Characterization of Shiga Toxin-producing Escherichia coli Isolated from Cattle Around Ulaanbaatar City, Mongolia.

Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0% to 40%. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 [stx1a (+), stx2a (+), ehxA (+) and saa (+)], was isolated from eight farms. This is the first report on the characterization of STEC in cattle in Mongolia, and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.

Prediction of PCR amplification from primer and template sequences using recurrent neural network.

We have developed a novel method to predict the success of PCR amplification for a specific primer set and DNA template based on the relationship between the primer sequence and the template. To perform the prediction using a recurrent neural network, the usual double-stranded formation between the primer and template nucleotide sequences was herein expressed as a five-lettered word. The set of words (pseudo-sentences) was placed to indicate the success or failure of PCR targeted to learn recurrent neural network (RNN). After learning pseudo-sentences, RNN predicted PCR results from pseudo-sentences which were created by primer and template sequences with 70% accuracy. These results suggest that PCR results could be predicted using learned RNN and the trained RNN could be used as a replacement for preliminary PCR experimentation. This is the first report which utilized the application of neural network for primer design and prediction of PCR results.

Research Note: Antimicrobial resistance of Campylobacter species isolated from chickens near Ulaanbaatar city, Mongolia.

There has been no report on the prevalence of Campylobacter spp. in farm animals in Mongolia. To uncover the prevalence of Campylobacter spp. in chickens in Mongolia and their antimicrobial resistance, in this study, we isolated and characterized Campylobacter spp. from chickens in Mongolia. We collected 71 cloacal swabs of chickens from 5 farms including 4 layer farms and one broiler farm near Ulaanbaatar city and isolated 25 Campylobacter jejuni and 6 Campylobacter coli isolates. All isolates were resistant to tetracycline, and 3 C. coli isolates were resistant to erythromycin. The C. coli isolates possessed either the erm(B) gene or nucleotide substitution at nt 2,075 of 23S rDNA, both of which are known to be associated with erythromycin resistance. Sixteen of the 31 C. jejuni/C. coli isolates (51.6%) were resistant to nalidixic acid and fluoroquinolones. All the fluoroquinolone-resistant isolates possessed amino acid substitution from threonine to isoleucine at codon 86 (nucleotide substitution: ACA to ATA). Multilocus sequence typing and phylogenetic analyses showed a variation in C. jejuni/C. coli in chickens in Mongolia. In addition, some of the C. jejuni isolates seemed to be phylogenetically close to isolates in Asian and Oceanian countries. This is the first report on the characterization of antimicrobial resistance of Campylobacter spp. in farm animals in Mongolia and is valuable for implementation of measures for a prudent use of antimicrobials in farm animals.

White nanolight source for optical nanoimaging.

Nanolight sources, which are based on resonant excitation of plasmons near a sharp metallic nanostructure, have attracted tremendous interest in the vast research fields of optical nanoimaging. However, being a resonant phenomenon, this ideally works only for one wavelength that resonates with the plasmons. Multiple wavelengths of light in a broad range confined to one spot within a nanometric volume would be an interesting form of light, useful in numerous applications. Plasmon nanofocusing can generate a nanolight source through the propagation and adiabatic compressions of plasmons on a tapered metallic nanostructure, which is independent of wavelength, as it is based on the propagation, rather than resonance, of plasmons. Here, we report the generation of a white nanolight source spanning over the entire visible range through plasmon nanofocusing and demonstrate spectral bandgap nanoimaging of carbon nanotubes. Our experimental demonstration of the white nanolight source would stimulate diverse research fields toward next-generation nanophotonic technologies.

Enhanced phosphorylation of PERK in primary cultured neurons as an autonomous neuronal response to prion infection.

Conversion of cellular prion protein (PrPC) into the pathogenic isoform of prion protein (PrPSc) in neurons is one of the key pathophysiological events in prion diseases. However, the molecular mechanism of neurodegeneration in prion diseases has yet to be fully elucidated because of a lack of suitable experimental models for analyzing neuron-autonomous responses to prion infection. In the present study, we used neuron-enriched primary cultures of cortical and thalamic mouse neurons to analyze autonomous neuronal responses to prion infection. PrPSc levels in neurons increased over the time after prion infection; however, no obvious neuronal losses or neurite alterations were observed. Interestingly, a finer analysis of individual neurons co-stained with PrPSc and phosphorylated protein kinase RNA-activated-like endoplasmic reticulum (ER) kinase (p-PERK), the early cellular response of the PERK-eukaryotic initiation factor 2 (eIF2α) pathway, demonstrated a positive correlation between the number of PrPSc granular stains and p-PERK granular stains, in cortical neurons at 21 dpi. Although the phosphorylation of PERK was enhanced in prion-infected cortical neurons, there was no sign of subsequent translational repression of synaptic protein synthesis or activations of downstream unfolded protein response (UPR) in the PERK-eIF2α pathway. These results suggest that PrPSc production in neurons induces ER stress in a neuron-autonomous manner; however, it does not fully activate UPR in prion-infected neurons. Our findings provide insights into the autonomous neuronal responses to prion propagation and the involvement of neuron-non-autonomous factor(s) in the mechanisms of neurodegeneration in prion diseases.

Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining.

We established abnormal isoform of prion protein (PrPSc)-specific double immunostaining using mAb 132, which recognizes aa 119-127 of the PrP molecule, and novel PrPSc-specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrPSc-specific double immunostaining, we analysed PrPSc in immortalized neuronal cell lines and primary cerebral-neuronal cultures infected with prions. The PrPSc-specific double immunostaining showed the existence of PrPSc positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrPSc species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrPSc staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrPSc stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrPSc stains were predominantly observed on the surface of the 22 L strain-infected primary cerebral neurons. Only 14 % of PrPSc in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrPSc in N2a-3 lack the N-terminal portion. In contrast, nearly half PrPSc detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrPSc that possesses the N-terminal portion of PrP. Further analysis of prion-infected primary neurons using PrPSc-specific immunostaining will reveal the neuron-specific mechanism for prion propagation.

Complement factors alter the amount of PrP(Sc) in primary-cultured mouse cortical neurons associated with increased membrane permeability.

We examined the effects of complement factors on primary-cultured neurons infected with prions. The amount of protease K (PK)-resistant abnormal form of prion protein (PrP(Sc)) reached a maximum level at 12 and 16 days post exposure (dpe) in 22L- and Chandler-infected neurons, respectively. In Chandler-infected neurons, the reaction of complement factors C1q, C3 and C9 significantly increased membrane permeability. This was followed by a decrease of PK-resistant PrP(Sc) at 16 and 20dpe. In contrast, in 22L-infected neurons, the effects of complement factors were observed at 12 and 16dpe, but not at 20dpe. Membrane permeability also increased in 22L-infected neurons by reaction of complement factor C3, but interestingly, the amount of PK-resistant PrP(Sc) initially decreased, and then increased. These results suggest that the reactivity of complement factors in prion-infected neurons depends on the amount of PrP(Sc) and the prion strain.

Cell observation method under near-living conditions by scanning electron microscopy.

Most cells of multicellular organisms have "primary cilia", which are single, non-motile, and sensory cilia. They have been reported to detect mechanical stimulation and transform it into internal cell, but the mechanisms are not still well known. Dermal papilla (DP) cells, which locate in the skin and regulate hair follicle development and hair cycle, were reported to have their primary cilia by immune-fluorescent method [1], but their detailed structure and function is unclear.For observation by scanning electron microscopy (SEM), biological specimens are conventionally fixed with glutaraldehyde and dehydrated in 30%, 50%, 70%, 90% and 100% ethanol. Then specimens are dried by butyl alcohol and coated with gold. It takes several days to prepare these specimens. Using many chemical reagent and many steps in this way may lead to destroy biological specimens structure. Here we attempted a recently proposed method using ionic liquid to prepare cell samples in near- living conditions observed the structure of DP cells (2D and clumps) with primary cilia.This time, we used ionic liquid for preparing specimens. First, cultured cells were fixed in glutaraldehyde, and immersed in ionic liquid. Next, the specimens were coated with gold and observed by SEM. Thus, it takes shorter time due to fewer step than conventional method and the process has no drying step. In a conventional way, we got the micrographs of 2D cultured DP cells and observed the cilium of DP cells (200-nm in diameter and 1.5um in length) on nucleus (15-um). In addition we could observe the clumps of DP cells and the cilia-like structure (∼12-um), but they do not attach to scaffoldings of the surface, probably due to drying. In observation using ionic liquid, we got the micrographs of 2D cultured DP cells and observed the cilium- like structure (200-nm in diameter and 2.1-um in length) on nucleus (30-um), as well. In this case, we could not find the cilia- like structure in the clumps of DP cells yet, but they well attached to the scaffoldings and kept the extending structure such as filopodia, too.We here observed DP cells and their cilia in near-living conditions. Unfortunately, we could not primary cilia in clumps of DP cells immersed in ionic liquid yet, but we could reduce damage receiving in the process of specimen's preparation, especially drying. In addition, we are challenging the observation using not only ionic liquid but also nano-suits by detergents [2] and the observation the cilia by SEM after identifying them by fluorescence microscopy, such as CLEM.