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Allosteric inhibitors of mutant IDH1 or IDH2 induce terminal differentiation of the mutant leukemic blasts and provide durable clinical responses in approximately 40% of acute myeloid leukemia (AML) patients with the mutations. However, primary resistance and acquired resistance to the drugs are major clinical issues. To understand the molecular underpinnings of clinical resistance to IDH inhibitors (IDHi), we perform multipronged genomic analyses (DNA sequencing, RNA sequencing and cytosine methylation profiling) in longitudinally collected specimens from 60 IDH1- or IDH2-mutant AML patients treated with the inhibitors. The analysis reveals that leukemia stemness is a major driver of primary resistance to IDHi, whereas selection of mutations in RUNX1/CEBPA or RAS-RTK pathway genes is the main driver of acquired resistance to IDHi, along with BCOR, homologous IDH gene, and TET2. These data suggest that targeting stemness and certain high-risk co-occurring mutations may overcome resistance to IDHi in AML. The regulation of resistance to IDH inhibitors in acute myeloid leukaemia is not completely understood. Here the authors reveal with integrative multi-omics analyses that stemness features are major drivers of primary resistance, while high-risk mutations drive acquired resistance.

Clonal diversity is a consequence of cancer cell evolution driven by Darwinian selection. Precise characterization of clonal architecture is essential to understand the evolutionary history of tumor development and its association with treatment resistance. Here, using a single-cell DNA sequencing, we report the clonal architecture and mutational histories of 123 acute myeloid leukemia (AML) patients. The single-cell data reveals cell-level mutation co-occurrence and enables reconstruction of mutational histories characterized by linear and branching patterns of clonal evolution, with the latter including convergent evolution. Through xenotransplantion, we show leukemia initiating capabilities of individual subclones evolving in parallel. Also, by simultaneous single-cell DNA and cell surface protein analysis, we illustrate both genetic and phenotypic evolution in AML. Lastly, single-cell analysis of longitudinal samples reveals underlying evolutionary process of therapeutic resistance. Together, these data unravel clonal diversity and evolution patterns of AML, and highlight their clinical relevance in the era of precision medicine., Understanding the evolutionary trajectory of cancer samples may enable understanding resistance to treatment. Here, the authors used single cell sequencing of a cohort of acute myeloid leukemia tumours and identify features of linear and branching evolution in tumours.

Until recently, our understanding of chromosome organization in higher eukaryotic cells has been based on analyses of large-scale, low-resolution changes in chromosomes structure. More recently, CRISPR-Cas9 technologies have allowed us to “zoom in” and visualize specific chromosome regions in live cells so that we can begin to examine in detail the dynamics of chromosome organization in individual cells. In this review, we discuss traditional methods of chromosome locus visualization and look at how CRISPR-Cas9 gene-targeting methodologies have helped improve their application. We also describe recent developments of the CRISPR-Cas9 technology that enable visualization of specific chromosome regions without the requirement for complex genetic manipulation.

Background The Rh complex contributes to cell membrane structural integrity of erythrocytes. Rhnull syndrome is characterized by the absence of the Rh antigen on the erythrocyte membrane, resulting in chronic hemolytic anemia. We recently came across 3 Rhnull phenotype probands within two families with the same novel RHAG mutation in the Japanese population. Materials and methods Detailed Rh phenotyping by hemagglutination was performed using monoclonal and polyclonal anti-D, -C, -c, -E, and -e; monoclonal and polyclonal anti-Rh17 antibodies; and polyclonal anti-Rh29 antibodies. RHAG mRNA transcripts were analyzed by reverse transcription-polymerase chain reaction, and the mutation was verified by genomic sequencing. Results The genomic region spanning exon 6 contained a G > A transition in the invariant GT motif of the 5' donor splice-site of Intron 6 (c.945+1G>A). The Rhnull phenotype was caused by an autosomal recessive mutation in Probands 1 and 2, determined by family history. Regarding clinical features, the degree of hemolysis varied slightly between these individuals, with Proband 3 displaying acute hemolytic anemia with an infection. While no standard therapy has been established, the condition of the patient in this study improved with conservative treatment, including hydration and antibiotics. Conclusion The mechanisms of hemolysis due to the Rhnull phenotype can vary, but our findings indicate that acute hemolytic crisis caused by the Rhnull syndrome could be associated with infection.

Doodhi et al. reconstitute the yeast kinetochore–microtubule interface in vitro and compare the relative strength of distinct kinetochore–microtubule interaction modes. Their results suggest how Aurora B kinase promotes the exchange of kinetochore–microtubule interactions to eliminate aberrant interactions and establish chromosome biorientation., To establish chromosome biorientation, aberrant kinetochore–microtubule interaction must be resolved (error correction) by Aurora B kinase. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment, respectively). However, it is still unclear how kinetochore–microtubule interactions are exchanged during error correction. Here, we reconstituted the budding yeast kinetochore–microtubule interface in vitro by attaching the Ndc80 complexes to nanobeads. These Ndc80C nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. This in vitro assay enabled the direct comparison of lateral and end-on attachment strength and showed that Dam1 phosphorylation by Aurora B makes the end-on attachment weaker than the lateral attachment. Similar reconstitutions with purified kinetochore particles were used for comparison. We suggest the Dam1 phosphorylation weakens interaction with the Ndc80 complex, disrupts the end-on attachment, and promotes the exchange to a new lateral attachment, leading to error correction.

Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting (dark) Pr state and the light activated Pfr state, in which light signals are received and transduced as structural change through the entire protein to modulate the activity of the protein. It is unknown how the Pr-to-Pfr interconversion occurs as the structure of intermediates remain notoriously elusive. Here, we present short-lived crystal structures of the classical phytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray Free Electron Laser 5 ns and 33ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway. The snapshots show with atomic precision how the signal progresses from the chromophore towards the output domains, explaining how plants, bacteria and fungi sense red light.

