Bayesian reconstruction of a vancomycin-resistant Enterococcus transmission route using epidemiologic data and genomic variants from whole genome sequencing.

BACKGROUND: Outbreaks of vancomycin-resistant enterococcus (VRE) are a serious problem in hospitals. Inferring the transmission route is an important factor to institute appropriate infection control measures; however, the methodology has not been fully established. AIM: To reconstruct and evaluate the transmission model using sequence variants extracted from whole genome sequencing (WGS) data and epidemiological information from patients involved in a VRE outbreak. METHODS: During a VRE outbreak in our hospital, 23 samples were collected from patients and environmental surfaces and analysed using WGS. By combining genome alignment information with patient epidemiological data, the VRE transmission route was reconstructed using a Bayesian approach. With the transmission model, evaluation and further analyses were performed to identify risk factors that contributed to the outbreak. FINDINGS: All VREs were identified as Enterococcus faecium belonging to sequence type 17, which consisted of two VRE genotypes: vanA (N = 8, including one environmental sample) and vanB (N = 15). The reconstruction model using the Bayesian approach showed the transmission direction with posterior probability and revealed transmission through an environmental surface. In addition, some cases acting as VRE spreaders were identified, which can interfere with appropriate infection control. Vancomycin administration was identified as a significant risk factor for spreaders. CONCLUSION: A Bayesian approach for transmission route reconstruction using epidemiologic data and genomic variants from WGS can be applied in actual VRE outbreaks. This may contribute to the design and implementation of effective infection control measures.

Magnetic resonance spectroscopy of an atomically thin material using a single-spin qubit.

Two-dimensional (2D) materials offer a promising platform for exploring condensed matter phenomena and developing technological applications. However, the reduction of material dimensions to the atomic scale poses a challenge for traditional measurement and interfacing techniques that typically couple to macroscopic observables. We demonstrate a method for probing the properties of 2D materials via nanometer-scale nuclear quadrupole resonance (NQR) spectroscopy using individual atomlike impurities in diamond. Coherent manipulation of shallow nitrogen-vacancy (NV) color centers enables the probing of nanoscale ensembles down to approximately 30 nuclear spins in atomically thin hexagonal boron nitride (h-BN). The characterization of low-dimensional nanoscale materials could enable the development of new quantum hybrid systems, combining atomlike systems coherently coupled with individual atoms in 2D materials.

Superlattice-Induced Insulating States and Valley-Protected Orbits in Twisted Bilayer Graphene.

Twisted bilayer graphene (TBLG) is one of the simplest van der Waals heterostructures, yet it yields a complex electronic system with intricate interplay between moiré physics and interlayer hybridization effects. We report on electronic transport measurements of high mobility small angle TBLG devices showing clear evidence for insulating states at the superlattice band edges, with thermal activation gaps several times larger than theoretically predicted. Moreover, Shubnikov-de Haas oscillations and tight binding calculations reveal that the band structure consists of two intersecting Fermi contours whose crossing points are effectively unhybridized. We attribute this to exponentially suppressed interlayer hopping amplitudes for momentum transfers larger than the moiré wave vector.

Tunable symmetry breaking and helical edge transport in a graphene quantum spin Hall state.

Low-dimensional electronic systems have traditionally been obtained by electrostatically confining electrons, either in heterostructures or in intrinsically nanoscale materials such as single molecules, nanowires and graphene. Recently, a new method has emerged with the recognition that symmetry-protected topological (SPT) phases, which occur in systems with an energy gap to quasiparticle excitations (such as insulators or superconductors), can host robust surface states that remain gapless as long as the relevant global symmetry remains unbroken. The nature of the charge carriers in SPT surface states is intimately tied to the symmetry of the bulk, resulting in one- and two-dimensional electronic systems with novel properties. For example, time reversal symmetry endows the massless charge carriers on the surface of a three-dimensional topological insulator with helicity, fixing the orientation of their spin relative to their momentum. Weakly breaking this symmetry generates a gap on the surface, resulting in charge carriers with finite effective mass and exotic spin textures. Analogous manipulations have yet to be demonstrated in two-dimensional topological insulators, where the primary example of a SPT phase is the quantum spin Hall state. Here we demonstrate experimentally that charge-neutral monolayer graphene has a quantum spin Hall state when it is subjected to a very large magnetic field angled with respect to the graphene plane. In contrast to time-reversal-symmetric systems, this state is protected by a symmetry of planar spin rotations that emerges as electron spins in a half-filled Landau level are polarized by the large magnetic field. The properties of the resulting helical edge states can be modulated by balancing the applied field against an intrinsic antiferromagnetic instability, which tends to spontaneously break the spin-rotation symmetry. In the resulting canted antiferromagnetic state, we observe transport signatures of gapped edge states, which constitute a new kind of one-dimensional electronic system with a tunable bandgap and an associated spin texture.

