Lipidomic changes in mouse oocytes vitrified in PEG 8000-supplemented vitrification solutions.

Cryopreserved oocytes are inevitably exposed to cold stress, which negatively affects the cellular aspects of the oocytes. Lipidomic analysis of the oocytes reveals quantitative changes in lipid classes under conditions of cold stress, leading to potential freezing-vulnerability. We had previously shown that specific phospholipids are significantly downregulated in vitrified-warmed mouse oocytes compared to those in fresh oocytes. In this study, we examined whether supplementation of polyethylene glycol 8000 (PEG 8000) during vitrification influences the lipidome of the oocytes. We used liquid chromatography with tandem mass spectrometry (LC-MS/MS) to study the alteration in the lipidome in three groups of mouse oocytes: fresh, vitrified-warmed, and vitrified with PEG 8000-warmed during vitrification. In these groups, we targeted to analyze 21 lipid classes. We profiled 132 lipid species in the oocytes and statistical analyses revealed lipid classes that were up- or downregulated in these groups. Overall, our data revealed that several classes of lipids were affected during vitrification, and that oocytes vitrified with PEG 8000 to some extent alleviated the levels of changes in phospholipid and sphingolipid contents during vitrification. These results suggest that phospholipids and sphingolipids are influenced by PEG 8000 during vitrification and that PEG 8000 can be considered as a potential candidate for preserving membrane integrity during oocyte cryopreservation.

Molecular analysis of lipid uptake- and necroptosis-associated factor expression in vitrified-warmed mouse oocytes.

BACKGROUND: We had previously demonstrated that vitrification reduces the levels of certain phospholipid classes, and that oocytes from aged mice show a similar lipidome alteration, even without vitrification. In the current investigation, we examined if vitrification-warming of mouse oocytes from young and aged mice causes any changes in molecular aspects of lipid-associated features. METHODS: Metaphase II (MII) stage oocytes were harvested from young (10-14-week-old) and aged (45-54-week-old) mice by a superovulation regime with PMSG followed by hCG. We examined the status of the intracellular lipid pool and the integrity of the plasma membrane by staining oocytes with BODIPY 500/510 and CellMask live dyes. Expression of lipid uptake- and necroptosis-associated genes was assessed by quantitative PCR analyses, in oocytes from young and old mice, before and after vitrification. Localization patterns of two crucial necroptosis proteins, phosphorylated MLKL (pMLKL) and phosphorylated RIPK1 (pRIPK1) were examined in mouse oocytes by immunofluorescence staining. Necrostain-1 (Nec1), an inhibitor of RIPK1, was used to examine if RIPK1 activity is required to maintain oocyte quality during vitrification. RESULTS: We confirmed that vitrified-warmed oocytes from aged mice showed noticeable decrease in both CellMask and BODIPY 500/510 dyes. Among the lipid uptake-associated genes, Cd36 expression was higher in oocytes from aged mice. Necroptosis is a type of programmed cell death that involves damage to the plasma membrane, eventually resulting in cell rupture. The expression of necroptosis-associated genes did not significantly differ among groups. We observed that localization patterns of pMLKL and pRIPK1 were unique in mouse oocytes, showing association with microtubule organizing centers (MTOCs) and spindle poles. pMLKL was also localized on kinetochores of MII chromosomes. Oocytes treated with Nec1 during vitrification showed a decreased survival rate, indicating the importance of RIPK1 activity in oocyte vitrification. CONCLUSIONS: We report that oocytes from aged mice show differential expression of CD36, which suggests that CD36-mediated lipid uptake may be influenced by age. We also show for the first time that pMLKL and pRIPK1 exhibit unique localization pattern in mouse oocytes and this may suggest role(s) for these factors in non-necroptosis-associated cellular processes.

Comparative toxicity of silver nanoparticles on oxidative stress and DNA damage in the nematode, Caenorhabditis elegans.

This study examined the effects of polyvinylpyrrolidone (PVP) surface coating and size on the organismal and molecular toxicity of silver nanoparticles (AgNPs) on the nematode, Caenorhabditis elegans. The toxicity of bare AgNPs and 8 and 38 nm PVP-coated AgNPs (PVP8-AgNPs, PVP38-AgNPs) were compared. The toxicity of AgNO3 was also tested because ion dissolution and particle-specific effects are often important characteristics determining Ag nanotoxicity. Comparative toxicity across AgNO3 and the three different types of AgNPs was first evaluated using a C. elegans mortality test by a direct comparison of the LC50 values. Subsequently, mutant screening followed by oxidative stress, mitochondrial toxicity and DNA damage assays were carried out at equitoxic (LC10 and LC50) concentrations to further assess the toxicity mechanism of AgNO3 and AgNPs. AgNO3 and bare AgNPs had similar toxicities, whereas PVP coating reduced the toxicity of the AgNPs significantly. Of the PVP-AgNPs, the smaller NPs were more toxic. Different groups of mutants responded differently to AgNO3 and AgNPs, which indicates that their toxicity mechanism might be different. AgNO3 and bare AgNPs induced mitochondrial membrane damage. None of the silver materials tested caused detectable polymerase-inhibiting DNA lesions in either the nucleus or mitochondria as measured by a quantitative PCR assay, but AgNO3, bare AgNPs and PVP8-AgNPs induced oxidative DNA damage. These results show that coatings on the AgNPs surface and the particle size make a clear contribution to the toxicity of the AgNPs, and oxidative stress-related mitochondrial and DNA damage appear to be potential mechanisms of toxicity.