Satellite cell proliferation is an essential step in proper skeletal muscle development and muscle regeneration. However, the mechanisms regulating satellite cell proliferation are relatively unknown compared to the knowledge associated with the differentiation of satellite cells. Moreover, it is still unclear whether overload muscle fiber hypertrophy is dependent on satellite cell proliferation. In general, cell proliferation is regulated by the activity of cell cycle regulators, such as cyclins and cyclin-dependent kinases (CDKs). Despite recent reports on the function of CDKs and CDK inhibitors in satellite cells, the physiological role of Cdk1 in satellite cell proliferation remains unknown. Herein, we demonstrate that Cdk1 regulates satellite cell proliferation, muscle regeneration, and muscle fiber hypertrophy. Cdk1 is highly expressed in myoblasts and is downregulated upon myoblast differentiation. Inhibition of CDK1 activity inhibits myoblast proliferation. Deletion of Cdk1 in satellite cells leads to inhibition of muscle recovery after muscle injury due to reduced satellite cell proliferation in vivo. Finally, we provide direct evidence that Cdk1 expression in satellite cells is essential for overload muscle fiber hypertrophy in vivo. Collectively, our results demonstrate that Cdk1 is essential for myoblast proliferation, muscle regeneration, and muscle fiber hypertrophy. These findings could help to develop treatments for refractory muscle injuries and muscle atrophy, such as sarcopenia.

Background: NPM1 mutations (NPM1 +) occur in ~30% of acute myeloid leukemia (AML), often with co-occurring mutations including isocitrate dehydrogenase mutations (IDH +). Previous studies suggest early acquisition and expansion of IDH1/2 mutations (IDH1 +/IDH2 +) prior to NPM1 + development increase relapse risk. Yet the clonal hierarchy of NPM1 +/IDH + AML, dynamics of IDH + during therapy, and predictive value of IDH1 +/2 + in NPM1 + AML in response to standard intensive (IC) or venetoclax-based (VEN) treatment is less studied. Methods: We identified 71 patients with newly diagnosed NPM1+ AML (index cohort). Longitudinal molecular assessment of bone marrow samples at diagnosis, remission, and relapse was performed using next-generation sequencing (NGS) of genes frequently mutated in AML. Clonality within each sample was inferred using variant allele frequency (VAF) of individual mutations compared with NPM1 +(co-dominant: VAF difference of < 10%; sub-clonal: VAF ≥10% lower). Measurable residual disease (MRD) was assessed using flow cytometry (FC) (MRD FC; lower limit of sensitivity 10 -3-10 -4) and NGS. To validate findings in the index cohort, clinical and genomic data were collected from a publicly available dataset of 39 patients with NPM1 + AML who underwent single cell DNA sequencing (scDNAseq) at our institution. Categorical and continuous variables were assessed using the fisher exact test and the Wilcoxon rank sum test, respectively. Adjustment for multiple comparisons utilized the Benjamini-Horchberg procedure. Time-to-event outcomes were assessed by the log-rank method. Results: Median patient age was 64 years (range 19-84). Thirty-eight patients (54%) received IC (IC: n=30, IC+VEN: n= 8); 33 (46%) received lower-intensity therapy (lower-intensity: n= 6, lower-intensity+VEN: n=27). Common co-occurring mutations at diagnosis included DNMT3A (62%), FLT3 (52%), TET2 (27%), IDH2 (23%), IDH1 (18%), PTPN11 (18%), and NRAS (17%). SRSF2, IDH2, TET2, ASXL1, and DNMT3A mutations preceded or were co-dominant with NPM1 +, while NRAS, PTPN11, and FLT3 mutations were sub-clonal. IDH1 +was sub-clonal in 40% of cases (Fig. 1A). In the scDNAseq cohort IDH1 + was sub-clonal in 40% (n=3/8); IDH2 + preceded NPM1 +in 75% (n=9). Compared with IDH1 +, IDH2 + was more likely to precede or be co-dominant with NPM1 + (95% vs. 60%, p-value: 0.017). DTA (DNMT3A, TET2, ASXL1) mutations were detected more often in remission versus IDH + (88% vs. 55%, p-value: 0.01). Persistent IDH1 + and IDH2 + were identified in 36% (n=4) and 73% (n=11) of patients. Concurrent MRD FC confirmed clearance of the previous AML clone in 100% of patients, with a pre-leukemic/CH immunophenotype identified in 50% and 80% of cases with persistent IDH1 +or IDH2 +, respectively Patients with IDH + often developed new or persistent mutations in remission compared with patients with wild-type IDH(54% vs. 12%, p-value Patients with NPM1 +/IDH + compared with NPM1 +/IDH - AML in complete remission (N=91) had improved event-free (EFS; median 65 vs. 28 months, p-value: 0.002) and overall survival (OS; median 82 months vs. NR, p-value: 0.02) (Fig. 1B and C), largely driven by NPM1 +/IDH2 + cases, and patients receiving IC. No survival difference was observed based on clonal hierarchy of IDH + (clonal vs sub-clonal) compared with NPM1 + or persistent IDH + in remission with concurrent clearance of NPM1 + and MRD FC. FLT3 mutations were identified at similar frequencies in the NPM1 +/IDH +and NPM1 +/IDH - groups (50% vs. 53%, p-value: 0.84; ITD: 81% vs. 69%, p-value: 0.49), and a similar number of patients receiving VEN (48% vs.43%, p-value: 0.38). Conclusion: IDH1 + and IDH2 + develop at differing timepoints during leukemogenesis in NPM1 + AML. IDH2 + frequently precedes NPM1 and persists in remission, while IDH1 + is more often sub-clonal and cleared in remission. Persistent IDH1 + or IDH2 + frequently correlate with detection of a CH phenotype by MFC in remission, and in the absence of NPM1 + and MRD FC do not influence relapse. Improved EFS and OS was observed with NPM1 +/IDH + AML, identifying a molecular subgroup associated with favorable outcomes to both VEN and IC treatment regimens Figure 1 Figure 1. Disclosures DiNardo: Foghorn: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios/Servier: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Wang: Stemline Therapeutics: Honoraria. Kadia: Ascentage: Other; AstraZeneca: Other; Liberum: Consultancy; Genfleet: Other; Pfizer: Consultancy, Other; Astellas: Other; Novartis: Consultancy; BMS: Other: Grant/research support; Jazz: Consultancy; Dalichi Sankyo: Consultancy; Genentech: Consultancy, Other: Grant/research support; Cure: Speakers Bureau; Amgen: Other: Grant/research support; Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support; Cellonkos: Other; Sanofi-Aventis: Consultancy; Pulmotech: Other. Daver: Gilead Sciences, Inc.: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Hanmi: Research Funding; Sevier: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Novimmune: Research Funding; Glycomimetics: Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Short: NGMBio: Consultancy; Takeda Oncology: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; AstraZeneca: Consultancy; Astellas: Research Funding; Novartis: Honoraria; Amgen: Consultancy, Honoraria. Khoury: Angle: Research Funding; Kiromic: Research Funding; Stemline Therapeutics: Research Funding. Konopleva: F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Agios: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; KisoJi: Research Funding; Sanofi: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Cellectis: Other: grant support; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Ablynx: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding. Ravandi: AbbVie: Honoraria, Research Funding; Xencor: Honoraria, Research Funding; Taiho: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria; Amgen: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Prelude: Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria. Takahashi: Celgene/BMS: Consultancy; Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Novartis: Consultancy. Loghavi: Abbvie: Current equity holder in publicly-traded company; Curio Sciences: Honoraria; Gerson Lehrman Group: Consultancy; Guidepoint: Consultancy; Peerview: Honoraria; Qualworld: Consultancy.

