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Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define metabolic differences between steady-state and stress conditions in HSCs and elucidate their regulatory mechanisms. Through quantitative13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of adenosine triphosphate (ATP) levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression ofPfkfb3induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss ofPfkfb3suppressed them. This study reveals the flexible and multilayered regulation of HSC metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.Key PointsCombined isotope tracing, mathematical modeling, and single cell ATP analysis enable high-resolution evaluation of blood cell metabolism.Under stress, HSCs quickly accelerate glycolysis to meet ATP demands and maintain hematopoiesis via context-dependent PFKFB3 activation.

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX–CBFβ-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system. The transcription factor IRF8 is required for both DC and monocyte differentiation from common myeloid progenitors. Tamura and colleagues identify an enhancer (+56 kb) in the Irf8 locus that regulates early myeloid lineage choice.

Dissemination of extended-spectrum cephalosporin (ESC)-resistant Salmonella is a public health concern in the egg production industry. ESC-resistant Salmonella often acquires the bla gene via insertion sequences (ISs). Therefore, this study aimed to assess antimicrobial resistance in Salmonella from Japanese layer breeding chains and egg processing chains, and determine the genetic profiles of IS-like elements in ESC-resistant Salmonella. Antimicrobial susceptibility testing was performed on 224 isolates from 49 facilities involving layer breeder farms, hatcheries, pullet-rearing farms, and layer farms in breeding chains along with egg processing chains. ESC-resistant Salmonella strains were whole-genome sequenced. Among them, 40 (17.9%) were resistant to at least streptomycin, tetracycline, ampicillin, chloramphenicol, cefpodoxime, nalidixic acid, ciprofloxacin, and/or kanamycin despite lacking resistance to azithromycin and meropenem. Moreover, 15 were ESC-resistant Salmonella harboring blaCMY-2 (Salmonella enterica serovar Ohio, n=12; S. Braenderup, n=1; untypeable with O7:b:-, n=1) and blaCTX-M-14 (S. Cerro, n=1). IncA/C2 plasmids containing ISEcp1, IS26, and multiple antimicrobial resistance genes (including blaCMY-2) were identified in S. Ohio isolates from pullet-rearing and layer farms belonging to the same company. Chromosomal integration of partial or whole IncA/C2 plasmids was seen with two S. Ohio isolates via ISEcp1 or IS26, respectively. Antimicrobial resistance genes such as blaCMY-2 might be transmitted among the upper and the lower levels of layer breeding chains via the replicon type IncA/C2 plasmids containing ISEcp1 and IS26.

DNA gyrase plays important roles in genome replication in various bacteria, including

Cancer stem cells are a sub-population of cancer cells with self-renewal activity that play key roles in tumor resistance to chemotherapy and radiation. Several cancer stem cell markers have been identified to correlate with clinical prognosis. However, which marker is associated with which cancer stem cell characteristic is unclear. The present study aimed to clarify the relationship between cancer stem cell markers associated with drug resistance acquisition and the characteristics of cancer stem cells. We generated cisplatin-resistant head and neck squamous cell carcinoma cells by culturing cells in increasing concentrations of cisplatin. The cisplatin-resistant head and neck squamous cell carcinoma cells also acquired multidrug resistance and were named resistant HSC-3 (R HSC-3) cells. R HSC-3 showed no differences in cell proliferation or cell cycle distributions compared with parental cells. R HSC-3 cells showed increased drug excretion ability and elevated expression of ATP-binding cassette subfamily G member 2 (ABCG2), a drug excretion pump. R HSC-3 cells also highly expressed CD44, a cancer stem cell marker, and exhibited enhanced cell invasion and spheroid formation abilities. Furthermore, the stem cell-related factor SRY-box transcription factor 9 (SOX9) was identified as increased in R HSC-3 cells by microarray analysis. Knockdown experiments showed that SOX9 and ABCG2 were involved in the drug excretion ability of R HSC3 cells and ABCG2 was involved in the spheroid formation ability of R HSC-3 cells. These results indicate that CD44, SOX9 and ABCG2 expression levels were enhanced in head and neck squamous cell carcinoma cells that acquired multidrug resistance and that these molecules are important for maintaining cancer stem cell characteristics. Overall, regulating CD44, SOX9 and ABCG2 may be a strategy to inhibit cancer stem cells.