Hypoxia inducible factor-1 (HIF-1)-flavin containing monooxygenase-2 (FMO-2) signaling acts in silver nanoparticles and silver ion toxicity in the nematode, Caenorhabditis elegans.

In the present study, nanotoxicity mechanism associated with silver nanoparticles (AgNPs) exposure was investigated on the nematode, Caenorhabditis elegans focusing on the hypoxia response pathway. In order to test whether AgNPs-induced hypoxia inducible factor-1 (HIF-1) activation was due to hypoxia or to oxidative stress, depletion of dissolved oxygen (DO) in the test media and a rescue effect using an antioxidant were investigated, respectively. The results suggested that oxidative stress was involved in activation of the HIF-1 pathway. We then investigated the toxicological implications of HIF-1 activation by examining the HIF-1 mediated transcriptional response. Of the genes tested, increased expression of the flavin containing monooxygenase-2 (FMO-2) gene was found to be the most significant as induced by AgNPs exposure. We found that AgNPs exposure induced FMO-2 activation in a HIF-1 and p38 MAPK PMK-1 dependent manner, and oxidative stress was involved in it. We conducted all experiments to include comparison of AgNPs and AgNO3 in order to evaluate whether any observed toxicity was due to dissolution or particle specific. The AgNPs and AgNO3 did not produce any qualitative differences in terms of exerting toxicity in the pathways observed in this study, however, considering equal amount of silver mass, in every endpoint tested the AgNPs were found to be more toxic than AgNO3. These results suggest that Ag nanotoxicity is dependent not only on dissolution of Ag ion but also on particle specific effects and HIF-1-FMO-2 pathway seems to be involved in it.

Comparison of real-time reverse transcriptase-polymerase chain reaction and nested or commercial reverse transcriptase-polymerase chain reaction for the detection of hepatitis E virus particle in human serum.

Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. The virus is the 7.5-kb single-stranded positive RNA virus and has been classified in the genus Herpevirus [corrected] of the [corrected] Herpeviridae [corrected] Recently, HEVs were identified from several countries worldwide from human and animals including swine. Studies on the genomic analysis of HEV isolates and seroprevalence of anti-HEV antibodies suggested that HEV has been considered as a potent zoonotic agent. The HEV infection has been diagnosed by detection of anti-HEV antibodies or virus by using reverse transcriptase-polymerase chain reaction (RT-PCR) methods in the blood or feces. However, these diagnostic methods were not quantitative and not enough to diagnose small amounts of target molecules. Moreover, these methods were not adequate during the incubation period or early acute phase. To overcome these problems, real-time RT-PCR method was developed with a cloned viral DNA and in vitro transcribed cRNA in this study. The sensitivity of the reaction was 1.68 x 10(1) copies per reaction. Correlation coefficient values of the reactions in the repeated experiments were over 0.99. Ranges of slopes and coefficient variation values were from 3.341 to 3.435 and from 1.20 to 5.98, respectively. In comparison of the real-time PCR with nested or commercial RT-PCR, HEV particles could be detected in the negative samples, which were determined by conventional nested RT-PCR.

In vivo induction of mucosal immune responses by intranasal administration of chitosan microspheres containing Bordetella bronchiseptica DNT.

In vitro immune-stimulating activities of Bordetella bronchiseptica dermonecrotoxin (BBD)-loaded in chitosan microspheres (CMs) were reported with a mouse alveolar macrophage cell line (RAW264.7). Based on the report, in vivo activity of immune-induction was investigated by intranasal administration of the BBD-loaded CMs into mice. BBD was loaded into the CMs prepared by an ionic gelation process with tripolyphosphate. Mice were immunized by direct administration of the BBD-loaded CMs into the nasal cavity. After immunization of the mice, BBD-specific immune responses (IgG and IgA titers) were measured in sera, nasal wash, and saliva by ELISA. BBD-specific IgA titers in the nasal cavity were time- and dose-dependently increased by the administration. Similar phenomena were observed in the analysis of systemic IgA and IgG in sera. However, the antibody in saliva was undetectable by ELISA. These results suggested that direct vaccination via the nasal cavity was effective for targeting nasal-associated lymphoid tissues, and that CMs were an efficient adjuvant in nasal mucosal immunity for atrophic rhinitis vaccine.

A predictive model for the level of sIgA based on IgG levels following the oral administration of antigens expressed in Saccharomyces cerevisiae.