A nanosecond pump–probe device for time-resolved serial femtosecond crystallography has been developed at SACLA., X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

Non-neural granular cell tumors (GCTs) are clinically rare, whereas cases arising in the uterine corpus are exceedingly rare. Only three uterine cervical cases of GCTs have been reported to date and, to the best of our knowledge, there are no reports of GCT of the uterine corpus in the literature. We herein describe the first case of non-neural GCT arising from the uterine corpus reported to date. A 55-year-old premenopausal woman was referred to the Department of Obstetrics and Gynecology of Hashimoto Municipal Hospital (Wakayama, Japan) with a suspected uterine tumor. The tumor presented as a uterine leiomyoma-like mass on radiological examinations, but the diagnosis of non-neural GCT was established based on pathological and immunohistochemical examinations. Microscopically, histological examination of the entire surgical specimen revealed large polygonal cells with abundant eosinophilic granular cytoplasm and round to oval nuclei. Immunohistochemistry revealed positive periodic acid-Schiff staining of the cytoplasmic granules, which was resistant to diastase. In addition, the tumor cells stained positive for CD68, but negative for S-100, neuron-specific enolase, cytokeratin, CD34, α-smooth muscle actin, desmin, estrogen receptor and progesterone receptor. It is important for gynecologists to be aware of the possibility of non-neural GCT of the uterine corpus, for which accurate diagnosis, complete resection and long-term follow-up are crucial, as it may be easily misdiagnosed as uterine leiomyoma.

Human noroviruses cause an estimated 685 million infections and 200 000 deaths annually worldwide. Although vaccines against GII.4 and GI.1 genotypes are under development, no information is available regarding vaccines or monoclonal antibodies to other noroviral genotypes. Here, we developed 2 variable-domain llama heavy-chain antibody fragment (VHHs) clones, 7C6 and 1E4, against GII.4 and GII.17 human noroviruses, respectively. Although 7C6 cross-reacted with virus-like particles (VLPs) of GII.17, GII.6, GII.3, and GII.4, it neutralized only GII.4 norovirus. In contrast, 1E4 reacted with and neutralized only GII.17 VLPs. Both VHHs blocked VLP binding to human induced pluripotent stem cell-derived intestinal epithelial cells and carbohydrate attachment factors. Using these 2 VHHs, we produced a heterodimeric VHH fragment that neutralized both GII.4 and GII.17 noroviruses. Because VHH fragments are heat- and acid-stable recombinant monoclonal antibodies, the heterodimer likely will be useful for oral immunotherapy and prophylaxis against GII.4 and GII.17 noroviruses in young, elderly, or immunocompromised persons.

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

To promote chromosome bi-orientation, Aurora B kinase weakens and disrupts aberrant kinetochore-MT interaction. It has long been debated how Aurora B halts this action when bi-orientation is established and tension is applied across sister kinetochores. Pertinent to this debate, it was shown that Bir1 (yeast Survivin), which recruits Ipl1-Sli15 (yeast Aurora B-INCENP) to centromeres, is dispensable for bi-orientation, raising the possibility that Aurora B localization at centromeres is not required for bi-orientation. Here, we show that the COMA inner kinetochore sub-complex physically interacts with Sli15, recruits Ipl1-Sli15 to the inner kinetochore and promotes chromosome bi-orientation, independently of Bir1, in budding yeast. Moreover, using an engineered recruitment of Ipl1-Sli15 to the inner kinetochore when both Bir1 and COMA are defective, we show that localization of Ipl1-Sli15 at centromeres/inner kinetochores is essential for bi-orientation, refuting the above possibility. Our results give important insight into how Aurora B disrupts kinetochore-MT interaction in a tension-dependent manner, to promote chromosome bi-orientation.

A total of 300 patients with nucleic acid test-confirmed rubella, mostly adults, were investigated to determine the clinical value of a rubella-specific IgM test using an EIA kit. IgM titers increased after rash onset, the median IgM titer being significantly higher 3 days post-onset than on previous days (P

BCOR, encoding BCL-6 corepressor (BCOR), is X-linked and targeted by somatic mutations in various hematological malignancies including myelodysplastic syndrome (MDS). We previously reported that mice lacking Bcor exon 4 (BcorΔE4/y) in the hematopoietic compartment developed NOTCH-dependent acute T-cell lymphoblastic leukemia (T-ALL). Here, we analyzed mice lacking Bcor exons 9 and 10 (BcorΔE9-10/y), which express a carboxyl-terminal truncated BCOR that fails to interact with core effector components of polycomb repressive complex 1.1. BcorΔE9-10/y mice developed lethal T-ALL in a similar manner to BcorΔE4/y mice, whereas BcorΔE9-10/y hematopoietic cells showed a growth advantage in the myeloid compartment that was further enhanced by the concurrent deletion of Tet2. Tet2Δ/ΔBcorΔE9-10/y mice developed lethal MDS with progressive anemia and leukocytopenia, inefficient hematopoiesis, and the morphological dysplasia of blood cells. Tet2Δ/ΔBcorΔE9-10/y MDS cells reproduced MDS or evolved into lethal MDS/myeloproliferative neoplasms in secondary recipients. Transcriptional profiling revealed the derepression of myeloid regulator genes of the Cebp family and Hoxa cluster genes in BcorΔE9-10/y progenitor cells and the activation of p53 target genes specifically in MDS erythroblasts where massive apoptosis occurred. Our results reveal a tumor suppressor function of BCOR in myeloid malignancies and highlight the impact of Bcor insufficiency on the initiation and progression of MDS.