Detection and control of flow separation is a key to improving the efficiency of fluid machinery. In this study, we developed a flexible MEMS (microelectromechanical systems) sensor for measuring the wall shear stress and flow angle in subsonic airflow. The developed sensor is made of a flexible polyimide film and a microheater surrounded by three temperature sensor pairs. The sensor measures the wall shear stress from the heater output and the flow angle from the temperature gradient around the heater. The geometry and design of the heater and temperature sensors were determined based on numerical simulations. To evaluate the validity of the sensor, we conducted an experiment to measure the wall shear stress and the flow angle in a wind tunnel in different velocities ranging from 30 m/s to 170 m/s, equivalent to Mach numbers from 0.1 to 0.5. The heater output was proportional to one-third power of the wall shear stress. Additionally, the bridge output correlating the temperature difference between two opposing temperature sensors showed sinusoidal variation depending on the flow angle. Consequently, we have clarified that the developed sensor can measure both the wall shear stress and flow direction in subsonic flow.

Zoonotic pathogen Escherichia albertii has been identified as the cause of several human disease outbreaks; however, factors such as the general symptoms and incubation period of E. albertii infect...

Our understanding of how each hereditary kidney cancer adapts to its tissue microenvironment is incomplete. Here, we present single-cell transcriptomes of 108,342 cells from patient specimens including from six hereditary kidney cancers. The transcriptomes displayed distinct characteristics of the cell of origin and unique tissue microenvironment for each hereditary kidney cancer. Of note, hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated kidney cancer retained some characteristics of proximal tubules, which were completely lost in lymph node metastases and present as an avascular tumor with suppressed T cells and TREM2-high macrophages, leading to immune tolerance. Birt-Hogg-Dubé (BHD)-associated kidney cancer exhibited transcriptomic intratumor heterogeneity (tITH) with increased characteristics of intercalated cells of the collecting duct and upregulation of FOXI1-driven genes, a critical transcription factor for collecting duct differentiation. These findings facilitate our understanding of how hereditary kidney cancers adapt to their tissue microenvironment.

The objective of this study is to investigate computed DWI (cDWI) as an alternative method to contrast-enhanced MRI in comparison with directory measured DWI (mDWI) and apparent diffusion coefficient (ADC) for differentiating synovial proliferation from joint effusion. Nine patients suspected with RA (5 women) were included in this study. A radiologist identified region of interest (ROI) based on STIR, and evaluated using a 5-point grading scale of 0 (fluid) to 4 (synovial proliferation) according to the degree of contrast enhancement within the ROI. cDWI was synthesized for b values from 1000 to 2000 at 200 s/mm(2) intervals using the combination of b values at mDWI. In addition to ADC values, contrast ratios were calculated using signal intensity for each ROI on the mDWI and cDWI. Visual assessment by a radiologist was conducted between pairs of STIR image and mDWI or cDWI. ROI grades were most significantly correlated with cDWI(2000) based on b values of 400-1000 s/mm(2) (r(s) = 0.405, p < 0.01). The area under the curve of cDWI(2000) based on b values of 400-1000 s/mm(2) (0.762) was larger than that of ADC values (0.570-0.608) when comparing low versus high contrast enhancement grades. Both cDWI(1800) (200-1000) and cDWI(2000) (400-1000) demonstrated high sensitivity and specificity in visual assessment (84.6% and 66.7%, respectively). The cDWI(2000) based on b values of 400-1000 s/mm(2) may be useful for noninvasive differentiation of synovial proliferation from joint effusion in hand arthritis.

DNA gyrase plays important roles in genome replication in various bacteria, including Pseudomonasaeruginosa. The gyrA gene encodes the gyrase subunit A protein (GyrA). Mutations in GyrA are associated with resistance to quinolone-based antibiotics. We performed a detailed molecular evolutionary analyses of the gyrA gene and associated resistance to the quinolone drug, ciprofloxacin, using bioinformatics techniques. We produced an evolutionary phylogenetic tree using the Bayesian Markov Chain Monte Carlo (MCMC) method. This tree indicated that a common ancestor of the gene was present over 760 years ago, and the offspring formed multiple clusters. Quinolone drug-resistance-associated amino-acid substitutions in GyrA, including T83I and D87N, emerged after the drug was used clinically. These substitutions appeared to be positive selection sites. The molecular affinity between ciprofloxacin and the GyrA protein containing T83I and/or D87N decreased significantly compared to that between the drug and GyrA protein, with no substitutions. The rate of evolution of the gene before quinolone drugs were first used in the clinic, in 1962, was significantly lower than that after the drug was used. These results suggest that the gyrA gene evolved to permit the bacterium to overcome quinolone treatment.

