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Objective: Portable laser methane detectors (LMDs) may be an economical means of estimating CH4 emissions from ruminants. We validated an LMD-based approach and then used that approach to evaluate CH4 emissions from indigenous dairy cows in a dryland area of Ethiopia. Methods: First, we validated our LMD-based approach in Simmental crossbred beef cattle (n = 2) housed in respiration chambers and fed either a high- or low-concentrate diet. From the results of the validation, we constructed an estimation equation to determine CH4 emissions from LMD CH4 concentrations. Next, we used our validated LMD approach to examine CH4 emissions in Fogera dairy cows grazed for 8 h/d (GG, n = 4), fed indoors on natural-grassland hay (CG1, n = 4), or fed indoors on Napier-grass (Pennisetum purpureum) hay (CG2, n = 4). All the cows were supplemented with concentrate feed. Results: The exhaled CH4 concentrations measured by LMD were linearly correlated with the CH4 emissions determined by infrared-absorption-based gas analyzer (r2 = 0.55). The estimation equation used to determine CH4 emissions (y, mg/min) from LMD CH4 concentrations (x, ppm m) was y = 0.4259x+38.61. Daily CH4 emissions of Fogera cows estimated by using the equation did not differ among the three groups; however, a numerically greater milk yield was obtained from the CG2 cows than from the GG cows, suggesting that Napiergrass hay might be better than natural-grassland hay for indoor feeding. The CG1 cows had higher CH4 emissions per feed intake than the other groups, without significant increases in milk yield and body-weight gain, suggesting that natural-grassland hay cannot be recommended for indoor-fed cows. Conclusion: These findings demonstrate the potential of using LMDs to valuate feeding regimens rapidly and economically for dairy cows in areas under financial constraint, while taking CH4 emissions into consideration.

Clinical isolates of drug-resistant (isoniazid and/or rifampicin-resistant) Mycobacterium tuberculosis were obtained from 254 patients diagnosed with drug-resistant tuberculosis in Japan from April 2015 to March 2017 in National Hospital Organization hospitals. The 254 patients were approximately 32% of all 795 patients who were diagnosed with culture-confirmed drug-resistant tuberculosis from 2015 to 2016 nationwide in Japan. The whole-genome sequences of all the isolates from the 254 patients and the lineages of these isolates were determined, and phylogenetic trees were constructed based on single nucleotide polymorphism concatemers. Of these patients, 202 (79.5%) were born in Japan and 52 (20.5%) were born elsewhere. Of the 254 drug-resistant isolates, 54 (21.3%) were multidrug resistant, being resistant to both isoniazid and rifampicin. The percentages of multidrug-resistant isolates were significantly higher in foreign-born (38.5% [20/52]) than Japanese-born patients (16.8% [34/202]). Of the 54 multidrug-resistant isolates, nine were extensively drug resistant, which were all obtained from Japanese-born patients. Five extensively drug-resistant isolates were obtained from patients with incipient tuberculosis. A significant number of multidrug-resistant M. tuberculosis strains were isolated from foreign-born patients from Asian countries that have a high tuberculosis burden. Foreign-derived isolates affect the nationwide genetic diversity of drug-resistant M. tuberculosis in Japan. Extensively drug-resistant M. tuberculosis isolates were transmitted among the Japanese population. IMPORTANCE The incidence rate of tuberculosis (TB) in Japan was 11.5 per 100,000 of the population in 2019. Of TB patients in Japan, 61.1% were aged >70 years, and 10.7% were born outside Japan, mostly in Asian countries with a high burden of tuberculosis. Of the tuberculosis patients in the present study, 5.4% and 1.0% showed resistance to isoniazid and rifampicin, respectively, and 0.7% were multidrug resistant. The objective of this study was to clarify the molecular epidemiological properties of drug-resistant tuberculosis in Japan. Molecular epidemiology provides several clues to inform potential measures to control drug-resistant tuberculosis in Japan.

