DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Bovine Lactoferrin Promotes Neurite Outgrowth in PC12 Cells via the TrkA Receptor.

Lactoferrin (LF) is a multifunctional protein abundant in breast milk that modulates the functions of neural stem cells. Recent studies have demonstrated the efficacy of bovine LF (bLF) in mitigating behavioral changes; however, the molecular mechanisms on the nervous system have not yet been elucidated. The presented study aimed to characterize the molecular mechanisms of bLF on nerve extension in PC12 cells. PC12 cells were treated with 0.01-1000 µg/mL of bLF, and cell viability was determined using the cell counting kit-8 assay after treatment for 24 h. Morphometric evaluation was performed after 24 or 72 h of treatment with 50 ng/mL nerve growth factor (NGF) or 100-500 µg/mL bLF. The molecular mechanisms were investigated using Western blotting and real-time quantitative PCR. Cell viability was significantly decreased after treatment with 600-1000 µg/mL bLF for 24 h compared with the control group. Morphometric evaluation revealed neurite outgrowth after 72 h of NGF treatment, with a significant increase in neurite outgrowth after treatment with 250 µg/mL bLF. The phosphorylated p44/42 expression ratio peaked at 5 min and persisted for up to 10 min. Quantitative real-time PCR revealed a significant decrease in MAP2 expression. Our findings suggested that bLF enhanced PC12 cell neurite outgrowth to a similar extent as NGF. These effects are thought to be mediated via the TrkA receptor and activated by the phosphorylated ERK signaling pathway. Therefore, this study demonstrates that bLF promotes neurite outgrowth via a pathway similar to that of NGF.

Host-Shift Speciation Proceeded with Gene Flow in Algae Covering Shells.

AbstractHost shifts represent the advancement of a novel niche and often lead to speciation in symbionts. However, its mechanisms are not well understood. Here, we focused on the alga Pseudocladophora conchopheria growing on the shells of intertidal snails. Previous surveys have shown that the alga has host specificity-only attaching to the shell of Lunella correensis-but we discovered that the alga attaches to the shells of multiple sympatric snails. A genome-wide single-nucleotide polymorphism analysis (MIG-seq) was performed to determine whether host-associated speciation occurred in the algae. As a result, there was no gene flow or limited gene flow among the algae from different hosts, and some algae were genetically differentiated among hosts. In addition, the demographic estimate revealed that speciation with gene flow occurred between the algae from different hosts. Therefore, these results support the idea that host-shift speciation gradually proceeded with gene flow in the algae, providing insight into the early evolution of host shifts.

[Fatal invasive pneumococcal disease developed 14 years after allogeneic hematopoietic stem cell transplantation in a patient with myelodysplastic syndrome].

Invasive pneumococcal diseases (IPDs) after allogeneic hematopoietic stem cell transplantation have high fatality rates and often develop late after transplantation. The patient was a 58-year-old female. Fourteen years ago, she underwent bone marrow transplantation from a HLA-DR 1-antigen mismatched unrelated donor for myelodysplastic syndrome. She developed pneumonia, chronic graft-versus-host disease, and hypogammaglobulinemia. She received 23-valent pneumococcal capsular polysaccharide vaccine 11 and 6 years earlier. She was presented to our emergency room with fever. Her blood culture was positive for pneumococcus, and she was diagnosed with an IPD. The patient received antibiotic treatment but died on the third day of hospitalization. Because of its seriousness, pneumococcal infection should receive attention even 10 or more years after transplantation. Preventive approaches such as vaccination and early intervention at the time of diagnosis are important.

Clinical characteristics and pregnancy outcomes of cases with an incarcerated gravid uterus.