Multiplex reverse transcription (RT)-polymerase chain reaction (PCR)-based assays involving fluorescent dye-labeled primers were modified to detect 10 types of gastroenteritis viruses by adding two further assays to a previously developed assay. Then, these assays were applied to clinical samples, which were collected between January 2006 and December 2013. All 10 types of viruses were effectively detected in the multiplex RT-PCR-based assays. In addition, various viral parameters, such as the detection rates and age distributions of each viral type, were examined. The frequency and types of mixed infections were also investigated. Among the 186 virus-positive samples, genogroup II noroviruses were found to be the most common type of virus (32.7%), followed by group A rotaviruses (10.6%) and parechoviruses (10.3%). Mixed infections were observed in 37 samples, and many of them were detected in patients who were less than 2 years old. These observations showed that the multiplex RT-PCR-based assays involving fluorescent dye-labeled primers were able to effectively detect the viruses circulating among pediatric acute gastroenteritis patients and contributed to the highly specific and sensitive diagnosis of gastroenteritis. J. Med. Virol. 89:791-800, 2017. © 2016 Wiley Periodicals, Inc.

SummaryWhen human cells enter mitosis, chromosomes undergo substantial changes in their organisation to resolve sister chromatids and compact chromosomes. Despite the fundamental importance of this phenomenon to genome stability, we still do not fully comprehend the timing and coordination of these events. To address these questions, we need to evaluate the progression of both sister chromatid resolution and chromosome compaction in one assay. We achieved this by analysing changes in configuration of marked chromosome regions over time, with high spatial and temporal resolution. This assay showed that sister chromatid resolution is an iterative process that begins in late G2 phase and completes in prophase. Cohesins and WAPL antagonistically regulate sister chromatid resolution in late G2 and prophase whilst local enrichment of cohesin on chromosomes prevents precocious sister chromatid resolution. Moreover, our assay allowed quantitative evaluation of the timing and efficiency of condensin II and I activities in promoting sister chromatid resolution and chromosome compaction, respectively. Thus, our real-time assay sheds new light on the dynamics of mitotic chromosome resolution and compaction.

Wheat (Triticum aestivum L.) and rice (Oryza sativa L.), which belong to the Poaceae family, are starch-grain crops, but wheat is adapted to cooler temperature conditions than rice. In this study, the difference between the two contrasting crops in grain-filling adaptability in response to temperature was investigated. Two spring wheat cultivars were grown in the Mediterranean-type environments of Western Australia and Southeast Turkey, and four temperate-zone rice cultivars were grown in several locations in Japan under irrigated conditions. Portions of the crops were enclosed under a plastic canopy to elevate the temperature after anthesis. Average temperatures during grain filling ranged from 14 to 24 °C for wheat and from 23 to 29 °C for rice. Grain yield varied from 280 to 599 g m-2 in wheat and 354 to 736 g m-2 in rice. When plant density was halved at flowering to estimate the potential grain-filling rate under an increased supply of assimilates, the grain-filling percentages [%F, observed grain weight (G)/potential grain weight (PG)] of both crops were represented by similar logistic curves of cumulative temperatures during the grain filling period. These results suggest that grain-filling responses to temperature scarcely differ between spring wheat and temperate-zone rice. G was estimated for the spring wheat and temperate-zone rice cultivars under different temperatures after anthesis using an assimilate-limited grain-potentiality model consisting of the following parameters: rate of whole-plant weight increase (?W/?t), rate of potential grain dry weight increase (?PG/?t) based on rate of%F (%F/?t) and PG, and the amount of stem reserves (SP). The observed data showed that the decrease in ?W/?t with an increase in temperature in wheat was greater than in rice. According to the model, G started to decrease at lower temperatures in wheat than in rice, and this decrease was accelerated by lower amounts of SP. Therefore, the difference in the optimal temperatures for G during grain filling between the two crops was suggested to mainly result from the sensitivity of assimilation to high temperatures. © 2017 Elsevier B.V. Commonwealth Scientific and Industrial Research Organisation Research Institute for Humanity and Nature Japan Society for the Promotion of Science Asthma Foundation of Western Australia Our thanks go to a project in Çukurova Unviersity, Turkey of the Impact of Climate Changes on Agricultural Production System in Arid Areas (ICCAP) in the Research Institute for Humanity and Nature, Japan, and CSIRO, Western Australia for the substantial support of, and contributions to, our research. We would like to thank Dr. Neil C. Turner, University of Western Australia for his comments and suggestions to our manuscript. We thank JSPS for their financial support of the project in Australia and Japan. Appendix A

The duration of viral shedding in the patients from two outbreaks and four sporadic cases of norovirus (NoV) infections was investigated. The longest period of viral shedding into feces was for 173 days in an inpatient from one case of outbreak. The VP1 sequence from two long-term viral shedding cases in the outbreak revealed four synonymous and one non-synonymous mutations in one inpatient at 26 days from the onset of illness, and nine synonymous and two non-synonymous mutations and a deletion, 10 synonymous mutations and a deletion in other inpatient at 29 days and 54 days from the onset of illness, respectively. Ten of the 11 amino acid positions detected in these two inpatients were in the outermost P2 domain of the viral capsid protein, and mutations at positions 295, 297, and 394 were shared in the inpatients. Mutations in the P2 domain were in epitopes A and D or near epitopes A, C, and E, suggesting that the long-term carrier state of norovirus infection contributes to the generation of escape mutants by host immunoselection.

For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles (bi-orientation). To establish bi-orientation, aberrant kinetochore-microtubule attachments are disrupted (error correction) by aurora B kinase (Ipl1 in budding yeast). Paradoxically, during this disruption, new attachments are still formed efficiently to enable fresh attempts at bi-orientation. How this is possible remains an enigma. Here we show that kinetochore attachment to the microtubule lattice (lateral attachment) is impervious to aurora B regulation, but attachment to the microtubule plus end (end-on attachment) is disrupted by this kinase. Thus, a new lateral attachment is formed without interference, then converted to end-on attachment and released if incorrect. This process continues until bi-orientation is established and stabilized by tension across sister kinetochores. We reveal how aurora B specifically promotes disruption of the end-on attachment through phospho-regulation of kinetochore components Dam1 and Ndc80. Our results reveal fundamental mechanisms for promoting error correction for bi-orientation.

The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.

Norovirus (NoV) infection is a major cause of food poisoning and food-related outbreaks of gastroenteritis that is being increasingly recognized as a public health problem. We developed the “PANtrap” method as a general protocol for detecting foodborne viruses in contaminated foods, including composite meals containing NoVs. This method is based on immune capture. The recovery rate of NoV GII.4 from potato salad with anti-NoV GII.4 serum was 78.3 ± 10.8%, whereas the recovery rate was only 0.33 ± 0.08% for that without antiserum. Similar results were obtained for stir-fried noodles. Because there is limited availability of NoV-specific antibodies, we also applied the PANtrap method to a commercially available human γ-globulin agent, which showed approximately the same detection rate. Also, other foodborne viruses, such as hepatitis A virus, sapovirus, and adenovirus, were detected. This newly developed method can be used to identify NoV contamination in composite foods, thus contributing to food safety.