Posttranslational protein modification through addition of the O‐linked β-N-acetyl-D-glucosamine (O-GlcNAc) moiety to serine or threonine residues, termed O-GlcNAcylation, is a highly dynamic process conserved throughout eukaryotes. O-GlcNAcylation is reversibly catalyzed by a single pair of enzymes, O-GlcNAc transferase and O-GlcNAcase, and it acts as a fundamental regulator for a wide variety of biological processes including gene expression, cell cycle regulation, metabolism, stress response, cellular signaling, epigenetics, and proteostasis. O-GlcNAcylation is regulated by various intracellular or extracellular cues such as metabolic status, nutrient availability, and stress. Studies over decades have unveiled the profound biological significance of this unique protein modification in normal physiology and pathologic processes of diverse cell types or tissues. In hematopoiesis, recent studies have indicated the essential and pleiotropic roles of O-GlcNAcylation in differentiation, proliferation, and function of hematopoietic cells including T cells, B cells, myeloid progenitors, and hematopoietic stem and progenitor cells. Moreover, aberrant O-GlcNAcylation is implicated in the development of hematologic malignancies with dysregulated epigenetics, metabolism, and gene transcription. Thus, it is now recognized that O-GlcNAcylation is one of the key regulators of normal and malignant hematopoiesis.

Chronic myeloid leukemia (CML) is a form of myeloproliferative neoplasm caused by the oncogenic tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitors have dramatically improved the prognosis of patients with CML, several problems such as resistance and recurrence still exist. Immunological control may contribute to solving these problems, and it is important to understand why CML patients fail to spontaneously develop anti-tumor immunity. Here, we show that differentiation of conventional dendritic cells (cDCs), which are vital for anti-tumor immunity, is restricted from an early stage of hematopoiesis in CML. In addition, we found that monocytes and basophils, which are increased in CML patients, express high levels of PD-L1, an immune checkpoint molecule that inhibits T cell responses. Moreover, RNA-sequencing analysis revealed that basophils express genes related to poor prognosis in CML. Our data suggest that BCR-ABL not only disrupts the “accelerator” (i.e., cDCs) but also applies the “brake” (i.e., monocytes and basophils) of anti-tumor immunity, compromising the defense against CML cells.

We developed high temperature in situ transmission electron microscopy using a high-density laser irradiation device (nominal maximum laser density ~9.4 GW/m

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis and frequent progression to leukemia. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually dominate the bone marrow space. Despite several studies implicating mesenchymal stromal or stem cells (MSCs), a principal component of the HSC niche, in the inhibition of normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. Here, we demonstrate that both human and mouse MDS cells perturb bone metabolism by suppressing the osteolineage differentiation of MSCs, which impairs the ability of MSCs to support normal HSCs. Enforced MSC differentiation rescues the suppressed normal hematopoiesis in both in vivo and in vitro MDS models. Intriguingly, the suppression effect is reversible and mediated by extracellular vesicles (EVs) derived from MDS cells. These findings shed light on the novel MDS EV-MSC axis in ineffective hematopoiesis.

Dissemination of extended-spectrum-cephalosporin (ESC)-resistant Salmonella, especially extended-spectrum-β-lactamase (ESBL)-producing Salmonella, is a concern worldwide. Here, we assessed Salmonella carriage by food workers in Japan to clarify the prevalence of ESC-resistant Salmonella harboring blaCTX-M. We then characterized the genetic features, such as transposable elements, of blaCTX-M-harboring plasmids using whole-genome sequencing. A total of 145,220 stool samples were collected from food workers, including cooks and servers from several restaurants, as well as food factory workers, from January to October 2017. Isolated salmonellae were subjected to antimicrobial susceptibility testing (disk diffusion method), and whole-genome sequencing was performed for Salmonella strains harboring blaCTX-M. Overall, 164 Salmonella isolates (0.113%) were recovered from 164 samples, from which we estimated that at least 0.113% (95% confidence interval [CI]: 0.096 to 0.132%) of food workers may carry Salmonella. Based on this estimation, 3,473 (95% CI = 2,962 to 4,047) individuals among the 3,075,330 Japanese food workers are likely to carry Salmonella. Of the 158 culturable isolates, seven showed resistance to ESCs: three isolates harbored blaCMY-2 and produced AmpC β-lactamase, while four ESBL-producing isolates harbored blaCTX-M-14 (n = 1, Salmonella enterica serovar Senftenberg) or blaCTX-M-15 (n = 3, S. enterica serovar Haardt). blaCTX-M-15 was chromosomally located in the S. Haardt isolates, which also contained ISEcp1, while the S. Senftenberg isolate contained an IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid plasmid carrying blaCTX-M-14 along with ISEcp1. This study indicates that food workers may be a reservoir of ESBL-producing Salmonella and associated genes. Thus, these workers may contribute to the spread of blaCTX-M via plasmids or mobile genetic elements such as ISEcp1. IMPORTANCE Antimicrobial-resistant Salmonella bacteria arise in farm environments through imprudent use of antimicrobials. Subsequently, these antimicrobial-resistant strains, such as extended-spectrum-β-lactamase (ESBL)-producing Salmonella, may be transmitted to humans via food animal-derived products. Here, we examined Salmonella carriage among food handlers in Japan. Overall, 164 of 145,220 fecal samples (0.113%) were positive for Salmonella. Among the 158 tested isolates, four were identified as ESBL-producing isolates carrying ESBL determinants blaCTX-M-15 or blaCTX-M-14. In all cases, the genes coexisted with ISEcp1, regardless of whether they were located on the chromosome or on a plasmid. Our findings suggest that food workers may be a reservoir of ESBL-producing strains and could contribute to the spread of resistance genes from farm-derived Salmonella to other bacterial species present in the human gut.

