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Post-bisulfite adaptor tagging (PBAT) is a concept that enables the preparation of an efficient sequencing library from bisulfite-treated DNA, and it also means the protocol implemented the concept. Although the previous PBAT or rPBAT was sensitive enough for single-cell methylome analysis, the protocol had several drawbacks owing to the repeated random priming reactions. To resolve these problems, we developed a unique single-strand DNA ligation technique, termed TACS ligation, and established a new protocol called tPBAT. With tPBAT, the data quality improved, with a longer insert and higher mapping rate than that obtained with rPBAT. In addition, paired-end sequencing and indexing were supported by the default. In this chapter, the tPBAT protocol is introduced, and a thorough description of its application to small samples is provided.

BACKGROUND The role and impact of radiation therapy (RT) on the development of herpes zoster (HZ) has not been well studied. The objective of this study was to investigate the association between RT and HZ. METHODS A propensity score-matched, retrospective cohort study was conducted using institutional cancer registry data and medical records from 2011 to 2015. The risk of developing HZ in the RT and non-RT groups was compared using a Cox proportional hazards model. Associations also were explored between the RT field and the anatomic location of HZ in patients who developed HZ after RT. The expected number of HZ events within the radiation field was calculated according to the RT received by each patient; then, this number was compared with the observed number of in-field events. RESULTS Of 17,655 patients, propensity score matching yielded 4350 pairs; of these, 3891 pairs were eligible for comparison. The cumulative incidence of HZ in the RT group (vs the non-RT group) during the first 5 years after the index date was 2.1% (vs 0.7%) at 1 year, 3.0% (vs 1.0%) at 2 years, 3.4% (vs 1.3%) at 3 years, 4.1% vs 1.7% at 4 years, and 4.4% vs 1.8% at 5 years. The RT group showed a significantly higher risk of HZ than the non-RT group (hazard ratio, 2.59, 95% CI, 1.84-3.66). In the 120 patients who developed HZ after RT, HZ events were observed significantly more frequently within the RT field than expected (74 vs 43.8 events; P

Background Hematologic malignancies occasionally cause serous retinal detachment (SRD); however, its pathogenesis remains unclear. Here we present the imaging characteristics of metastatic choroidal lymphoma masquerading as Vogt-Koyanagi-Harada (VKH) disease. Case presentation A 45-year-old Japanese woman was referred to our clinic because of bilateral SRD with blurred vision. Fluorescein angiography revealed multiple pinpoint leakage followed by pooling OU. Enhanced depth imaging optical coherence tomography showed marked choroidal thickening OU. Laser speckle flowgraphy detected choroidal circulation impairment OU. Although these results totally agreed with the inflammatory manifestations of acute VKH disease, indocyanine green angiography demonstrated various sizes of sharply marginated hypofluorescent lesions that seemed atypical for the finding of VKH disease, i.e., vaguely marginated hypofluorescent small dots. Cerebrospinal fluid pleocytosis was not detected. Blood tests revealed leukocytosis together with elevation of lactate dehydrogenase and soluble interleukin-2 receptor levels. Corticosteroid pulse therapy did not improve any ocular findings. Bone marrow biopsy was then performed, leading to a definite diagnosis of diffuse large B-cell lymphoma. After starting systemic chemotherapy, both SRD and choroidal thickening resolved rapidly with visual recovery. However, choroidal hypoperfusion persisted, which contrasted distinctly with the inflammatory pattern of VKH disease, i.e., the restoration of choroidal blood flow in parallel with normalization of choroidal thickness. Conclusions Our detailed multimodal observations highlighted the differential imaging features of choroidal lymphoma despite close resemblance to VKH disease especially at the initial stage. Impaired circulation in the thickened choroid marked the pseudo-inflammatory pathogenesis of SRD due to choroidal involvement with neoplastic, but not inflammatory cells.

Whole-genome bisulfite sequencing (WGBS) is the current gold standard of methylome analysis. Post-bisulfite adaptor tagging (PBAT) is an increasingly popular WGBS protocol because of high sensitivity and low bias. PBAT originally relied on two rounds of random priming for adaptor-tagging of single-stranded DNA (ssDNA) to attain high efficiency but at a cost of library insert length. To overcome this limitation, we developed terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation as an alternative to random priming. In this method, TdT attaches adenylates to the 3′-end of input ssDNA, which are then utilized by RNA ligase as an efficient connector to the ssDNA adaptor. A protocol that uses TACS ligation instead of the second random priming step substantially increased the lengths of PBAT library fragments. Moreover, we devised a dual-library strategy that splits the input DNA to prepare two libraries with reciprocal adaptor polarity, combining them prior to sequencing. This strategy ensured an ideal base–color balance to eliminate the need for DNA spike-in for color compensation, further improving the throughput and quality of WGBS. Adopting the above strategies to the HiSeq X Ten and NovaSeq 6000 platforms, we established a cost-effective, high-quality WGBS, which should accelerate various methylome analyses.