Enteric viruses are an important cause of viral food-borne disease. Shellfish, especially oysters, are well recognized as a source of food-borne diseases, and oyster-associated gastroenteritis outbreaks have on occasion become international occurrences. In this study, 286 fecal specimens from 88 oyster-associated gastroenteritis outbreaks were examined for the presence of 10 human enteric viruses using antigenic or genetic detection methods in order to determine the prevalence of these infections. All virus-positive patients were over 18 years old. The most common enteric virus in outbreaks (96.6%) and fecal specimens (68.9%) was norovirus (NoV), indicating a high prevalence of NoV infection associated with the consumption of raw or under-cooked oysters. Five other enteric viruses, aichiviruses, astroviruses, sapoviruses, enteroviruses (EVs), and rotavirus A, were detected in 30.7% of outbreaks. EV strains were characterized into three rare genotypes, coxsackievirus (CV) A1, A19, and EV76. No reports of CVA19 or EV76 have been made since 1981 in the Infectious Agents Surveillance Report by the National Infectious Diseases Surveillance Center, Japan. Their detection suggested that rare types of EVs are circulating in human populations inconspicuously and one of their transmission modes could be the consumption of contaminated oysters. Rapid identification of pathogens is important for the development of means for control and prevention. The results of the present study will be useful to establish an efficient approach for the identification of viral pathogens in oyster-associated gastroenteritis in adults.

Snapshots of bacteriorhodopsin Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used timeresolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport. Science , this issue p. 1552

Single-cell analyses in budding yeast reveal that neighboring replicons are assembled stochastically and stay associated to maintain stable replication factories., Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures.

Summary Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here, we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3–Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore, DDK at kinetochores independently recruits the Scc2–Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites., Graphical Abstract, Highlights • The Ctf19 complex recruits Cdc7-Dbf4 kinase (DDK) to kinetochores (KTs) in telophase • DDK at KTs recruits Sld3–Sld7 to pericentromeric origins, advancing replication timing • DDK at KTs recruits Scc2, enhancing cohesin loading and cohesion at pericentromeres • The DDK functions in replication timing and cohesion are independent of each other

The LIM-domain protein LMO2 is a T-cell oncogenic protein first recognized by gene activation through chromosomal translocations, but it is also responsible for leukaemias arising as secondary, adverse effects in an X-SCID gene therapy trial. There are no specific reagents currently available to analyse the LMO2 multiprotein complex or to combat LMO2-dependent leukaemias. Accordingly, we have isolated an anti-LMO2 single chain Fv antibody fragment to determine if intracellular interference with LMO2-protein complexes can avert LMO2-dependent functions in normal and cancer settings. The anti-LMO2 single chain Fv, obtained using Intracellular Antibody Capture (IAC) technology, is specific for LMO2 among the LIM-only protein family and binds LMO2 through the third and fourth LIM fingers. Using vector-mediated expression of anti-LMO2 scFv, we show inhibition of Lmo2-dependent erythropoiesis but not endothelial development. We also demonstrate inhibition of Lmo2-dependent leukaemia in a mouse T-cell tumourigenesis transplantation assay with retroviral-mediated expression of anti-LMO2 scFv. Our studies establish that interference with the LMO2 multiprotein complex inhibits both normal and tumourigenic roles. The antibody fragment is a tool for dissecting LMO2 function in haematopoiesis and leukaemia and is a lead for development of therapeutics against LMO2-dependent T-ALL.

Antibodies are now indispensable tools for all areas of cell biology and biotechnology as well as for diagnosis and therapy. Antigen-specific single immunoglobulin variable domains that bind to native antigens can be isolated and manipulated using yeast intracellular antibody capture technology but converting these to whole monoclonal antibody requires that complementary variable domains (VH or VL) bind to the same antigenic site. We describe a simple approach (CatcherAb) for specific isolation of such complementary single domains allowing the constitution of functional Fv, forming the basis of antigen-specific whole immunoglobulin and thus antibody production. We illustrate this approach by developing high-affinity Fv from single variable domains binding to RAS and LMO2 oncogenic proteins.

The discovery of chromosomal translocations in leukemia/lymphoma and sarcomas presaged a widespread discovery in epithelial tumors. With the advent of new-generation whole-genome sequencing, many consistent chromosomal abnormalities have been described together with putative driver and passenger mutations. The multiple genetic changes required in mouse models to assess the interrelationship of abnormalities and other mutations are severe limitations. Here, we show that sequential gene targeting of embryonic stem cells can be used to yield progenitor cells to generate chimeric offspring carrying all the genetic changes needed for cell-specific cancer. Illustrating the technology, we show that MLL–ENL fusion is sufficient for lethal leukocytosis and proof of genome integrity comes from germline transmission of the sequentially targeted alleles. This accelerated technology leads to a reduction in mouse numbers (contributing significantly to the 3Rs), allows fluorescence tagging of cancer-initiating cells, and provides a flexible platform for interrogating the interaction of chromosomal abnormalities with mutations. Cancer Res; 74(5); 1588–97. ©2014 AACR.

PURPOSE: Bcr-Abl, the causative agent of chronic myelogenous leukemia (CML), localizes in the cytoplasm where its oncogenic signaling leads to proliferation of cells. If forced into the nucleus Bcr-Abl causes apoptosis. To achieve nuclear translocation, binding domains for capture of Bcr-Abl were generated and attached to proteins with signals destined for the nucleus. These resulting proteins would be capable of binding and translocating endogenous Bcr-Abl to the nucleus. METHODS: Bcr-Abl was targeted at 3 distinct domains for capture: by construction of high affinity intracellular antibody domains (iDabs) to regions of Bcr-Abl known to promote cytoplasmic retention, via its coiled coil domain (CC), and through a naturally occurring protein-protein interaction domain (RIN1). These binding domains were then tested for their ability to escort Bcr-Abl into the nucleus using a "protein switch" or attachment of 4 nuclear localization signals (NLSs). RESULTS: Although RIN1, ABI7-iDab, and CCmut3 constructs all produced similar colocalization with Bcr-Abl, only 4NLS-CCmut3 produced efficient nuclear translocation of Bcr-Abl. CONCLUSIONS: We demonstrate that a small binding domain can be used to control the subcellular localization of Bcr-Abl, which may have implications for CML therapy. Our ultimate future goal is to change the location of critical proteins to alter their function.