Massive Dirac fermions and Hofstadter butterfly in a van der Waals heterostructure.

van der Waals heterostructures constitute a new class of artificial materials formed by stacking atomically thin planar crystals. We demonstrated band structure engineering in a van der Waals heterostructure composed of a monolayer graphene flake coupled to a rotationally aligned hexagonal boron nitride substrate. The spatially varying interlayer atomic registry results in both a local breaking of the carbon sublattice symmetry and a long-range moiré superlattice potential in the graphene. In our samples, this interplay between short- and long-wavelength effects resulted in a band structure described by isolated superlattice minibands and an unexpectedly large band gap at charge neutrality. This picture is confirmed by our observation of fractional quantum Hall states at ± 5/3 filling and features associated with the Hofstadter butterfly at ultrahigh magnetic fields.

Characterization of Toxoplasma gondii 5' UTR with encyclopedic TSS information.

The 5' UTR is widely involved in gene expression via post-transcriptional regulation. However, a detailed profile of the 5' UTR for Toxoplasma gondii has not yet been demonstrated. To investigate the issue, we compared the predicted open reading frames (ORFs) and transcription start sites (TSSs) of T. gondii obtained by TSS-seq, a method that enables analysis of encyclopedic TSSs with next-generation sequencers. As a result, it was demonstrated that the mode length of the 5' UTR is between 120 and 140 nucleotides (nts) when a subset of genes with predicted signal peptides was examined. However, when genes without the signal peptide were examined, the length was extended to approximately 600 nts. Because additional information on the predicted signal peptide generates increased reliability to the 5' end estimation of each ORF, we believe that the former value was more reliable as a representative of the 5' UTR length of T. gondii. The discrepancy suggests that current predictions of the 5' end of the ORF were less accurate and considerably more discordant with the natural status. The 5' untranslated region (5' UTR) is defined as that between the 5' end of the transcripts and just in front of a start codon of an ORF. Therefore, the 5' UTR does not contain any information for a protein sequence; however, it is involved in the control of protein expression via the modulation of translational efficiency (Kozak, 1991b; Hughes, 2006).

A case of exfoliative esophagitis with pemphigus vulgaris.

Autoimmune blistering skin diseases, including pemphigus vulgaris, rarely involve the esophagus. We report a case of exfoliative esophagitis with pemphigus vulgaris. A sloughing esophageal cast observed by endoscopy was dissected esophageal squamous epithelium in all layers. Our case is the fifth case of pemphigus vulgaris associated with esophageal cast formation recorded in the medical literature. Prednisolone was administered, and both the pemphigus vulgaris and exfoliative esophagitis improved. Upon findings of exfoliative esophagitis by endoscopic examination, we should consider the coexistence of blistering skin diseases, including pemphigus vulgaris.

Molecular characterizations of three distinct Babesia gibsoni rhoptry-associated protein-1s (RAP-1s).