Oral vaccination may be the most efficient way of inducing an immune response at the remote mucosal site through the common mucosal immune network. Antigen-specific secretory IgA (sIgA) is the major immunoglobulin type generally detected in the secretions of experimental animals following an effective oral immunization. Actinobacillus pleuropneumoniae causing disease in the lung of pig initially interacts, colonizes, and infects the host tissues at the mucosal surface of the respiratory tract. Also, importantly for A. pleuropneumoniae protection, the quantity of sIgA in the lung had merits associated with the mucosal immunity. However, there is no simple method to monitor the level of sIgA as an indicator for the induction of local immune responses by an oral vaccination in the target tissue. Therefore, the relationship between sIgA and IgG was analyzed to evaluate the induction of local immune responses by an oral immunization with Saccharomyces cerevisiae expressing the apxIA and apxIIA genes of A. pleuropneumoniae in this study. The correlation coefficient of determination (r2 x 100) for paired samples in both vaccinated and control groups showed a significant positive-relationship between IgG in sera and sIgA in the lung or intestine. These results indicated that IgG antibody titers in sera could be useful to indirectly predict local immune response, and sIgA, in the lung or intestine to evaluate the efficacy of an oral vaccination.

Development and use of a multiplex polymerase chain reaction assay based on Apx toxin genes for genotyping of Actinobacillus pleuropneumoniae isolates.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of a porcine pleuropneumonia that threatens the global swine industry. The major pathogenic toxins of A. pleuropneumoniae include ApxI, ApxII, ApxIII, and ApxIV, which are serotype or serovar specific. Several techniques have been developed for the identification and typing of A. pleuropneumoniae. Serological assays are used to identify and serotype A. pleuropneumoniae, but factors such as cross-reactivity limit their specificity. Labor, time, and the requirement for specific antibodies are also drawbacks of these assays. Multistep polymerase chain reaction (PCR) techniques based on apx genes have been reported for the identification and typing of A. pleuropneumoniae. This study developed multiplex PCR for the identification and genotyping of A. pleuropneumoniae based on apx genes. This multiplex PCR technique was successful in differentiating 11 of 15 reference serotypes. Five different primer sets were used to amplify the 4 apx genes from each serotype in a single-step reaction. The multiplex PCR reported in this study was further used in genotyping 51 field isolates of A. pleuropneumoniae from different regions of Korea. The concomitant amplification of all 4 apx genes makes multiplex PCR more specific and convenient for the diagnosis and genotyping of A. pleuropneumoniae.

Changes in patterns of antimicrobial susceptibility and class 1 integron carriage among Escherichia coli isolates.

The worldwide use of antimicrobials in different fields has created enormous pressure for the selection of resistance among opportunistic bacterial pathogen. One hundred four E. coli isolates were collected and identified from swine with diarrhea in Korea during the period of 2002. The isolates showed highly resistant to streptomycin (99.0%), tetracycline (97.1%), neomycin (91.3%) and carbenicillin (84.6%) in antimicrobial susceptibility test. Moreover, all of the isolates showed multiple antimicrobial resistant to more than 3, and 85% of them were resistant to more than 7 of total 14 antimicrobial agents. In comparison with isolates in 1998, resistance to antimicrobials was more frequent among the isolates in 2002. Presence of class 1 integrons was investigated through amplification of the gene with PCR, and could be classified 8 groups by pattern of 4 different amplicons. Class 1 integrons were observed in 67 strains (64.2%) of E. coli from swine in Korea. One and 1.6 kbp of amplicons were revealed to contain aadA1 and aadB-aadA1 gene cassettes respectively. Two kbp of amplicon had three different gene cassettes, dhfrXII-orfF-aadA2, and 3.0 kbp of amplicon includes aadB-cmlA1 gene cassettes.

Identification of novel human hepatitis E virus (HEV) isolates and determination of the seroprevalence of HEV in Korea.

Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. Recently, HEV isolates were subsequently identified in humans and swine in many countries, including Korea. Also, public concerns regarding HEV as a potential zoonotic agent have been increasing. Therefore, we attempted to identify HEV from Korean sera and compare the nucleotide sequences with those of previously identified HEV isolates from other countries. In our study, viral RNA was purified from 568 human sera collected from different regions of Korea. Nested PCR and reverse transcriptase PCR were developed based on the nucleotide sequences of open reading frame 2 (ORF 2) of U.S. and Japanese HEV isolates from humans and Korean HEV isolates from swine. After amplification of the HEV ORF 2 gene from 14 serum samples that were collected mainly from rural areas (2.64% prevalence of HEV viremia), the gene was cloned and sequenced. The isolates were classified into seven different strains, all of which belonged to genotype III. The human isolates we identified were closely related to three Korean swine isolates, with 99.2 to 92.9% nucleotide sequence homology. Our isolates were also related to the Japanese and U.S. HEV isolates, with 99.6 to 97.9% amino acid sequence homology. Human sera were collected from 361 individuals from community health centers and medical colleges. With respect to seroprevalence, 11.9% of the Korean population had anti-HEV immunoglobulin G (IgG). In individuals ranging in age from 40 to over 60 years, the prevalence of anti-HEV IgG was demonstrated by a seroprevalence of almost 15%, especially among populations in rural areas. This is the first report on the identification of human HEV in Korea. Overall, this study demonstrates that subclinical HEV infections may prevail in human populations in Korea and that there is a strong possibility that HEV is a zoonotic agent.