Mutations in the RAS family of oncogenes are highly prevalent in human cancer and, amongst its manifold effects, oncogenic RAS impairs the expression of components of the antigen presentation pathway. This allows evasion of cytotoxic T lymphocytes (CTL). CTL and natural killer (NK) cells are reciprocally regulated by MHC class I molecules and any gain in CTL recognition obtained by therapeutic inactivation of oncogenic RAS may be offset by reduced NK cell activation. We have investigated the consequences of targeted inactivation of oncogenic RAS on the recognition by both CTL and NK cells. Inactivation of oncogenic RAS, either by genetic deletion or inactivation with an inducible intracellular domain antibody (iDAb), increased MHC class I expression in human colorectal cell lines. The common RAS mutations, at codons 12, 13 and 61, all inhibited antigen presentation. Although MHC class I modulates the activity of both CTL and NK cells, the enhanced MHC class I expression resulting from inactivation of mutant KRAS did not significantly affect the in vitro recognition of these cell lines by either class of cytotoxic lymphocyte. These results show that oncogenic RAS and its downstream signalling pathways modulate the antigen presentation pathway and that this inhibition is reversible. However, the magnitude of these effects was not sufficient to alter the in vitro recognition of tumour cell lines by either CTL or NK cells.

The new GII.2 variant collected from May 2012–March 2014 consisted of GII.15 and GII.2 genomes, in which the putative recombination points found in the boundary region between ORF1 and ORF2. These findings suggested that the swapping of structural and non-structural proteins is a common mechanism for generating new epidemic variants in nature.

Sapovirus (SaV), a member of the family Caliciviridae, is an important cause of acute epidemic gastroenteritis in humans. Human SaV is genetically and antigenically diverse and can be classified into four genogroups (GI, GII, GIV, and GV) and 16 genotypes (7 GI [GI.1-7], 7 GII, [GII.1-7], 1 GIV and 1 GV), based on capsid sequence similarities. Monoclonal antibodies (MAbs) are powerful tools for examining viruses and proteins. PAI myeloma cells were fused with spleen cells from mice immunized with a single type of recombinant human SaV virus-like particles (VLPs) (GI.1, GI.5, GI.6, GII.3, GIV, or GV). Sixty-five hybrid clones producing MAbs were obtained. Twenty-four MAbs were characterized by ELISA, according to their cross-reactivity to each VLP (GI.1, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GIV, and GV). The MAbs were classified by this method into: (i) MAbs broadly cross-reactive to all GI, GII, GIV and GV strains; (ii) those reactive in a genogroup-specific; and (iii) those reactive in a genotype-specific manner. Further analysis of three broadly cross-reactive MAbs with a competitive ELISA demonstrated that at least two different common epitopes are located on the capsid protein of human SaVs in the four genogroups. The MAbs generated and characterized in this study will be useful tools for further study of the antigenic and structural topography of the human SaV virion and for developing new diagnostic assays for human SaV.

Twenty autopsy cases with 2009 pandemic influenza A (2009 H1N1) virus infection, performed between August 2009 and February 2010, were histopathologically analyzed. Hematoxylin-eosin staining, immunohistochemistry for type A influenza nucleoprotein antigen, and real-time reverse transcription-PCR assay for viral RNA were performed on formalin-fixed and paraffin-embedded specimens. In addition, the D222G amino acid substitution in influenza virus hemagglutinin, which binds to specific cell receptors, was analyzed in formalin-fixed and paraffin-embedded trachea and lung sections by direct sequencing of PCR-amplified products. There were several histopathological patterns in the lung according to the most remarkable findings in each case: acute diffuse alveolar damage (DAD) with a hyaline membrane (four cases), organized DAD (one case), acute massive intra-alveolar edema with variable degrees of hemorrhage (three cases), neutrophilic bronchopneumonia (five cases) and tracheobronchitis with limited histopathological changes in alveoli (four cases). In two cases, the main findings were due to preexisting disease. Influenza virus antigen was only detected in the respiratory tract in 10 cases by immunohistochemistry. The antigen was detected in type II pneumocytes (three cases) in the epithelial cells of the trachea, bronchi and glands (six cases), and in the epithelial cells in both of the above (one case). The four cases with acute DAD presented with antigen-positive type II pneumocytes. In one case, the D222G substitution was detected in the lung as a major sequence, although 222D was prominent in the trachea, suggesting that selection of the viral clones occurred in the respiratory tract. In five cases, the pathogenesis of 2009 H1N1 was confirmed to be viral infection in pneumocytes, which caused severe alveolar damage and fatal viral pneumonia. Further studies on both host and viral factors in autopsy or biopsy materials will be essential to elucidate the other pathogenic factors involved in influenza virus infection.

Norovirus GII.4 is a major etiological agent of acute viral gastroenteritis worldwide. We examined GII.4 evolution using 277 near-full-length GII.4 genome sequences from human stool specimens collected at 20 sites in Japan between May 2006 and March 2010. We found outbreaks of 8 monophyletic GII.4 subtypes, among which a single subtype, termed 2006b, had continually predominated (222/277: 80.7%). Four of the 8 GII.4 subtypes were chimera viruses of recently prevalent GII.4 subtypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF) 1 and ORF 2 (P

The CutA1 protein from Pyrococcus horikoshii (PhCutA1), a hyperthermophile, has an unusually high content of charged residues and an unusually high denaturation temperature. To elucidate the role of ion-ion interactions in protein stability, mutant proteins of PhCutA1 in which charged residues were substituted by noncharged residues were comprehensively examined. The denaturation temperatures (T(d)) for 13 of 53 examined mutant proteins were higher than that of the wild-type (148.5 °C at pH 7.0), among which E99Q had the highest T(d) at 154.9 °C. R25A had the largest decrease in T(d) among single mutants at ΔT(d) = -12.4 °C. The average decrease in T(d) of Lys or Arg mutants was greater than that of Glu or Asp mutants, and the average change in T(d) (ΔT(d)) of 21 Glu mutants was negligible, at 0.03 ± 2.05 °C. However, the electrostatic energy (-159.3 kJ·mol(-1)) of PhCutA1 was quite high, compared with that of CutA1 from Escherichia coli (-9.7 kJ·mol(-1)), a mesophile. These results indicate that: (a) many Glu and Asp residues of PhCutA1 should be essential for highly efficient interactions with positively charged residues and for generating high electrostatic energy, although they were forced to be partially repulsive to each other; (b) the changes in stability of mutant proteins with a T(d) value of ~140-150 °C were able to be explained by considering factors important for protein stability and the structural features of mutant sites; and (c) these findings are useful for the design of proteins that are stable at temperatures > 100 °C.

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites.