Several visual scoring methods are currently used to assess progression of rheumatoid arthritis (RA) on radiography. However, they are limited by its subjectivity and insufficient sensitivity. We have developed an original measurement system which uses a technique called phase-only correlation (POC). The purpose of this study is to validate the system by using a phantom simulating the joint of RA patients. A micrometer measurement apparatus that can adjust arbitrary joint space width (JSW) in a phantom joint was developed to define true JSW. The phantom was scanned with radiography, 320 multi detector CT (MDCT), high-resolution peripheral quantitative CT (HR-pQCT), cone beam CT (CBCT), and tomosynthesis. The width was adjusted to the average size of a women’s metacarpophalangeal joint, from 1.2 to 2.2 mm with increments of 0.1 mm and 0.01 mm. Radiographical images were analyzed by the POC-based system and manual method, and images from various tomographical modalities were measured via the automatic margin detection method. Correlation coefficients between true JSW difference and measured JSW difference were all strong at 0.1 mm intervals with radiography (POC-based system and manual method), CBCT, 320MDCT, HR-pQCT, and tomosynthesis. At 0.01 mm intervals, radiography (POC-based system), 320MDCT, and HR-pQCT had strong correlations, while radiography (manual method) and CBCT had low correlations, and tomosynthesis had no statistically significant correlation. The smallest detectable changes for radiography (POC-based system), radiography (manual method), 320MDCT, HR-pQCT, CBCT, and tomosynthesis were 0.020 mm, 0.041 mm, 0.076 mm, 0.077 mm, 0.057 mm, and 0.087 mm, respectively. We conclude that radiography analyzed with the POC-based system might sensitively detect minute joint space changes of the finger joint.

Background Detection of Clostridium perfringens enterotoxin (CPE) is critical for disease surveillance; however, commercial testing kits produce contrasting results. Methods We examined the cause of the differing results from a reversed passive latex agglutination (RPLA) assay (PET-RPLA Toxin Detection Kit) and an enzyme-linked immunosorbent assay (C. perfringens Enterotoxin ELISA Kit) using 73 human norovirus-positive fecal samples from gastroenteritis patients across 22 episodes in Japan. Results CPE was detected in 39/73 samples using the RPLA method; however, ELISA-based examination of 10 RPLA-positive samples produced negative results. Moreover, cpe was not detected in any of the RPLA-positive (n = 32) or -negative (n = 5) samples, and C. perfringens was only isolated from one RPLA-positive sample. Conclusions An ELISA-based testing approach may be more reliable than RPLA assays for CPE detection from human fecal samples. These findings may also be applicable to the detection of other foodborne diseases.

Hospital-acquired infections caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are a global problem. Healthy people can carry ESBL-producing E. coli in the intestines; thus, E. coli from healthy people can potentially cause hospital-acquired infections. Therefore, the transmission routes of ESBL-producing E. coli from healthy persons should be determined. A foodborne outbreak of human norovirus (HuNoV) GII occurred at a restaurant in Shizuoka, Japan, in 2018. E. coli O25:H4 was isolated from some of the HuNoV-infected customers. Pulsed-field gel electrophoresis showed that these E. coli O25:H4 strains originated from one clone. Because the only epidemiological link among the customers was eating food from this restaurant, the customers were concurrently infected with E. coli O25:H4 and HuNoV GII via the restaurant food. Whole genome analysis revealed that the E. coli O25:H4 strains possessed genes for regulating intracellular iron and expressing the flagellum and flagella. Extraintestinal pathogenic E. coli often express these genes on the chromosome. Additionally, the E. coli O25:H4 strains had plasmids harboring nine antimicrobial resistance genes. These strains harbored ESBL-encoding blaCTX-M-14 genes on two loci of the chromosome and had higher ESBL activity. Multilocus sequence typing and fimH subtyping revealed that the E. coli O25:H4 strains from the outbreak belonged to the subclonal group, ST131-fimH30R, which has been driving ESBL epidemics in Japan. Because the E. coli O25:H4 strains isolated in the outbreak belonged to a subclonal group spreading in Japan, foods contaminated with ESBL-producing E. coli might contribute to spreading these strains among healthy persons. The isolated E. coli O25:H4 strains produced ESBL and contained plasmids with multiple antimicrobial resistance genes, which may make it difficult to select antimicrobials for treating extraintestinal infections caused by these strains. HIGHLIGHTS