Three cDNAs encoding rhoptry-associated protein 1 (RAP-1) homologues were found in the Babesia gibsoni EST database. Based on similarities to BgRAP-1a, which was identified previously by serological screening of a cDNA merozoite library, the two new genes were designated BgRAP-1b (33.7%) and BgRAP-1c (57%). Mice antiserum raised against each recombinant protein reacted specifically with B. gibsoni parasites as determined by Western blotting, which showed native molecular sizes of the BgRAP-1a (51 kDa), BgRAP-1b (53 kDa) and BgRAP-1c (47 kDa) consistent with predictable molecular weights. Immunofluoresence using these antibodies revealed localization of all BgRAP-1s within the matrix of merozoites; however, BgRAP-1a appeared to diverge from the other two when it was found secreted into the cytoplasm of infected erythrocytes. Apical localization of all 3 BgRAP-1s during the extracellular stage of the parasite combined with their ability to bind a canine erythrocyte membrane fraction was suggestive of a role for these proteins in erythrocyte attachment. Lastly, the ability of these recombinant proteins to be used as diagnostic reagents was tested by ELISA and the sensitivities of BgRAP-1a and BgRAP-1c were found increased through N-terminal truncation. Taken together, our data suggest divergent roles for the 3 BgRAP-1s in the merozoite stage of B. gibsoni.

Molecular and immunological characterization of Babesia gibsoni and Babesia microti heat shock protein-70.

Serological immunoscreening was used to identify a gene encoding heat shock protein-70 from Babesia gibsoni (BgHSP-70) that showed high homology with HSP-70s from other apicomplexan parasites. This gene corresponded to a full-length cDNA containing an open reading frame of 1968 bp predicted to result in a 70-kDa mature protein consisting of 656 amino acids. Analysis of the expression levels of BgHSP-70 indicated elevated transcription from cultured parasites incubated at 40 degrees C for 1 h, but not at 30 degrees C. Interestingly, antiserum raised against recombinant BgHSP-70 protein reacted specifically not only with a 70-kDa protein of B. gibsoni but also with a corresponding native protein of B. microti (BmHSP-70), indicating the high degree of conservation of this protein. The BmHSP-70 gene was then isolated and characterized and the immunoprotective properties of recombinant BgHSP-70 (rBgHSP-70) and rBmHSP-70 were compared in vitro and in vivo. Both proteins had potent mitogenic effects on murine and canine mononuclear cells as evidenced by high proliferative responses and IFN-gamma production after stimulation. Immunization regimes in BALB/c and C57BL/6 mice using rBgHSP-70 and rBmHSP-70 elicited high antibody levels, with concurrent significant reductions in peripheral parasitaemias. Taken together, these results emphasize the potential of HSP-70s as a molecular adjuvant vaccine.

Characterization of a leucine aminopeptidase of Babesia gibsoni.

Peptidases of parasitic protozoa are currently under intense investigation in order to identify novel virulence factors, drug targets, and vaccine candidates, except in Babesia. Leucine aminopeptidases in protozoa, such as Plasmodium and Leishmania, have been identified to be involved in free amino acid regulation. We report here the molecular and enzymatic characterization, as well as the localization of a leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from B. gibsoni (BgLAP). A functional recombinant BgLAP (rBgLAP) expressed in Escherichia coli efficiently hydrolysed synthetic substrates for aminopeptidase, a leucine substrate. Enzyme activity of the rBgLAP was found to be optimum at pH 8.0 and at 37 degrees C. The substrate profile was slightly different from its homologue in P. falciprum. The activity was also strongly dependent on metal divalent cations, and was inhibited by bestatin, which is a specific inhibitor for metalloprotease. These results indicated that BgLAP played an important role in free amino acid regulation.

Flowering response of rice to photoperiod and temperature: a QTL analysis using a phenological model.