Summary Proper chromosome segregation in mitosis relies on correct kinetochore-microtubule (KT-MT) interactions. The KT initially interacts with the lateral surface of a single MT (lateral attachment) extending from a spindle pole and is subsequently anchored at the plus end of the MT (end-on attachment) [1]. The conversion from lateral to end-on attachment is crucial because end-on attachment is more robust [2–4] and thought to be necessary to sustain KT-MT attachment when tension is applied across sister KTs upon their biorientation [1]. The mechanism for this conversion is still elusive. The Ndc80 complex is an essential component of the KT-MT interface [1, 5], and here we studied a role of the Ndc80 loop region, a distinct motif looping out from the coiled-coil shaft of the complex [6], in Saccharomyces cerevisiae. With deletions or mutations of the loop region, the lateral KT-MT attachment occurred normally; however, subsequent conversion to end-on attachment was defective, leading to failure in sister KT biorientation. The Ndc80 loop region was required for Ndc80-Dam1 interaction and KT loading of the Dam1 complex, which in turn supported KT tethering to the dynamic MT plus end [3, 7]. The Ndc80 loop region, therefore, has an important role in the conversion from lateral to end-on attachment, a crucial maturation step of KT-MT interaction., Graphical Abstract Highlights ► Kinetochores must be anchored at the dynamic microtubule plus ends in early mitosis ► The Ndc80 loop region is crucial in kinetochore anchoring at the microtubule ends ► The Ndc80 loop region facilitates the interaction with the Dam1 complex ► Ndc80-Dam1 interaction ensures kinetochore anchoring at the microtubule plus ends

Interfering intracellular antibodies are valuable for biological studies as drug surrogates and as potential macromolecular drugs per se. Their application is still limited because of the difficulty of acquisition of functional intracellular antibodies. We describe the use of the new intracellular antibody capture procedure (IAC3) to facilitate direct isolation of functional single domain antibody fragments using four independent target molecules (LMO2, TP53, CRAF1, and Hoxa9) from a set of diverse libraries. Initially, these have variability in only one of the three antigen-binding CDR regions of VH or VL and first round single domains are affinity matured by iterative randomization of the two other CDRs and reselection. We highlight the approach using a single domain binding to LMO2 protein. Our results show that interfering with LMO2 protein function demonstrates a role specifically in erythroid differentiation, confirm a necessary and sufficient function for LMO2 as a cancer therapy target in T-cell neoplasia and allowed for the first time production of soluble recombinant LMO2 protein by co-expression with intracellular domain antibodies. Co-crystallization of LMO2 and the anti-LMO2 VH protein was successful. These results demonstrate that this third generation IAC3 offers a robust toolbox for various biomedical applications and consolidates functional features of the LMO2 protein complex, which includes the importance of Lmo2-Ldb1 protein interaction.

Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus has caused significant outbreaks worldwide. A previous one-step real-time reverse transcription-PCR (rRT-PCR) assay for detecting A/H1N1pdm virus (H1pdm rRT-PCR assay) was improved since the former probe had a low melting temperature and low tolerance to viral mutation. To help with the screening of the A/H1N1pdm virus, rRT-PCR assays were also developed for detecting human seasonal A/H1N1 (H1 rRT-PCR assay) and A/H3N2 influenza viruses (H3 rRT-PCR assay). H1pdm, H1, and H3 rRT-PCR assays were evaluated using in vitro-transcribed control RNA, isolated viruses, and other respiratory pathogenic viruses, and were shown to have high sensitivity, good linearity (R2 = 0.99), and high specificity. In addition, the improved H1pdm rRT-PCR assay could detect two viral strains of A/H1N1pdm, namely, A/Aichi/472/2009 (H1N1)pdm and A/Sakai/89/2009 (H1N1)pdm, which have mutation(s) in the probe-binding region of the hemagglutinin gene, without loss of sensitivity. Using the three rRT-PCR assays developed, 90 clinical specimens collected between May and October 2009 were then tested. Of these, 26, 20, and 2 samples were identified as positive for A/H1pdm, A/H3, and A/H1, respectively, while 42 samples were negative for influenza A viruses. The present results suggest that these highly sensitive and specific H1pdm, H1, and H3 rRT-PCR assays are useful not only for diagnosing influenza viruses, but also for the surveillance of influenza viruses.

Introduction: Glycosaminoglycans (GAGs), such as heparan sulfate and hyaluronic acid, have been implicated in several hematopoietic processes. GAGs are abundant in the extracellular matrix (ECM) and interact with several cell surface proteins and chemokines. However, the effects of chondroitin sulfate (CS), another species of GAG, in hematopoiesis remain unclear. We examined CS in hematopoiesis by genetically reducing CS in mice by disruption of a gene encoding the rate-limiting CS-synthesizing enzyme N-acetylgalactosaminyltransferase-1 (T1). Methods: T1 knockout (T1KO) mice were generated from the C57BL/6N strain (WT). We evaluated hematopoietic recovery after sublethal irradiation (a 5 Gy dose) to understand the role of CS in hematopoiesis after radiation stress. In addition, we evaluated the effects of each CS on hematopoietic cells and on the stromal microenvironment by creating conditions of CS deficiency in hematopoietic cells or in the stromal microenvironment using hematopoietic stem cell transplantation. In particular, BM cells from WT or T1KO mice were transplanted into 8-10-week-old recipient WT or T1KO mice irradiated at a dose of 9 Gy, and mice were analyzed 5 weeks after transplantation. Furthermore, we examined the role of CS on long-term reconstructive function using a CRU assay in serial transplantation. BM cells from WT or T1KO (CD45.2) mice were transplanted into recipient mice (CD45.1) irradiated at a dose of 9 Gy with BM competitor cells from CD45.1 mice, and PB and BM cell chimerism were analyzed 6 weeks and 12 weeks after transplantation. For serial transplantation, BM cells were collected from recipient mice 12 weeks after transplantation and were transplanted into CD45.1 mice irradiated at a dose of 9 Gy without competitor cells. For evaluating the effect of CS on the stromal microenvironment, BM cells from WT mice were serially transplanted into WT or T1KO recipient mice irradiated at a dose of 9 Gy 12 weeks after transplantation. Results: The amount of CS in BM of T1KO mice was 50-66% of that in WT mice. At steady state, there were no significant differences in the number of PB cells, such as neutrophils, lymphocytes, RBCs and platelets, and total BM cells in T1KO and WT mice. T1KO mice had a significantly higher number of BM LSK cells compared to that of WT mice (WT: 0.213 ± 0.044%; T1KO: 0.282 ± 0.046%, p < 0.01). The corresponding number of CFU-GM of BM cells was also higher in the T1KO mice group (WT: 29.6 ± 3.60; T1KO: 45.4 ± 2.37, p < 0.01). However, hematopoietic recovery (PB cells, total BM cells, and LSK cells) after sublethal irradiation was significantly delayed in T1KO mice. CS deficiency in hematopoietic cells resulted in a lower number of LSK cells compared to that of WT hematopoietic cells after transplantation (WT: 0.176 ± 0.078%; T1KO: 0.131 ± 0.046% p < 0.05). Conversely, no significant difference was observed in mice with CS-reduced stroma. To reveal the effect of CS in hematopoietic cells on long-term reconstructive function, we evaluated the chimerism of PB Gr1+CD11b+cells, B220+ cells, CD3+ cells, and BM LSK cells by a CRU assay. In the first transplantation, there were no significant differences in short-term reconstitution (after 6 weeks) and long-term reconstitution (after 12 weeks). In the second transplantation, hematopoietic cells derived from T1KO mice had lower chimerism in all PB cell lineages. Next, we evaluated the role of CS on the stromal microenvironment by serial transplantation. In the first transplantation, there were no significant differences between PB and BM cells. In the second transplantation, the proportion of BM LSK cells was higher in T1KO recipient mice (CS deficiency in the stroma). Conclusion: CS may have an important role in hematopoiesis. CS in hematopoietic cells and the stromal microenvironment had different effects on BM hematopoiesis. Disclosures No relevant conflicts of interest to declare.