Dissemination of extended-spectrum-cephalosporin (ESC)-resistant Salmonella, especially extended-spectrum-β-lactamase (ESBL)-producing Salmonella, is a concern worldwide. Here, we assessed Salmonella carriage by food workers in Japan to clarify the prevalence of ESC-resistant Salmonella harboring bla(CTX-M). We then characterized the genetic features, such as transposable elements, of bla(CTX-M)-harboring plasmids using whole-genome sequencing. A total of 145,220 stool samples were collected from food workers, including cooks and servers from several restaurants, as well as food factory workers, from January to October 2017. Isolated salmonellae were subjected to antimicrobial susceptibility testing (disk diffusion method), and whole-genome sequencing was performed for Salmonella strains harboring bla(CTX-M). Overall, 164 Salmonella isolates (0.113%) were recovered from 164 samples, from which we estimated that at least 0.113% (95% confidence interval [CI]: 0.096 to 0.132%) of food workers may carry Salmonella. Based on this estimation, 3,473 (95% CI = 2,962 to 4,047) individuals among the 3,075,330 Japanese food workers are likely to carry Salmonella. Of the 158 culturable isolates, seven showed resistance to ESCs: three isolates harbored bla(CMY-2) and produced AmpC β-lactamase, while four ESBL-producing isolates harbored bla(CTX-M-14) (n = 1, Salmonella enterica serovar Senftenberg) or bla(CTX-M-15) (n = 3, S. enterica serovar Haardt). bla(CTX-M-15) was chromosomally located in the S. Haardt isolates, which also contained ISEcp1, while the S. Senftenberg isolate contained an IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid plasmid carrying bla(CTX-M-14) along with ISEcp1. This study indicates that food workers may be a reservoir of ESBL-producing Salmonella and associated genes. Thus, these workers may contribute to the spread of bla(CTX-M) via plasmids or mobile genetic elements such as ISEcp1. IMPORTANCE Antimicrobial-resistant Salmonella bacteria arise in farm environments through imprudent use of antimicrobials. Subsequently, these antimicrobial-resistant strains, such as extended-spectrum-β-lactamase (ESBL)-producing Salmonella, may be transmitted to humans via food animal-derived products. Here, we examined Salmonella carriage among food handlers in Japan. Overall, 164 of 145,220 fecal samples (0.113%) were positive for Salmonella. Among the 158 tested isolates, four were identified as ESBL-producing isolates carrying ESBL determinants bla(CTX-M-15) or bla(CTX-M-14). In all cases, the genes coexisted with ISEcp1, regardless of whether they were located on the chromosome or on a plasmid. Our findings suggest that food workers may be a reservoir of ESBL-producing strains and could contribute to the spread of resistance genes from farm-derived Salmonella to other bacterial species present in the human gut.

We performed detailed genetic analyses of the partial hemagglutinin–neuraminidase (HN) gene in 34 human respirovirus 3 (HRV3) strains from children with acute respiratory illness during 2013–2015 in Iwate Prefecture, Japan. In addition, we performed analyses of the evolutionary timescale of the gene using the Bayesian Markov chain Monte Carlo (MCMC) method. Furthermore, we analyzed pairwise distances and performed selective pressure analyses followed by linear B-cell epitope mapping and N-glycosylation and phylodynamic analyses. A phylogenetic tree showed that the strains diversified at around 1939, and the rate of molecular evolution was 7.6 × 10−4 substitutions/site/year. Although the pairwise distances were relatively short (0.03 ± 0.018 [mean ± standard deviation, SD]), two positive selection sites (Cys544Trp and Leu555Ser) and no amino acid substitutions were found in the active/catalytic sites. Six epitopes were estimated in this study, and three mouse monoclonal antibody binding sites (amino acid positions 278, 281, and 461) overlapped with two epitopes belonging to subcluster C3 strains. Bayesian skyline plot analyses indicated that subcluster C3 strains have been increasing from 2004, whereas subcluster C1 strains have declined from 2004. Based on these results, Iwate strains were divided into two subclusters and each subcluster evolved independently. Moreover, our results suggested that some predicted linear epitopes (epitopes 3 and 5) are candidates for an HRV3 vaccine motif. To better understand the details of the molecular evolution of HRV, further studies are needed.

