environment and in patients].

BACKGROUND: Antimicrobial susceptibility of Legionella spp. has rarely been studied in Korea. Therefore, we aimed to determine the susceptibility of Legionella spp. to various antibiotics. METHODS: We assessed the antimicrobial susceptibility of 66 environmental and clinical Legionella isolates collected between January 2001 and December 2008 from Korea and Japan. The minimum inhibitory concentrations (MICs) of 6 antibiotics, namely, azithromycin, ciprofloxacin, clarithromycin, clindamycin, gatifloxacin, and gemifloxacin were determined by the broth microdilution method using buffered starch yeast extract broth. RESULTS: The MIC ranges of the 6 antibiotics used against the Legionella isolates were as follows: 0.004-0.062 microg/mL (azithromycin), 0.002-0.5 microg/mL (ciprofloxacin), 0.004-0.5 microg/mL (clarithromycin), 0.12-4 microg/mL (clindamycin), 0.002-0.12 microg/mL (gatifloxacin), and 0.008-1 microg/mL (gemifloxacin). CONCLUSIONS: Legionella spp. isolates from Korea and Japan were most susceptible to gatifloxacin. Azithromycin, clarithromycin, ciprofloxacin, and gemifloxacin were also effective for treating legionellosis.

[Comparison of the frequencies and distributions of unexpected antibodies based on different calculation criteria].

BACKGROUND: The frequencies and distributions of unexpected antibodies have been reported using two different criteria, based on either number of persons tested or number of tests performed. But there has been no study that compared the results of analyses based on these two different criteria using the same data set. METHODS: Unexpected antibody tests performed in a University Hospital during recent 6 yr (January 2002-December 2007) were retrospectively analyzed: 76,985 tests (59,503 persons) for screening and 875 tests (749 persons) for identification. Data were analyzed using two different criteria, based on 'persons tested' and 'tests performed'. Antibodies had been screened and identified using LISS/Coombs gel cards with DiaMed-ID system (DiaMed AG, Switzerland). RESULTS: Frequencies of unexpected antibodies based on 'persons tested' and 'tests performed' were 1.32% and 1.34%, respectively (P=0.88). For frequently detected as well as rarely detected antibodies, there were no significant differences in the frequencies based on two different criteria. However, for rarely detected antibodies (anti-Xg(a) and Anti-E & D), the frequencies based on 'tests performed' were higher than those based on 'persons tested', affecting a change in the order of frequencies of antibodies detected. CONCLUSIONS: As there were no significant differences in the frequencies of unexpected antibodies calculated using two different criteria, both criteria can be used together for the patient population in our hospital. However, two criteria should be compared to validate the results for other populations.