A new oligosaccharide citric acid derivative was isolated by hot aqueous extraction at 85 °C from Vigna angularis seeds, and it was purified using anion exchange chromatography DE-52. The structure was inferred by using UV, 1 D and 2 D NMR spectroscopy and HR-MALDI/MS, and identified as α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyle-(1→2)-β-D-fructofuranosyle citric acid. The isolated oligosaccharide citric acid derivative revealed promising anti-influenza A virus with 50% inhibition concentration (IC50) of 0.162 nM/mL and 50% cytotoxic dose (CC50) of >2.38 nM/mL.

Objective There has been little research on human herpesvirus 6B (HHV-6B) in healthy adults and prevalences in different age groups have been unclear. Therefore, the major objective of this study was to evaluate seroprevalence to HHV-6 antibodies in ordinary working people and examine the effect of aging on seroprevalence. Also, as HHV-6B is reactivated in saliva, another objective was to investigate an association between age and HHV-6B reactivation based on measured salivary HHV-6 DNA levels. Methods Our subjects were 77 ordinary office workers who underwent a health checkup. In this population, we measured anti-HHV-6 antibody titers using enzyme-linked immunosorbent assay and salivary HHV-6 DNA levels. In addition to examining an association with age, we examined associations with body mass index, smoking habit, and alcohol consumption as confounding factors. Results There was a significant decrease in the seropositivity of HHV-6 antibodies in subjects of 50 years and older, and age was significantly negatively correlated with anti-HHV-6 antibody titers. Age and salivary HHV-6 DNA levels were also significantly negatively correlated but there were no significant correlations with other factors. Conclusions Our results suggest that HHV-6B reactivation is attenuated by aging. Thus, HHV-6 antibodies steadily decrease in the body with aging.

Objective In dryland areas of China, alfalfa hay (AH) is a possible substitute for concentrate feed for beef cattle. To evaluate the potential benefits of this substitution, we studied the effect of the ratio of AH intake to total dry matter (DM) intake on average daily body-weight gain (ADG), dietary energy utilization status, and economic benefit in Gansu province. Methods In each of two feeding trials in 2016 (trial 1 [T1], July 3 to 17; trial 2 [T2], August 15 to September 23), crossbred male Simmental calves were allocated to low AH (LA), medium AH (MA), and high AH (HA) feeding groups (n = 4 per group). The target ADG was set as 1 kg for both trials. In a one-way-layout design based on conventional feeding practices in the province, calves received diets containing the different AH amounts, with a constant ratio of corn stover:total DM and decreasing rations of concentrate feed proportional to the increase in AH. Calves in T1 received AH at 15% (T1-LA), 23% (T1-MA), or 31% (T1-HA) of their dietary DM allowances; those in T2 received 9% (T2-LA), 24% (T2-MA), or 34% (T2-HA) AH. Results Among the T1 groups, both ADG and economic benefit were highest in T1-LA; whereas in T2, they were higher in the T2-LA and T2-MA groups than in T2-HA. Energy digestibility did not significantly differ among the groups in either trial. The dietary AH inclusion ratios of 14% in the warm season and 8% to 21% in the cool season appeared to yield optimal ADG, metabolizable energy intake, and economic benefit. Conclusion Low-level inclusion of AH, ranging from 8% to 21%, is a practical approach for beef cattle feeding. This modified feeding regimen likely will promote increased growth performance during the fattening stage of beef steers in dryland areas of Gansu province, China.