Letter to the editor

Section 4 of the FM14 focus on the outreach action and advocacy in the context of IAUs 2020-2030 Strategic Plan. This paper also contains supplementary materials that point to contributed talks and poster presentations that can be found online.

Next-generation sequencing of single-stranded DNA (ssDNA) is attracting increased attention from a wide variety of research fields. Accordingly, various methods are actively being tested for the efficient adaptor-tagging of ssDNA. We conceived a novel chemo-enzymatic method termed terminal deoxynucleotidyl transferase (TdT)-assisted, copper-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated ssDNA ligation (TCS ligation). In this method, TdT is used to incorporate a single 3'-azide-modified dideoxyribonucleotide onto the 3'-end of target ssDNA, followed by CuAAC-mediated click ligation of the azide-incorporated 3'-end to a 5'-ethynylated synthetic adaptor. This report presents the first proof-of-principle application of TCS ligation with its use in the preparation of a next-generation sequencing library.

Purpose: Although stereotactic body radiation therapy is one of the standard treatments for stage I nonsmall cell lung cancer, in the case of central tumors it carries the risk of severe adverse events for serial organs. Accelerated hypofractionated radiotherapy is considered a reasonable alternative to treat central tumors. We have been treating central tumors with accelerated hypofractionated radiotherapy using a 75 Gy/25 fr/5 weeks regimen, and we compared the results with those of stereotactic body radiation therapy using 48 Gy/4 fr/1 week. Methods: Patients with central tumors and/or unfit for 1-hour fixation were candidates for accelerated hypofractionated radiotherapy. Based on the proximity to the biologically effective dose at 10 Gy, above accelerated hypofractionated radiotherapy regimen was adopted. Results: From October 2003 to December 2010, 159 patients, who received either accelerated hypofractionated radiotherapy (103 cases) or stereotactic body radiation therapy (56 cases), were included in the analysis. In the accelerated hypofractionated radiotherapy group, 40 (39%) cases were central tumors, whereas all cases were peripheral tumors in the stereotactic body radiation therapy group. Overall 5-year local control and survival rates were 81.9% (95% confidence interval 73.6%-90.1%) and 46.5% (95% confidence interval 36.7%-56.2%), respectively for the accelerated hypofractionated radiotherapy group, and 75.4% (95% confidence interval 63.0%-87.8%) and 44.6% (95% confidence interval 31.6%-57.7%), respectively for the stereotactic body radiation therapy group (n.s.). Among central tumors, ultracentral tumors (21 cases) and the remaining central tumors (19 cases) were similar in both local control and survival. On multivariate analysis, hazard ratios for accelerated hypofractionated radiotherapy versus stereotactic body radiation therapy were

The relationships between large-scale circulation and foehns observed during midsummer in Hokuriku district, located on the Japan Sea side of central Japan, are examined using Japanese long-term reanalysis project data, with additional data from the Japan Meteorological Agency climate data assimilation system. All foehn events are classified into two types: a tropical cyclone (TC)-induced foehn and an extratropical cyclone (EC)induced foehn. The occurrence of the TC type is attributed to a combination of a typhoon and its induced teleconnection pattern, the Pacific-Japan (PJ) pattern, in the lower troposphere. Local intensification of the North Pacific high just east of Japan, accompanied by the dominance of the PJ pattern, can in turn force a typhoon track to shift westward. The northward migration of the typhoon along the western periphery of the locally enhanced high strengthens a zonal pressure gradient across central Japan, thus producing a foehn. In contrast, an upper-level teleconnection along the Asian jet serves as a prominent trigger of the occurrence of an EC-type foehn. Stationary Rossby wave packets propagating eastward along the upper-level Asian waveguide facilitate not only the westward development of the North Pacific high but also the development of an extratropical cyclone in the vicinity of the Japan Sea by leading to the equatorward advection of higher potential vorticity from high latitudes. Both developments are crucial for the reinforcement of a northwest-southeast pressure gradient in the lower troposphere around Japan, thus providing a favorable condition for a foehn event.