This study aimed to clarify the appropriate management of gravid uterus incarceration through a retrospective analysis of 10 women with an incarcerated gravid uterus who delivered at our hospital between 2000 and 2019. The incidence of an incarcerated gravid uterus was one in 2000 cases. Nine cases were diagnosed during pregnancy (15-30 gestational weeks) and one during caesarean section (performed at 37 gestational weeks on suspicion of placenta previa). Two women underwent manual reduction at 19 and 20 gestational weeks, respectively; the procedure failed in one case, and intrauterine foetal death occurred after the procedure. Among the remaining eight cases, spontaneous reduction was observed in three women at 31, 33 and 34 gestational weeks, respectively. The prevalence of incarcerated gravid uteri may be higher than that previously reported, cases may more likely recover spontaneously, and spontaneous reduction could occur in the third trimester.Impact StatementWhat is already known on this subject? Gravid uterine incarceration is a rare condition that occurs in one in 3000-10,000 cases. In addition, previous reports have suggested that manual reduction should not be attempted after the 20th week of gestation.What do the results of this study add? In the current study, the incidence of an incarcerated gravid uterus was one in 2000 cases, higher than that mentioned in previous reports. In addition, spontaneous reductions occurred in three of eight cases in which manual reductions were not performed at 31, 33 and 34 gestational weeks.What are the implications of these findings for clinical practice and/or further research? The prevalence of an incarcerated gravid uterus and the rate of spontaneous reduction might be higher than those reported in previous studies, and spontaneous reduction could occur in later gestational weeks than that previously reported. Further prospective multicentre studies are necessary to establish new evidence for the management of gravid uterine incarceration.

[Successful outcome of allogeneic hematopoietic stem cell transplantation for systemic mastocytosis complicated with acute myeloid leukemia].

A 60-year-old woman developed a fifth thoracic spine fracture with progressive paraplegia and underwent posterior spine fusion in June 2018. Based on the histopathological analysis of the surgical specimen, she was diagnosed with KIT D816V-positive systemic mastocytosis (SM). In June 2019, peripheral blood examination revealed remarkable eosinophilia. She was given prednisolone, which resulted in the resolution of eosinophilia. In May 2020, she developed acute myeloid leukemia (AML). Induction therapy was initiated and complete remission achieved. Subsequently, she received one course of consolidation therapy and allogeneic hematopoietic stem cell transplantation (allo-SCT). Although the residual mast cell tumor aggravated during chemotherapy for AML, the tumor regressed after allo-SCT, suggesting a graft-versus-mastocytosis effect. Nine months after the transplantation, the patient is alive and healthy without recurrence of AML and SM.

Towards understanding the effect of cedar roof covering application on firebrand production in large outdoor fires.

Due to the increased concern for the environment, sustainable construction materials are getting increased attention. Wood is considered a renewable, sustainable construction material. The problem with wood is that it is a fire-prone material. With an increasing number of wildland-urban interface (WUI) fires recently, it is important to consider that wood buildings are in danger to be ignited as well as may produce new sources of ignition due to firebrand production. Experiments were performed to investigate cedar roof covering effects of firebrand production from roof assemblies. Two wind speeds, 6 m/s and 8 m/s were selected for this experiment for a comparison with literature. The wood materials used were untreated cedar shingles, untreated cedar shakes, fire retardant cedar shingles and fire retardant cedar shakes. The roof assembly with fire retardant cedar shakes applied produced little or no firebrands under both wind speeds tested. Roof coverings fitted with cedar siding produced more variety of firebrands compared to only base sheathing materials. The mass and the projected area of firebrands were found to have a linear relationship. The firebrand coefficient was used for comparison. With attendant increase in wind speed, the firebrand coefficient increased in this study.

Role of Firebrand Combustion in Large Outdoor Fire Spread.