The nutritionally imbalanced poor-quality diet feeding is the major constraint of dairy production in tropical regions. Hence, alternative high-quality roughage-based diets are required to improve milk yield and reduce methane emission (CH4). Thus, we tested the effects of feeding natural pasture hay, improved forage grass hays (Napier and Brachiaria Hybrid), and treated crop residues (Eragrostis tef straw) on nutrient digestibility, milk yield, nitrogen balance, and methane emission. The eight lactating Fogera cows selected for the experiment were assigned randomly to a 4 &times, 4 Latin square design. Cows were housed in well-ventilated individual pens and fed a total mixed ration (TMR) comprising 70% roughage and 30% concentrate. The four roughage-based basal dietary treatments supplemented with formulated concentrate were: Control (natural pasture hay (NPH)), treated teff straw silage (TTS), Napier grass hay (NGH), and Brachiaria hybrid grass hay (BhH). Compared with the control diet, the daily milk yield increased (p &lt, 0.01) by 31.9%, 52.9%, and 71.6% with TTS, NGH, and BhH diets, respectively. Cows fed BhH had the highest dry matter intake (8.84 kg/d), followed by NGH (8.10 kg/d) and TTS (7.71 kg/d), all of these intakes were greater (p = 0.01) than that of NPH (6.21 kg/d). Nitrogen digestibility increased (p &lt, 0.01) from the NPH diet to TTS (by 27.7%), NGH (21.7%), and BhH (39.5%). The concentration of ruminal ammonia nitrogen was higher for cows fed NGH than other diets (p = 0.01) and positively correlated with plasma urea nitrogen concentration (R&sup2, = 0.45). Feeding TTS, NGH, and BhH hay as a basal diet changed the nitrogen excretion pathway from urine to feces, which can help protect against environmental pollution. Estimated methane yields per dry matter intake and milk yield were decreased in dairy cows fed BhH, NGH, and TTS diets when compared to cows fed an NPH diet (p &lt, 0.05). In conclusion, feeding of TTS, NGH, and BhH roughages as a basal diet to lactating dairy cows in tropical regions improved nutrient intake and digestibility, milk yield, nitrogen utilization efficiency, and reduced enteric methane emission.

Summary Little is known about the effect of latent-phase herpesviruses on their host. Human herpesvirus 6B (HHV-6B) is one of the most ubiquitous herpesviruses, and olfactory astrocytes are one of the most important sites of its latency. Here, we identified SITH-1, an HHV-6B latent protein specifically expressed in astrocytes. Mice induced to produce SITH-1 in their olfactory astrocytes exhibited olfactory bulb apoptosis, a hyper-activated hypothalamic-pituitary-adrenal (HPA) axis and depressive symptoms. The binding of SITH-1 to the host protein calcium-modulating ligand (CAML) to form an activated complex promoted the influx of extracellular calcium. The serum antibody titers for depressive patients with respect to this activated complex were significantly higher than for normal controls (p = 1.78 × 10−15), when the antibody positive rates were 79.8% and 24.4%, respectively, and the odds ratio was 12.2. These results suggest that, in the latent phase, HHV-6B may be involved in the onset of depression., Graphical Abstract, Highlights • We identified SITH-1, a new protein specific to HHV-6B latent infection • Mice expressing SITH-1 at HHV-6B latent infection site had depressive symptoms • Depressive symptoms due to SITH-1 were associated with a hyper-activated HPA axis • SITH-1-specific antibody detection significantly greater in depressive patients, Molecular Biology; Behavioral Neuroscience; Virology

The human herpesvirus 6B (HHV-6B) U79/80 gene belongs to the early gene class and appears as early as 3 hr postinfection. It is one of the most abundantly expressed transcripts and a useful diagnostic marker for viral reactivation. However, the expression mechanisms of the U79/80 gene remain unclear. To identify the viral factor(s) that activates the U79/80 promoter along with other HHV-6B core early gene promoters, p41, DNA polymerase, and U41, we examined the activities of U79/80 and other early gene promoters. In HHV-6B-infected MT-4 cells, U79/80 promoter activity was the highest among early gene promoters. In addition, we identified that HHV-6B immediate-early (IE)2B protein is one of the viral proteins involved in the activation of the U79/80 and other early gene promoters. Although the IE2B could independently activate these early gene promoters, the presence of IE1B significantly augmented the activities of early gene promoters. We also found that IE2B bound three human cytomegalovirus IE2-binding consensus, cis repression signal (CRS), within the U79/80 promoter. Moreover, the U79/80 promoter was activated by cellular factors, which are highly expressed in MT-4 cells, instead of HeLa cells because it was upregulated by mock infection and in the absence of IE2B. These results suggested that the activation mechanism of the U79/80 gene differs from other HHV-6B core early genes, apparently supporting its rapid and abundant expression. Therefore, the U79/80 early gene is an actually suitable biomarker of HHV-6B reactivation.