Background The loop-mediated isothermal amplification (LAMP) method is a novel technique for the amplification of specific DNA sequences. Objectives To establish the LAMP method for amplifying Epstein–Barr virus (EBV) DNA and to examine its reliability for the detection of EBV DNA in clinical specimens. Study design Sera from 108 patients, who were initially suspected of primary EBV infection, were tested by the EBV LAMP method, and the results were compared with those of the real-time PCR assay. Serological examination was regarded as the standard diagnostic method. Results To diagnose primary EBV infection, the sensitivity of LAMP was 86.4% and the specificity was 100%. The sensitivity of the real-time PCR assay was 84.1% and the specificity was 98.4%. Longitudinal analysis showed that the detection rate of EBV DNA in serum by the LAMP method decreased with time in accordance with the decrease of the EBV load. EBV DNA could not be detected in serum 40 days after onset of symptoms. Conclusions These results indicate that the sensitivity and specificity of the LAMP method are comparable to those of real-time PCR and that detecting EBV DNA in serum by this method is potentially useful for diagnosing primary EBV infection.

Epstein-Barr virus (EBV) is known to be a causative agent of hemophagocytic lymphohistiocytosis (HLH). To investigate association of apoptosis in the pathogenesis of EBV-associated HLH, the serum EBV loads, and serum concentrations of soluble tumor necrosis factor receptor 1 (sTNF-R1), soluble Fas ligand, and cytochrome c were examined in 15 patients with EBV-associated HLH and 24 patients with infectious mononucleosis (IM). Levels of sTNF-R1 are known to reflect the biological activity of TNF-alpha and cytochrome c is a specific marker of apoptosis. EBV loads, and concentrations of sTNF-R1 and cytochrome c were significantly higher in patients with EBV-associated HLH than in patients with IM. On the other hand, there were no statistically significant differences in the concentrations of soluble Fas ligand. In patients with EBV-associated HLH, EBV loads, concentrations of sTNF-R1, and cytochrome c were correlated with each other. These results suggest that apoptosis, which is dependent on the EBV load and could be mediated by TNF-alpha, plays a major role in the pathophysiology of EBV-associated HLH.

Chronic active Epstein–Barr virus (EBV) infection is a severe systemic disease associated with high rates of mortality and morbidity. Recent studies suggest that the clonal expansion of EBV-infected T or natural killer cells plays a crucial role in the pathogenesis of chronic active EBV infection. However, it is not clear whether chronic active EBV infection is truly a monoclonal disorder that originates from one cell. The clonality of EBV was investigated by sequence variation of the latent membrane protein 1 (LMP1) gene, which has a high degree of sequence heterogeneity. Peripheral blood mononuclear cells were obtained from nine Japanese patients with chronic active EBV infection and four with infectious mononucleosis. A carboxyl-terminal region of the LMP1 gene was analyzed by polymerase chain reaction (PCR). The amplified PCR products were subcloned, and 18 clones from each sample were sequenced. Patients with chronic active EBV infection each had two to five different LMP1 nucleotide sequences, whereas patients with infectious mononucleosis each had one to seven different sequences. Patients with chronic active EBV infection and infectious mononucleosis both had one dominant sequence. Longitudinal analysis was performed in four patients with chronic active EBV infection, in whom the dominant strains were found to have remained unchanged for several years. The results suggest that EBV in patients with chronic active EBV infection was polyclonal, although clonal expansion may occur. Collectively, these findings are critical to clarify further the pathogenesis of chronic active EBV infection and aid in the development of effective treatment strategies. J. Med. Virol. 78:770–779, 2006. © 2006 Wiley-Liss, Inc.

Infantile hepatitis is occasionally seen in apparently healthy children. In most cases, the etiology of the infection is uncertain. However, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV-7), human parvovirus B19, and TT virus (TTV) are considered to be associated with hepatitis in children. The objective of this study was to investigate the correlations between these viruses and infantile hepatitis. Twenty-six children from 1 to 24 months old (median age, 7 months) who had liver dysfunction of unknown etiology were enrolled in this study. Plasma samples were examined by a real-time PCR assay for CMV, EBV, HHV-6, HHV-7, parvovirus B19, and TTV DNA. The DNA of CMV was detected in the plasma of four patients (15.4%) and was detected significantly more often in the patient group than in the control group. The CMV-infected patients were 1 to 3 months old, which was significantly younger than the remaining patients. The serological findings did not always correlate with the results of the real-time PCR assay. The DNA of TTV was detected in four patients (15.4%), while human parvovirus B19 DNA was detected in three (11.5%). However, the detection frequencies of these viral DNAs were not significantly different from those in the control groups, and some of these patients had co-infections. These results indicate that CMV might be one of the major pathogens responsible for infantile hepatitis; however, serological tests have limited utility for the diagnosis of CMV infection in young children.