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes deregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old) and aged (20-month-old) mice to gain insight into the dynamics of HSC aging. scRNA-seq analysis revealed three major clusters (A, B, C) and three minor clusters (D, E, F) in young HSCs. Of interest, aged HSCs also showed similar cluster formation. One of the minor clusters was characterized by gene signature associated with inflammatory response and significantly increased with age, while the remaining two minor clusters, which showed cell cycle gene signature, did not change in proportion. One of the major clusters, Cluster C, which showed the strongest expression of HSC-specific gene sets compared with other clusters, moderately increased with age. Our scRNA-seq analysis confirmed upregulation of age-related genes previously reported, such as Selp, Mt1, and Vwf, in a considerable portion of aged HSCs. von Willebrand factor (Vwf) encodes a blood glycoprotein produced by megakaryocytes and endothelial cells. Several groups have reported that Vwf-expressing HSCs show myeloid/platelet-biased differentiation (Joana C et al., Nature 2013; Sandra P et al., Developmental Cell 2018). Importantly, our scRNA-seq data identified that Clusterin (Clu) is rarely expressed in young HSCs and is dramatically upregulated in aged HSCs. Clu expression was predominantly increased in one of the major clusters, Cluster C. Clusterin encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis. We hypothesized that Clu would be useful as a marker of a unique subpopulation of aged HSCs, and thus conducted further analysis by using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory sequences of the BAC gene. Clu/GFP was preferentially expressed in HSCs and at lower frequencies in MPP1 than HSCs in Lineage -Sca-1 +c-Kit + (LSK) cell fraction. Clu-positive HSCs expressed high levels of CD150 and were detected in 10% and 60% of HSCs in 10-week-old young mice and 8-month-old middle-aged mice, respectively, indicating that Clu-positive HSCs increase with aging. We next assessed the function of Clu-positive HSCs in young mice. Clu-positive young HSCs established significantly lower chimerism than Clu-negative young HSCs and preferentially differentiated into myeloid cells in competitive transplantation assays. RNA-seq analysis of Clu-positive and Clu-negative HSCs from young mice confirmed that Clu-positive HSCs show the gene signature of myeloid-biased HSCs. Characterization of Clu-positive and negative subpopulations in aged HSCs is currently underway. These results suggest that Clu-positive HSCs represent myeloid-biased HSCs which expand with aging, thus Clu expression serves as a novel marker to monitor the alterations in HSC heterogeneity with aging. Disclosures Iwama: Nissan Chemical Corporation: Research Funding.

Escherichia albertii is a recently recognized human enteropathogen that is closely related to Escherichia coli . In many Gram-negative bacteria, including E. coli , O-antigen variation has long been used for the serotyping of strains. In E. albertii , while eight O-serotypes unique to this species have been identified, some strains have been shown to exhibit genetic or serological similarity to known E. coli / Shigella O-serotypes. However, the diversity of O-serotypes and O-antigen biosynthesis gene clusters (O-AGCs) of E. albertii remains to be systematically investigated. Here, we analysed the O-AGCs of 65 E. albertii strains and identified 40 E. albertii O-genotypes (EAOgs) (named EAOg1–EAOg40). Analyses of the 40 EAOgs revealed that as many as 20 EAOgs exhibited significant genetic and serological similarity to the O-AGCs of known E. coli / Shigella O-serotypes, and provided evidence for the inter-species horizontal gene transfer of O-AGCs between E. albertii and E. coli . Based on the sequence variation in the wzx gene among the 40 EAOgs, we developed a multiplex PCR-based O-genotyping system for E. albertii (EAO-genotyping PCR) and verified its usefulness by genotyping 278 E. albertii strains from various sources. Although 225 (80.9 %) of the 278 strains could be genotyped, 51 were not assigned to any of the 40 EAOgs, indicating that further analyses are required to better understand the diversity of O-AGCs in E. albertii and improve the EAO-genotyping PCR method. A phylogenetic view of E. albertii strains sequenced so far is also presented with the distribution of the 40 EAOgs, which provided multiple examples for the intra-species horizontal transfer of O-AGCs in E. albertii .