In this study we have attempted to quantify the thermal and photoperiodical responses of rice (Oryza sativa L.) flowering time QTLs jointly by a 'date-of-planting' field experiment of a mapping population, and a 'phenological model' analysis that separately parameterizes the two responses, based on daily temperature, daily photoperiod and flowering date. For this purpose, the 'three-stage Beta model', which parameterizes the sensitivity to temperature (parameter alpha), the sensitivity to photoperiod (parameter beta), and earliness under optimal conditions (10 h photoperiod at 30 degrees C) (parameter G), was applied to 'Nipponbare' x 'Kasalath' backcross inbred lines that were transplanted on five dates. QTLs for the beta value were detected in the four known flowering time QTL (Hd1, Hd2, Hd6 and Hd8) regions, while QTLs for the G value were detected only in the Hd1 and Hd2 regions. This result was consistent with previous reports on near-isogenic lines (NILs) of Hd1, Hd2 and Hd6, where these loci were involved in photoperiod sensitivity, and where Hd1 and Hd2 conferred altered flowering under both 10 and 14 h photoperiods, while Hd6 action was only affected by the 14 h photoperiod. Hd8 was shown to control photoperiod sensitivity for the first time. Interestingly, Hd1 and Hd2 were associated with a QTL for the alpha value, which might support the previous hypothesis that the process of photoinduction depends on temperature. These results demonstrate that our approach can effectively quantify environmental responses of flowering time QTLs without controlled environments or NILs.

Bacterial population dynamics and community structure in a pharmaceutical manufacturing water supply system determined by real-time PCR and PCR-denaturing gradient gel electrophoresis.

AIMS: To control bacteria in the pharmaceutical water supply system. METHODS AND RESULTS: Bacteria were enumerated by conventional culture method and fluorescent vital staining. Activated carbon treatment and storage in a tank provided favourable environments for bacterial growth. The bacterial population of the water in both the post-activated carbon treatment and the tank was analysed by denaturing gradient gel electrophoresis (DGGE) with PCR-amplified 16S rDNA fragments including V6, -7, and -8 regions. The bacterial community structure in activated carbon treated water was stable throughout the year. Several kinds of bacteria such as genus Aquaspirillum and Methylobacterium were found in the water after activated carbon treatment. The bacterial community structure was changed and other bacteria such as mycobacteria were detected after storage. Mycobacteria were quantified in water samples using real-time PCR targeting the 16S rDNA gene. Mycobacteria were also detected in tap water and their number was increased 10(3)-10(4)-fold higher after storage. CONCLUSION: These data suggest the importance of culture-independent methods for quality control of water used in pharmaceutical manufacturing. SIGNIFICANCE AND IMPACT OF THE STUDY: Critical steps and specified bacteria that should be controlled in the water supply system were recognized by culture-independent methods. These data will enable effective control of water used in the pharmaceutical industry.

QTLs for branching, floret formation, and pre-flowering floret abortion of rice panicle in a temperate japonica x tropical japonica cross.

A large panicle with numerous florets is essential for improving rice ( Oryza sativa L.) yield. Rice panicle size is determined by such underlying morphogenetic processes as: (1) primary branch formation on the panicle axis; (2) floret formation on the primary branches (mainly determined by the secondary branch formation); and (3) pre-flowering abortion of florets in the panicle. We examined QTLs for these processes to understand how they are integrated into panicle size. We developed 106 backcross-inbred lines (BC1F4) from a cross between 'Akihikari' (a temperate japonica) and 'IRAT109' (a tropical japonica) and constructed a genetic map. One QTL detected on chromosome 2, with a large effect (R=0.30) on the number of florets per panicle, affected both primary branch formation on the panicle axis and floret formation on the primary branches. In addition, three QTLs that affect only one of these two processes were identified on chromosomes 4, 9, and 11, each having a subsidiary effect on the number of florets per panicle (R2=0.04-0.07). QTLs for pre-flowering floret abortion were detected at three different regions of the genome (chromosomes 1, 10, and 11). This is the first report on QTLs for pre-flowering floret abortion in grasses. The absence of a co-location between QTLs suggests that floret formation and abortion are not directly linked causally. These results demonstrate that studying the partitioning of panicle size into these underlying morphogenetic components would be helpful in understanding the complicated genetic control of panicle size.

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

Color/power Doppler sonographic differential diagnosis of superficial lymphadenopathy: metastasis, malignant lymphoma, and benign process.