From August to September 2015 (trial 1 [T1]) and September to November 2015 (trial 2 [T2]), the effects of replacing concentrate feed (C) with alfalfa hay (AH) on the daily gain (DG), dietary energy utilization status and the economic advantage of AH feeding for growing beef cattle were studied in crossbred male Simmental calves (n = 18) in Gansu Province, China. The target DG was set as 1 kg for both trials. Animals in each trial were allocated to a conventional feeding group (CTRL), a low-level AH feeding group (LA), and a high-level AH feeding group (HA). In a one-way-layout design, they were fed iso-energetic experimental diets comprising harvested corn stover (CS) and C (T1-CTRL, T2-CTRL), diets replacing 22% (T1-LA) or 44% (T1-HA) of the quantity of C for T1-CTRL with AH, and diets replacing 13% (T2-LA) or 25% (T2-HA) of the quantity of C for T2-CTRL with AH. Measurements of feed intake and DG, respiration and metabolism trials were performed for 49 and 41 days in T1 and T2, respectively. Average DG did not reach the target value for HA in T1 and CTRL in T2. Energy metabolizability was slightly greater for CTRL than for LA and HA in T1 and significantly greater for CTRL than for the other groups in T2. There was no marked difference in energy metabolizability between LA and HA in both trials. Dietary substituting AH for C did not impair the feed intake of the animals, but it did not improve feed efficiency. In terms of economic feasibility, low-level AH inclusion in the diets of growing beef cattle appeared more profitable at the 1-kg DG level as compared with CTRL and high-level AH inclusion, and should be practiced in the drylands of Gansu Province, China.

Extracts of the sponge Hyattella aff. intestinalis showed moderate inhibition against adenovirus. Chromatographic separation of the extracts followed by analysis of spectroscopic data allowed us to elucidate the structures of three new metabolites as 2α-hydroxyspongia-13(16),14-diene-3-one (4), 3β-hydroxyspongia-13(16),14-diene-2-one (7), and 2α,3α-diacetoxy-17,19-dihydroxyspongia-13(16),14-diene (8) and to identify six known ones 1-3, 5, 6 and 9. Among the molecules, compounds 1 and 3 showed antiviral activity at IC50 17.0 and 52.0 µM.

Objective: Both infected subdural hematoma (ISH) and Edwardsiella tarda infections are rare in humans. E. tarda is a motile, facultative anaerobic, gram-negative rod bacterium, which is isolated from fresh or brackish water, but not usually from humans. Extra-intestinal E. tarda infections are rare and might cause severe clinical symptoms. However, ISH caused by E. tarda has not been reported previously. We report the first case of ISH due to E. tarda. Patient: A 76-year-old man was admitted to our hospital with a headache, loss of appetite, and nausea. Computed tomography revealed bilateral subdural hematoma. Results: We performed burr hole drainage. A hematoma with pus was found on the left side and chronic hematoma was found on the right side. Consequently, we diagnosed him with ISH on the left side and chronic subdural hematoma on the right side. E. tarda was detected in a culture from the hematoma with pus on the left side. As postoperative antibiotic therapy, we administered ceftriaxone and metronidazole for 47 days. The patient was discharged with no residual neurological deficit. Conclusion: Our case implied that favorable outcomes can be obtained by drainage and appropriate antibiotic therapy for ISH caused by E. tarda.