The crystal structure of an uncharacterized protein TTHA0061 from Thermus thermophilus HB8, was determined and refined to 1.8 A by a single wavelength anomalous dispersion (SAD) method. The structural analysis and comparison of TTHA0061 with other existing structures in the Protein Data Bank (PDB) revealed a novel fold, suggesting that this protein may belong to a translation initiation factor or ribosomal protein family. Differential scanning calorimetry analysis suggested that the thermostability of TTHA0061 increased at pH ranges of 5.8–6.2, perhaps due to the abundance of glutamic acid residues.

Summary Sister chromatid separation is initiated at anaphase onset by the activation of separase, which removes cohesins from chromosomes. However, it remains elusive how sister chromatid separation is completed along the entire chromosome length. Here we found that, during early anaphase in Saccharomyces cerevisiae, sister chromatids separate gradually from centromeres to telomeres, accompanied by regional chromosome stretching and subsequent recoiling. The stretching results from residual cohesion between sister chromatids, which prevents their immediate separation. This residual cohesion is at least partly dependent on cohesins that have escaped removal by separase at anaphase onset. Meanwhile, recoiling of a stretched chromosome region requires condensins and generates forces to remove residual cohesion. We provide evidence that condensins promote chromosome recoiling directly in vivo, which is distinct from their known function in resolving sister chromatids. Our work identifies residual sister chromatid cohesion during early anaphase and reveals condensins' roles in chromosome recoiling, which eliminates residual cohesion to complete sister chromatid separation., Graphical Abstract Highlights ► Sister chromatid cohesion partially persists after anaphase onset in budding yeast ► Residual cohesion is partly dependent on cohesins and causes chromosome stretching ► Condensins directly promote recoiling of stretched chromosomes during early anaphase ► Chromosome recoiling generates forces to remove residual sister chromatid cohesion

Since the realisation that the antigen-binding regions of antibodies, the variable (V) regions, can be uncoupled from the rest of the molecule to create fragments that recognise and abrogate particular protein functions in cells, the use of antibody fragments inside cells has become an important tool in bioscience. Diverse libraries of antibody fragments plus in vivo screening can be used to isolate single chain variable fragments comprising VH and VL segments or single V-region domains. Some of these are interfering antibody fragments that compete with protein-protein interactions, providing lead molecules for drug interactions that until now have been considered difficult or undruggable. It may be possible to deliver or express antibody fragments in target cells as macrodrugs per se. In future incarnations of intracellular antibodies, however, the structural information of the interaction interface of target and antibody fragment should facilitate development of binding site mimics as small drug-like molecules. This is a new dawn for intracellular antibody fragments both as macrodrugs and as precursors of drugs to treat human diseases and should finally lead to the removal of the epithet of the 'undruggable' protein-protein interactions.

Kinetochore capture and transport by spindle microtubules plays a crucial role in high-fidelity chromosome segregation, although its detailed mechanism has remained elusive. It has been difficult to observe individual kinetochore–microtubule interactions because multiple kinetochores are captured by microtubules during a short period within a small space. We have developed a method to visualize individual kinetochore–microtubule interactions in Saccharomyces cerevisiae, by isolating one of the kinetochores from others through regulation of the activity of a centromere. We detail this technique, which we call ‘centromere reactivation system’, for dissection of the process of kinetochore capture and transport on mitotic spindle. Kinetochores are initially captured by the side of microtubules extending from a spindle pole, and subsequently transported poleward along them, which is an evolutionarily conserved process from yeast to vertebrate cells. Our system, in combination with amenable yeast genetics, has proved useful to elucidate the molecular mechanisms of kinetochore–microtubule interactions. We discuss practical considerations for applying our system to live cell imaging using fluorescence microscopy.

Sapovirus (SaV) is an important pathogen of human acute gastroenteritis. A gastroenteritis outbreak occurred at a wedding hall in October 2007 in Ehime Prefecture, Japan. One hundred nine people who had either attended wedding parties or had eaten a box lunch at a conference held at the same hall complained of gastroenteritis symptoms. Among these 109 people, stool specimens from 56 patients were available for pathogen screening, and 20 (35.7%) of these specimens were positive for SaV, of whom 18 showed symptoms. The numbers of cDNA copies of the specimens ranged from 2.36 x 10(6) to 3.03 x 10(10) for symptomatic patients, and 2.19 x 10(6) and 1.18 x 10(9) per gram of stool for two asymptomatic food handlers. The incubation periods of the 18 symptomatic patients ranged from 14.5 to 99.5 hr. Identical nucleotide sequence types of SaV; that is, a single synonymous nucleotide difference (transition) or microheterogeneity, was detected in stool specimens from the symptomatic patients and the asymptomatic food handlers, with the direct nucleotide sequence of approximately 2.3 kb 3' end of the genome. Based on the phylogenetic analysis with the complete capsid nucleotide sequence, these strains were clustered into genogroup IV. This outbreak was thought to be caused by a single source, and underscores the importance of proper hygiene in the environment and/or in food-handling practices to control SaV outbreaks.