OBJECTIVE: Differentiation of lymph nodes as metastasis, malignant lymphoma, and benign lesions by gray scale and color/power Doppler ultrasonography has been reported. In this study we sought to determine patterns of abnormal vascularity, the relationship between diagnostic criteria and node size, and which vessel in the node was best suited for Doppler spectral analysis. Extrahilar vessels, defined as vessels entering through the lymphatic surface and not through the hilus, were also investigated. METHODS: One hundred twenty-nine nodes were examined by color/power Doppler ultrasonography. RESULTS: Extrahilar vessels were seen in 85.4% of metastatic, 40.5% of lymphomatous, and 7.7% of benign nodes regardless of node size. The mean pulsatility index and resistive index values of metastatic nodes were higher than those of benign nodes regardless of their size. Those of lymphoma were intermediate compared with the others. CONCLUSIONS: A pulsatility index greater then 1.3 and a resistive index greater than 0.72 suggested malignancy as measured from any vessel.

Tomographic imaging by two-wave mixing with a wavelength-scanning laser diode.

A method of tomographic imaging is proposed in which two-wave mixing in a photorefractive crystal is used with wavelength scanning of a laser diode and phase modulation of the pump beam. This method provides full optical processing and is effective for weak light from objects because of the use of two-wave mixing. The depth resolution of the method was ~1 cm when the wavelength-scanning width was ~0.02 nm .

Genome organization and mRNA structure of Periplaneta fuliginosa densovirus imply alternative splicing involvement in viral gene expression.

We determined the complete nucleotide sequence of an infectious clone of the cockroach small spherical virus (CSSV) genome. Analysis of the genome organization and the predicted viral protein sequences showed clearly that this virus should be classified as a new member of the subfamily Densovirinae, genus Densovirus, and should be designated as PfDNV. However, our data revealed some differences between the gene expression strategies used by PfDNV and other DNVs. An internal promoter, in addition to the promoter (p3) at the genome terminus, was observed at map unit 18 (p18), implying transcriptional regulation of generation of the nonstructural proteins of PfDNV. Furthermore, the structural analysis of cDNAs complementary to mRNAs from the region coding for structural proteins suggested alternative splicing and polyadenylation as means for generation of the structural proteins of PfDNV.

Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli.

The outer membrane protein OpcP1 of Burkholderia (formerly Pseudomonas) cepacia is one of the subunits forming the porin oligomer OpcPO by non-covalent association. OpcP1 was cleaved with lysyl endopeptidase and the N-terminal amino acid (aa) sequences of the polypeptide fragments were determined. Based on the sequence information, we cloned the opcP gene on a 10-kb EcoRI DNA fragment of the B. cepacia ATCC25416 chromosome. Nucleotide (nt) sequencing revealed a 1086-bp open reading frame (ORF), encoding a 361-aa polypeptide with a signal sequence of 20 residues. The predicted opcP gene encoded a mature protein of Mr 35,696, which agrees well with the value observed previously on SDS-PAGE. The opcP was sub-cloned into pTrc99A and introduced into Escherichia coli. Immunoblot analysis using murine antiserum specific to OpcP1 visualized the protein expressed in the E. coli cells after induction by isopropyl beta-D-thiogalactopyranoside (IPTG).

Detection of Fc receptor genes from Staphylococcus aureus and streptococci by polymerase chain reaction.

A method based upon the polymerase chain reaction (PCR) for detecting genes encoding the Fc receptors of Staphylococcus aureus and streptococci is described. Primers were designed from the nucleotide sequences of the five Fc receptor genes encoding protein A, protein G, protein H, FcRA and protein V. Amplification products corresponding in size to the protein A and protein G genes were detected in S. aureus strain Cowan 1 and Streptococcus pyogenes strain G148, respectively, as expected. Str. pyogenes strain AR1 was shown to possess the type H receptor gene. Two clinical isolates of Str. pyogenes, strains IP-28 and ES-21L, were shown to possess genes for Fc receptor types FcRA and protein G, respectively. The identification of all these products was confirmed by restriction endonuclease analysis. Amplification of protein H genes from two other clinical isolates of streptococci, MS-4 and MS-38, yielded a product larger than expected and with a different restriction fragment pattern to strain AR1, indicating a new type of Fc receptor gene. This PCR method provides a DNA-based method for the determination of Fc receptor type in S. aureus and streptococci.