Large outdoor fires are an increasing danger to the built environment. Wildfires that spread into communities, labeled as Wildland-Urban Interface (WUI) fires, are an example of large outdoor fires. Other examples of large outdoor fires are urban fires including those that may occur after earthquakes as well as in informal settlements. When vegetation and structures burn in large outdoor fires, pieces of burning material, known as firebrands, are generated, become lofted, and may be carried by the wind. This results in showers of wind-driven firebrands that may land ahead of the fire front, igniting vegetation and structures, and spreading the fire very fast. Post-fire disaster studies indicate that firebrand showers are a significant factor in the fire spread of multiple large outdoor fires. The present paper provides a comprehensive literature summary on the role of firebrand mechanisms on large outdoor fire spread. Experiments, models, and simulations related to firebrand generation, lofting, burning, transport, deposition, and ignition of materials are reviewed. Japan, a country that has been greatly influenced by ignition induced by firebrands that have resulted in severe large outdoor fires, is also highlighted here as most of this knowledge remains not available in the English language literature. The paper closes with a summary of the key research needs on this globally important problem.

Discovery of a Novel Class of State-Dependent NaV1.7 Inhibitors for the Treatment of Neuropathic Pain.

The discovery of a novel class of state-dependent voltage-gated sodium channel (NaV)1.7 inhibitors is described. By the modification of amide or urethane bond in NaV1.7 blocker III, structure-activity relationship studies that led to the identification of novel NaV1.7 inhibitor 2i (DS01171986) were performed. Compound 2i exhibited state-dependent inhibition of NaV1.7 without NaV1.1, NaV1.5 or human ether-a-go-go related gene (hERG) liabilities at concentrations up to 100 µM. Further biological profiling successfully revealed that 2i possessed potent analgesic properties in a murine model of neuropathic pain (ED50: 3.4 mg/kg) with an excellent central nervous system (CNS) safety margin (> 600 fold).

Garnering understanding into complex firebrand generation processes from large outdoor fires using simplistic laboratory-scale experimental methodologies.

A simple laboratory-scale experimental method was developed to study firebrand generation processes. As part of these experiments, Japanese wind facilities were used to elucidate the effect of wind speed on firebrand generation from structural materials. It was found that very simple experimental methodologies developed as part of this study for mock-ups of full-scale roofing assemblies yielded important understanding into firebrand generation processes for both real-scale structure combustion processes as well as available firebrand information from urban and wildland-urban interface (WUI) fires.

Spindle pole body movement is affected by glucose and ammonium chloride in fission yeast.

The complexity of chromatin dynamics is orchestrated by several active processes. In fission yeast, the centromeres are clustered around the spindle pole body (SPB) and oscillate in a microtubule- and adenosine triphosphate (ATP)-dependent manner. However, whether and how SPB oscillation are affected by different environmental conditions remain poorly understood. In this study, we quantitated movements of the SPB component, which colocalizes with the centromere in fission yeast. We found that SPB movement was significantly reduced at low glucose concentrations. Movement of the SPB was also affected by the presence of ammonium chloride. Power spectral analysis revealed that periodic movement of the SPB is disrupted by low glucose concentrations. Measurement of ATP levels in living cells by quantitative single-cell imaging suggests that ATP levels are not the only determinant of SPB movement. Our results provide novel insight into how SPB movement is regulated by cellular energy status and additional factors such as the medium nutritional composition.

Investigating Effect of Wind Speeds on Structural Firebrand Generation in Laboratory Scale Experiments.

Firebrands generated from structures are known to be a source of rapid flame spread within communities in large outdoor fires, such as wildland-urban (WUI) fires, and urban fires. It is important to better understand firebrand generation mechanism to prevent structure ignitions by firebrands. Though the wind plays an important role during the large outdoor fires, little known is the influence of wind speeds on firebrand production. To this end, a series of experiments were performed using mock-ups of full-scale wall assemblies exposed to wind. The objective of this study was to examine if experiments with mock-ups of full-scale wall assemblies may provide insight into firebrand generation from structures. Specifically, generated firebrands were collected and compared with those collected from full-scale components and a full-scale structure. The relationship between projected area and mass of firebrands were compared with previous experimental data. It was found that the projected area of firebrands was proportional to the firebrand mass in this study, which is the same as those from experimental studies performed for full-scale components and a full-scale structure. The slope of the relationship of the projected area and the mass of firebrands was the same under the same wind speed and was affected by the applied wind speed within this experimental range.