Escherichia albertii is a recently recognized human enteropathogen that is closely related to Escherichia coli . In many Gram-negative bacteria, including E. coli , O-antigen variation has long been used for the serotyping of strains. In E. albertii , while eight O-serotypes unique to this species have been identified, some strains have been shown to exhibit genetic or serological similarity to known E. coli / Shigella O-serotypes. However, the diversity of O-serotypes and O-antigen biosynthesis gene clusters (O-AGCs) of E. albertii remains to be systematically investigated. Here, we analysed the O-AGCs of 65 E. albertii strains and identified 40 E. albertii O-genotypes (EAOgs) (named EAOg1–EAOg40). Analyses of the 40 EAOgs revealed that as many as 20 EAOgs exhibited significant genetic and serological similarity to the O-AGCs of known E. coli / Shigella O-serotypes, and provided evidence for the inter-species horizontal gene transfer of O-AGCs between E. albertii and E. coli . Based on the sequence variation in the wzx gene among the 40 EAOgs, we developed a multiplex PCR-based O-genotyping system for E. albertii (EAO-genotyping PCR) and verified its usefulness by genotyping 278 E. albertii strains from various sources. Although 225 (80.9 %) of the 278 strains could be genotyped, 51 were not assigned to any of the 40 EAOgs, indicating that further analyses are required to better understand the diversity of O-AGCs in E. albertii and improve the EAO-genotyping PCR method. A phylogenetic view of E. albertii strains sequenced so far is also presented with the distribution of the 40 EAOgs, which provided multiple examples for the intra-species horizontal transfer of O-AGCs in E. albertii .

Zoonotic pathogen

Escherichia albertii, a zoonotic enteropathogen, is responsible for outbreaks of disease in humans. Identifying strains of E. albertii by phenotypic characterization tests is difficult because of its poorly defined properties. Screening its phenotypic characteristics is, nevertheless, a necessary prerequisite for further genetic analysis of its properties, and species-specific polymerase chain reaction (PCR) analysis can be used to type the pathogen. While two E. albertii biogroups (1 and 2) have been described, strains with characteristics divergent from both biogroups have been reported worldwide. The aim of the present study was to evaluate the characteristics of non-biogroup 1 or 2 strains, and discern the characteristics common to all of the E. albertii strains from this study. Altogether, 107/414 field isolates were selected for examination based on pulsed-field gel electrophoresis analysis. The 107 strains were isolated from 92 sources, including humans and pigeon feces, other wild birds, and retail chicken livers. All strains were then examined using various culture-based, biochemical (API 50CHE tests, API Zym test, and others) and molecular (virulence gene screening, multi-locus sequence analysis) testing methods. Our results revealed that all field strains (n = 107) showed non-biogroup 1 or 2 characteristics, with multiple sequence differences. Variations in indole production and the lysine decarboxylase activity profiles among the isolates made identification of E. albertii very difficult. Therefore, we propose that non-biogroup 1 or 2 of E. albertii should be assigned to biogroup 3 to make screening of them easier in public health and clinical laboratory settings. Clearly, having group criteria for indole-negative/lysine-positive, indole-positive/lysine-negative, and indole-positive/lysine-positive E. albertii biogroups 1, 2, and 3 strains, respectively, should provide for more accurate identification of E. albertii isolates. Based on our findings, we recommend that isolates displaying phenotype mobility-negativity (sulfide-indole-motility medium, 37°C), hydrogen sulfide production-negativity (triple sugar iron medium), acid production-negativity from xylose, negative β-glucuronidase activity properties, and showing indole production and lysine decarboxylase activity profiles in accordance with one of the three biogroups, should be further assessed using an E. albertii-specific PCR assay.

After allogeneic hematopoietic stem cell transplantation (HSCT), human herpesvirus-6 (HHV-6) can cause serious central nervous system (CNS) disorder and typically presents as encephalitis. Another manifestation of HHV-6 is myelitis, which has not been fully evaluated. In this study, we retrospectively analyzed 19 patients who developed HHV-6 myelitis after allogeneic HSCT. Median onset was 20 days after transplantation (range, 13-31), with a cumulative incidence of 4.1% at day 40 after transplantation. Median age at transplant was 50 years (range, 17-61). Median copy number of HHV-6 DNA was 3000 copies/ml in cerebrospinal fluid (CSF; range, 200-100,000). The most common symptoms were pruritus, pain of the extremities/back, and numbness. Three patients subsequently developed encephalitis in the clinical course of myelitis; their HHV-6 copy numbers in CSF had been higher than 10,000 copies/ml at the onset of myelitis. Antiviral agents were initiated shortly after onset in all patients, resulting in recovery. These results suggest that myelitis would be an important subtype of HHV-6-associated CNS disorders after allogeneic HSCT, whose prognosis could be favorable by an early intervention. Transplant physicians should recognize early posttransplant neurological symptoms such as pruritus, pain, or numbness as possible signs of HHV-6 myelitis, which could also progress to encephalitis.