A low nitrogen utilization efficiency (NUE, the ratio of retained N to N intake) and high methane (CH4) emissions of ruminants can lead to potentially high diet protein wastage and directly contribute to global warming. Diet manipulation is the most effective way to improve NUE or reduce CH4 emissions. This study investigated how replacing oat hay with alfalfa hay (AH) or common vetch hay (CVH) with different proportions (20% (20) and 40% (40) of the total dry matter (DM) allowance) affects the body weight gain (BWG), NUE, and CH4 emissions of crossbred Simmental cattle. The forage dry matter intake (DMI) and the total DMI of cattle fed on a CVH40 diet were significantly higher than the values for those fed on AH20 or AH40 diets (p &lt, 0.05). There were no differences in the BWG for the four treatments observed, however, nutrient digestibility significantly decreased in the AH40 diet as compared with the AH20 diet (p &lt, 0.05). The NUE was significantly lower in AH40 than in CVH20. The CH4 emissions were significantly lower for the CVH40 diet than with the AH20 diet (p &lt, 0.05). Our findings suggest that a 20% AH and 40% CVH substitution for oat hay are the optimal proportions to maintain the BWG, NUE, nutrient digestibility, and reduce the CH4 emissions of crossbred Simmental cattle. Overall, CVH has a greater potential to reduce CH4 emissions than AH.

Several studies have noted an effect of DNA methylation on the pathogenesis of Alzheimer's disease (AD). We have already reported that DNA methylation levels in the NCAPH2/LMF2 promoter region can be a useful biomarker for the diagnosis of AD and amnesic mild cognitive impairment (aMCI). However, there is still uncertainty about the mechanism by which NCAPH2/LMF2 methylation affects the pathogenesis of AD and aMCI. In this study, we investigated relationships between NCAPH2/LMF2 methylation and other factors. AD (n=30) and aMCI (n=28) subjects were included in this study. NCAPH2/LMF2 methylation levels were measured by pyrosequencing. Correlations between methylation levels and other factors including age at onset, sex, duration of disease, education, mini-mental state examination (MMSE) and frontal assessment battery (FAB) scores, APOE genotype, degree of hippocampal atrophy, and total brain atrophy were measured. Degrees of hippocampal atrophy and total brain atrophy were measured by VSRAD (Voxel-Based Specific Regional Analysis System for Alzheimer's Disease). Regression analysis revealed that only hippocampal atrophy according to VSRAD is a significant dependent variable correlated with NCAPH2/LMF2 methylation levels. Our results suggest that DNA methylation in the NCAPH2/LMF2 promoter region is associated with hippocampal atrophy through apoptosis.

Influenza A and B virus infections are serious public health concerns globally. However, the concerns regarding influenza B infection have been underestimated. The currently used anti-influenza drugs have not provided equal efficacy for both influenza A and B viruses. Susceptibility to neuraminidase (NA) inhibitors has been observed to be lower for influenza B viruses than for influenza A viruses. Moreover, the emergence of resistance to anti-influenza drugs underscores the need to develop new drugs. Recently, we reported that methylglyoxal (MGO) suppressed influenza A virus replication in a strain-independent manner. Therefore, we hypothesize that MGO exhibits anti-influenza activity against B strains. This study aimed to evaluate the anti-influenza viral activity of MGO against influenza B strains by using Madin-Darby canine kidney (MDCK) cells. Several types of influenza B viruses were used to determine the activity of MGO. The susceptibilities of influenza A and B viruses to NA inhibitors were compared. MGO inhibited influenza B virus replication, with 50% inhibitory concentrations ranging from 23-140 μM, which indicated greater sensitivity of influenza B viruses than influenza A viruses. Our results show that MGO has potent inhibitory activity against influenza B viruses, including NA inhibitor-resistant strains.