Influence of Board Spacing on Mitigating Wood Decking Assembly Ignition.

As part of recent building code change discussions, it has been suggested that by increasing the spacing of boards, it may be possible to mitigate ignition of wood decking assemblies from wind-driven firebrand showers. An experimental series was undertaken to vary the board spacing from 0 mm (no gaps), 5 mm, and 10 mm, to determine if it was possible to observe reduced ignition propensity of full-scale wood decking assemblies fitted to a reentrant corner wall assembly. In these experiments, three common wood types were used and firebrand showers were directed at the wall/decking assemblies using wind speeds of 8 m/s generated using a realistic-scale wind tunnel. Based on the results of these experiments, it was observed that board spacing significantly influenced ignition propensity of these assemblies. Ignition events were observed for all board spacing considered and in particular, more ignition points were observed for a board spacing of 10 mm.

Initial Study on Thatched Roofing Assembly Ignition Vulnerabilities to Firebrand Showers.

Structures fitted with thatched roofing assemblies are prone to ignition during the course of large outdoor fires. Experiments with thatched roofing assemblies were performed by using a reduced-scale continuous-feed firebrand generator in a wind facility to investigate fundamental ignition mechanisms. The wind speed was varied from 3 m/s to 6 m/s to observe the ignition and flame spread of thatched roofing assemblies. It was observed that firebrands penetrated into the thatched roofing assembly, sometimes unseen from the outside, resulting in ignition and ultimately rapid flame penetration. Information obtained in this study would be useful to evaluate and develop effective counter measures to protect historical structures with thatched roofing assemblies, especially for historical buildings, such as The United Nations Educational, Scientific and Cultural Organization (UNESCO)'s world heritage sites in Japan.

[Chronic myeloid leukemia harboring T315I and F317L mutations successfully treated with interferon-α and ponatinib].

Our patient was diagnosed with chronic myeloid leukemia (CML) in chronic phase (CP) when he was 40 years old. Although dasatinib (DAS) was prescribed during his clinical course, he was poorly compliant with the treatment. In November 20XX, at 65 years of age, he visited our hospital with leukocytosis. He was diagnosed with CML in CP and recommenced DAS at 50 mg/day, achieving a complete hematological response after 2 months. However, DAS was increased to 100 mg/day because only minimum cytogenetic response was evident even after 9 months, but CML progressed to the accelerated phase after 18 months. The ABL kinase domain mutations T315I and F317L were detected. Ponatinib (PON) was not yet approved, and he declined allogeneic stem cell transplantation therapy. He commenced interferon-α (IFN-α) in addition to DAS, and the F317L mutation (only) disappeared after 7 months; the patient achieved a major cytogenetic response. In January 20XX＋4, he commenced PON monotherapy (the drug was approved by this time) and achieved a major molecular response after 8 months. The T315I mutation disappeared during PON therapy. Although IFN-α is rarely used in the treatment of CML, this case suggests that IFN-α should be re-considered in patients with CML who exhibit tyrosine kinase inhibitor resistance.

Infraorbital Nerve Located Medially to Postoperative Maxillary Cysts: A Risk of Endonasal Surgery.

BACKGROUND/AIMS: This study aimed to examine variations in the location of the infraorbital nerve relative to postoperative maxillary cysts to assess the potential risk of nerve injury during endonasal marsupialization. METHODS: Coronal computed tomography images of 130 patients (162 sides) with postoperative maxillary cysts who visited our clinic between 2003 and 2014 were reviewed from the viewpoint of the anatomical relationship between the infraorbital nerves and cysts. RESULTS: The proportions of the six locations were as follows: upside 45.1% (n = 73), separate 13.0% (n = 21), medial 5.6% (n = 9), lateral 14.2% (n = 23), in-between 7.4% (n = 12), and unevaluable 14.8% (n = 24). The proportion of the cases with a potential risk of infraorbital nerve damage during endoscopic marsupialization, including medial, in-between, and unevaluable locations, was 27.8%. Retrospective chart review revealed that 2 patients with a postoperative maxillary cyst that were unevaluable complained of persistent postoperative hypoesthesia of the cheek. CONCLUSION: The anatomical relationship between the infraorbital nerve and postoperative maxillary cysts varied among patients, with approximately one-fourth of the patients being at risk of infraorbital nerve injury even during endoscopic procedures.