Ensuring the proper use of drugs is an important role of pharmacists in the medical team. In the present paper, we describe two support tools for this purpose made for cancer chemotherapy, one of them having the title “Basic Knowledge ofCancer Chemotherapy” and the other “Cancer Chemotherapy Work Sheet”. They are designed to help maintain safety incancer chemotherapy and can be used in many different hospitals. Our objectives in making these tools were to standardizepharmaceutical care and improve its quality, in the process of promoting the proper use of drugs. First, we prepared our Basic Knowledge of Cancer Chemotherapy sheet for 4 types of cancer (stomach cancer, ovary cancer, malignant lymph adenoma, lung cancer) and then g Cancer Chemotherapy Work Sheets for various chemotherapy regimens : Tegafur-gimeraciloteracil+Cisplatin (TS-1+CDDP) (2), Paclitaxel+Carboplatin (TAXOL+CBDCA) (2), Cyclophosphamide+Doxorubicin+Vincristine (CHOP), bi-weekly Cyclophosphamide+Doxorubicin+Vincristine (bi-weeklyCHOP), Gemcitabine+Cisplatin (GEM+CDDP), Weekly-Paclitaxel+Carboplatin (Weekly-TAXOL+CBDCA), Cisplatin+Etoposide (CDDP+VP16). We evaluated the Cancer Chemotherapy Work Sheets through a questionnaire sent to hospital pharmacists. Most pharmacists (91%) responded that the sheets were “useful” and over half (69%) thought that their purpose was “understandable”.In order to expand the use of our sheets among hospital pharmacists, we made them available through an internet website. We believe that these tools will contribute to the standardization of the pharmaceutical care in cancer chemotherapy and raising its quality.

Cytomegalovirus (CMV) and Epstein–Barr virus (EBV) usually cause primary and latent infections during childhood; thus, coinfection with these viruses occurs occasionally in children. However, its clinical impact has not been established, and may be underestimated. Three cases of coinfection involving these two viruses in toddlers are described: a 14-month-old male with infectious mononucleosis, an 18-month-old female with hemophagocytic lymphohistiocytosis, and a 13-month-old female with acute hepatitis. All three patients had prolonged illnesses. Serial serological testing and quantitation of viral DNA for CMV and EBV using peripheral blood from the patients suggested primary infections with both viruses. In all three cases, the viral load of EBV and CMV in the early stage of disease exceeded 6.4 × 103 and 8.8 × 102 copies/ml of whole blood, respectively, suggesting that the viruses were associated with the clinical condition. Recognizing that coinfection with these viruses may modulate the clinical course of disease is important. J. Med. Virol. 81:1399–1402, 2009. © 2009 Wiley-Liss, Inc.

BACKGROUND Chronic active Epstein-Barr virus (EBV) infection is characterized by recurrent infectious mononucleosis-like symptoms, and infected patients have high viral loads in their peripheral blood. Standard therapy for the disease has not yet been established. Recently, hematopoietic stem cell transplantation (HSCT) has been introduced and has the potential to become a standard treatment, although guidelines for HSCT to treat chronic active EBV infection have not yet been proposed. METHODS Fifteen patients were retrospectively analyzed, both clinically and virologically, to investigate the factors associated with prognosis of chronic active EBV infection treated with HSCT. RESULTS After HSCT, 7 patients died after survival periods that ranged from 1 to 16 months (mean duration of survival after HSCT, 5 months). Three patients were considered to have died of transplantation-related complications. The duration between infection onset and diagnosis was significantly longer in patients who died than in those who survived. Five of the 7 patients who died experienced > or =3 life-threatening complications. The plasma concentrations of interferon-gamma, interleukin-10, thrombomodulin, and soluble E-selectin did not differ significantly between the groups of patients. With regard to sequence variations in the EBV latent membrane protein 1 gene, no specific patterns were found in the patients who died. Importantly, the plasma EBV load at diagnosis was significantly higher in patients who died than in living patients. Moreover, plasma viral load was shown to be an important factor to monitor during follow-up for patients after HSCT. CONCLUSIONS The number of life-threatening complications and plasma viral load are indicative of the stage of disease progression and may be useful factors for predicting the outcome of HSCT.

Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus-6 (HHV-6) cause symptomatic diseases in liver transplant recipients. The loads of these viruses, the associations between viral DNAemia, serologic status, and acute rejection reactions were investigated in a group of 17 juvenile and 17 adult recipients of living donor liver transplantation (LDLT) for a median of 8 weeks posttransplantation. At least 1 plasma sample from 15/34 (44.1%) patients was positive for CMV DNA. For most of the CMV-positive patients, the CMV DNA appeared in the second week of LDLT, and disappeared by the eighth week. A minimum of 200 EBV DNA copies/mug peripheral blood mononuclear cell DNA (defined as positive for EBV) was detected in 5/34 (14.7%) patients, and the number of EBV-positive children was significantly greater than the number of EBV-positive adults. In most of the EBV-positive patients, the EBV loads increased after 4 weeks posttransplantation. Plasma HHV-6 was detected in 7/34 (20.6%) patients. HHV-6 DNA appeared for a short period from the second week of LDLT. In addition, 8 of the 19 virus-positive recipients carried 2 viruses, with the combination of CMV and HHV-6 being the most frequent. Serologic status seemed to be an important factor for all 3 viral infections. The rate of acute cellular rejection was not significantly higher in the CMV-, EBV-, or HHV-6-positive groups. Simultaneous monitoring for 3 herpesviruses revealed the impact of these viruses on LDLT recipients.

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by recurrent infectious mononucleosis-like symptoms and has high mortality and morbidity. To clarify the mechanisms of CAEBV, the gene-expression profiles of peripheral blood obtained from patients with CAEBV were investigated. Twenty genes were differentially expressed in 4 patients with CAEBV. This microarray result was verified using a real-time reverse-transcriptase polymerase chain reaction assay in a larger group of patients with CAEBV. Eventually, 3 genes were found to be significantly upregulated: guanylate binding protein 1, tumor necrosis factor-induced protein 6, and guanylate binding protein 5. These genes may be associated with the inflammatory reaction or with cell proliferation.

The JALT CALL Journal

Infections of T cells and natural killer (NK) cells play a central role in the pathogenesis of chronic active Epstein-Barr virus (CAEBV) infection. To characterize the virologic and cytokine profiles of T cell-type and NK cell-type infection, 39 patients with CAEBV infection were analyzed. Patients with T cell-type infection had higher titers of immunoglobulin G against early and late EBV antigens, suggesting lytic cycle infection. However, the pattern of EBV gene expression was latency type II; BZLF1, which is a hallmark of lytic cycle infection, could not be detected in any patients, regardless of infection type. Patients with CAEBV infection had high concentrations of proinflammatory, T helper cell type 1, and anti-inflammatory cytokines. The cytokine profile in patients with NK cell-type infection was similar to that in patients with T cell-type infection, but the concentration of IL-13 was high in patients with NK cell-type infection. These findings should help to clarify the pathogenesis of CAEBV infection and facilitate the development of more-effective treatments.

The role of virulence factor of elastolytic proteinase produced by Aspergillus fumigatus was investigated in mice immunocompromised with cyclophosphamide (CY) to block neutrophil and lymphocyte functions or with carrageenan (CA) to block the phagocytic activity of macrophages. These mice were infected through inhalation with the spores of elastolytic proteinase producing or nonproducing strains of A. fumigatus, and then were observed daily for 15 days or until death. Compared to CY-immunocompromised mice treated with the spores of proteinase nonproducing strains, CY-immunocompromised mice treated with the spores of proteinase producing strains had a significantly shorter survival rate. Furthermore, when treated with the spores of proteinase producing strains, CA-immunocompromised mice survived significantly longer than did CY-immunocompromised mice. Assessment of the effect of elastolytic proteinase on rat lung showed that this enzyme induced hemorrhage of the lung, alveolar septal edema and alveolar hypertrophy due to blood neutrophil infiltration. Neither fusion nor denaturation of the elastic fiber was observed, however. These studies have suggested that when infected with A. fumigatus, a severe lesion in bronchioli or alveoli is induced by the proteinase producing strains, and that these strains are more virulent than the proteinase nonproducing strains.