Escherichia albertii, a zoonotic enteropathogen, is responsible for outbreaks of disease in humans. Identifying strains of E. albertii by phenotypic characterization tests is difficult because of its poorly defined properties. Screening its phenotypic characteristics is, nevertheless, a necessary prerequisite for further genetic analysis of its properties, and species-specific polymerase chain reaction (PCR) analysis can be used to type the pathogen. While two E. albertii biogroups (1 and 2) have been described, strains with characteristics divergent from both biogroups have been reported worldwide. The aim of the present study was to evaluate the characteristics of non-biogroup 1 or 2 strains, and discern the characteristics common to all of the E. albertii strains from this study. Altogether, 107/414 field isolates were selected for examination based on pulsed-field gel electrophoresis analysis. The 107 strains were isolated from 92 sources, including humans and pigeon feces, other wild birds, and retail chicken livers. All strains were then examined using various culture-based, biochemical (API 50CHE tests, API Zym test, and others) and molecular (virulence gene screening, multi-locus sequence analysis) testing methods. Our results revealed that all field strains (n = 107) showed non-biogroup 1 or 2 characteristics, with multiple sequence differences. Variations in indole production and the lysine decarboxylase activity profiles among the isolates made identification of E. albertii very difficult. Therefore, we propose that non-biogroup 1 or 2 of E. albertii should be assigned to biogroup 3 to make screening of them easier in public health and clinical laboratory settings. Clearly, having group criteria for indole-negative/lysine-positive, indole-positive/lysine-negative, and indole-positive/lysine-positive E. albertii biogroups 1, 2, and 3 strains, respectively, should provide for more accurate identification of E. albertii isolates. Based on our findings, we recommend that isolates displaying phenotype mobility-negativity (sulfide-indole-motility medium, 37°C), hydrogen sulfide production-negativity (triple sugar iron medium), acid production-negativity from xylose, negative β-glucuronidase activity properties, and showing indole production and lysine decarboxylase activity profiles in accordance with one of the three biogroups, should be further assessed using an E. albertii-specific PCR assay.

[Aim] To determine genotypes of Mycobacterium tuberculosis strains in Fukuoka Prefecture, Japan. [Methods] A total of 296 isolates from 296 tuberculosis patients is tested using 24-locus variable-number tandem- repeat (VNTR) typing. We also determined whether these isolates and a further 10 were Beijing lineage. [Results] The 296 isolates were classified into 264 VNTR types, and re-classified into 25 clusters when each cluster was defined as isolates being identical to VNTR types in 24 regions, or in 23 regions with the exception of one hypervariable region. Two clusters were shown to be identical to that of the Kansai regional epidemic. Regarding regional diversity, hypervariable regions showed relatively higher variation of isolate types. The Beijing lineage accounted for 78.1% of all isolates, which was similar to the value obtained from Kobe (78.5% in 2009) in the Kansai region. [Discussion] Six isolates from Fukuoka Prefecture over- lapped with those from Kansai region with respect to domi- nant VNTR type, while clusters from Fukuoka Prefectural isolates were unique, which may be a feature of Fukuoka prefectural isolates. [Conclusion] These data are likely to be useful for public health measures in the area.

Summary O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.

Acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) with both inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and monosomy 7 defines an extremely aggressive myeloid cancer whose molecular pathogenesis and optimal therapeutic strategy still remain unclear. We established a new MDS/AML cell line, YCU-AML1, and its patient-derived xenograft (PDX) model from a high-risk MDS patient who later transformed into AML harboring both t(3;3)(q21;q26.2) and monosomy 7. YCU-AML1 cells propagated in co-culture system with stromal cells in granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent manner. CD34+ bone marrow cells derived from our PDX model showed high EVI1 and low GATA2 expression. Moreover, mutational profile of our MDS/AML model was consistent with recently published mutational spectrum of myeloid malignancies with inv(3)/t(3;3). These data suggest that YCU-AML1 cells and its MDS/AML model strongly mimics a high-risk human myeloid cancer with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and monosomy 7 in terms of both clinical phenotype and molecular basis. We believe our model can be used as a feasible tool to further explore molecular pathogenesis and novel treatment strategy of high-risk MDS/AML with t(3;3)(q21;q26.2) and monosomy 7.