Summary of Workshop Large Outdoor Fires and the Built Environment.

Large outdoor fires present a risk to the built environment. Wildfires that spread into communities, referred to as Wildland-Urban Interface (WUI) fires, have destroyed communities throughout the world, and are an emerging problem in fire safety science. Other examples are large urban fires including those that have occurred after earthquakes. Research into large outdoor fires, and how to potentially mitigate the loss of structures in such fires, lags other areas of fire safety science research. At the same time, common characteristics between fire spread in WUI fires and urban fires have not been fully exploited. In this paper, an overview of the large outdoor fire risk to the built environment from each region is presented. Critical research needs for this problem in the context offire safety science are provided. The present paper seeks to develop the foundation for an international research needs roadmap to reduce the risk of large outdoor fires to the built environment.

[Acquisition of IgH/CCND1 translocation during the natural disease course in a patient with chronic lymphocytic leukemia].

A 69-year-old man visited a doctor because of systemic lymphadenopathy. Peripheral blood examination revealed leukocytosis, anemia, and decreased platelet count (WBC, 103,060/µl; lymph, 92.2%; Hb, 8.9 g/dl; and Plt, 4.1×104/µl). Bone marrow biopsy revealed that approximately 70% of nucleated cells were small, mature lymphoid cells with positive immunostaining for CD5, CD20, and CD23. He was diagnosed with chronic lymphocytic leukemia (CLL). The IgH/CCND1 translocation and ATM locus loss in 20% and 95% peripheral cells, respectively, were detected by fluorescence in situ hybridization. Immunostaining revealed that cyclin D1 was positive in approximately 30% bone marrow cells. As the positive rate of CCND1 fusion signal was low, the diagnosis of mantle cell lymphoma was excluded. In contrast, signals of ATM locus deletion were detected in most tumor cells. Therefore, we assessed that IgH/CCND1 translocations occurred during the natural clinical course of CLL with ATM locus deletion from the onset of disease. The secondary IgH/CCND1 translocation in CLL is rare, and all reported cases with such translocations received treatments with alkylating agents. This is the first report regarding secondary IgH/CCND1 translocation during the natural clinical course of CLL and may provide insights into CLL pathogenesis.

Experimental Investigation of Firebrand Accumulation Zones in Front of Obstacles.

It is well accepted that as structures are exposed to wind, stagnation planes are produced around structures. Past work by the authors demonstrated for the first-time that wind-driven firebrand showers may accumulate in these stagnation planes. While those experiments demonstrated this important phenomenon, due to the limited duration of firebrand showers of the original NIST Batch-Feed Firebrand Generator, it was not possible to perform a more systematic study. To this end, a series of detailed experiments were performed using the recently developed NIST Continuous-Feed Firebrand Generator capable of firebrand showers of unlimited duration. Full-scale walls of varying size were placed downstream of the device and the wind speed was varied in increments up to 10 m/s. The experiments were conducted in the Building Research Institute's Fire Research Wind Tunnel Facility (FRWTF). For a given wall size exposed to specific firebrand size/mass distribution, it was observed that wind speed influences not only the spatial location and extent of the accumulated firebrands in the stagnation plane in front of the wall, but also the nature of the smoldering combustion intensity of the accumulated firebrands. The experiments demonstrated that higher wind speeds (10 m/s) did not promote accumulation of firebrands in stagnation planes in front of walls. The data may be used to provide guidance to appropriate separation distances that combustibles should be placed near structures and is also of great use to develop and validate numerical models of firebrand accumulation.