As described in Japanese essay (Hojoki), written around 1200, various disasters such as big fires, earthquakes, and famines have occurred in Japan. Asian countries have been suffering from the disasters; furthermore, natural disasters are increasing due to global warming. Because tropical-infectious diseases are often disaster-related infectious diseases (DRIDs), the strategies against the former kind of disease could be applicable to DRIDs. Meteorological analysis of the occurrence of DRIDs using a method of time series analysis is important. In situations of disasters, it is desirable if you can identify the pathogen and identify disease severity simultaneously. A dipstick DNA chromatography assay termed as Single-Tag Hybridization—Printed Array Strip (STH—PAS) system was developed based on the DNA sequences of various mosquito-borne diseases. The plasma levels of matricellular proteins including galectin-9 (Gal-9) and osteopontin (OPN) were found to reflect the disease severities in the dengue virus and other DIRDs. Because both proteins have been reported to be immune-check molecules, their inhibition might enhance the immune system against pathogens. We found that brefelamide derivatives could inhibit OPN and other inflammatory molecules synthesis. Very recently, different derivatives were found to inhibit PD-L1 transcription. Applications of these agents should be considered as multi-step strategies against DRIDs.

Background: There is little information on molecular epidemiology of drug-resistant tuberculosis including multidrug-resistant (MDR) TB in Japan. We have recently developed a web server, comprehensive analysis server for Mycobacterium tuberculosis complex (CASTB), which can analyze whole genome sequencing data generated by next-generation sequencers. Objectives: To analyze molecular epidemiology of drug-resistant TB in Japan. Methods: Whole genome sequences of 178 M. tuberculosis isolates with isoniazid- or rifampicin-resistance (n=149) and MDR (n=29) obtained from 22 National Hospital Organization Hospitals in Japan, were analyzed using CASTB. Results: The 178 drug-resistant M. tuberculosis isolates clustered into four clades; Lineage 2 (83.1%), Lineage 4 (10.1%), Lineage 1 (5.6%) and Lineage 3 (1.1%), indicating that the majority of drug-resistant M. tuberculosis isolates in Japan belonged to Lineage 2. Of the isolates classified in Lineage 2, 76.4% were of the ancestral Beijing sub-genotype. MDR-TB isolates were frequently seen in the modern Beijing sub-genotype, whereas few in Lineage 4. Among the Lineage 2 ancestral isolates, 5.3% were from foreign-born patients, whereas among the Lineage 2 modern isolates, 28.6% were from foreign-born patients. We detected a sub-clade consisting of isolates mainly from patients born in Asian high TB-burden countries in the Lineage 2 modern genotype, suggesting the introduction of globally disseminated isolates into Japan. Conclusions: Whole genome analysis of drug-resistant M. tuberculosis isolates in Japan using CASTB revealed the genetic diversity of M. tuberculosis isolates, as well as the impact of foreign-born patients.

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from patients in Japan, was conducted by Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases and Japanese Society for Clinical Microbiology in 2010.The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period from January and April 2010 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical and Laboratory Standard Institutes using maximum 45 antibacterial agents.Susceptibility testing was evaluable with 954 strains (206 Staphylococcus aureus, 189 Streptococcus pneumoniae, 4 Streptococcus pyogenes, 182 Haemophilus influenzae, 74 Moraxella catarrhalis, 139 Klebsiella pneumoniae and 160 Pseudomonas aeruginosa). Ratio of methicillin-resistant S.aureus was as high as 50.5%, and those of penicillin-intermediate and -resistant S.pneumoniae were 1.1% and 0.0%, respectively. Among H.influenzae, 17.6% of them were found to be β-lactamase-non-producing ampicillin (ABPC)-intermediately resistant, 33.5% to be β-lactamase-non-producing ABPC-resistant and 11.0% to be β-lactamase-producing ABPC-resistant strains. Extended spectrum β-lactamase-producing K.pneumoniae and multi-drug resistant P.aeruginosa with metallo β-lactamase were 2.9% and 0.6%, respectively.Continuous national surveillance of antimicrobial susceptibility of respiratory pathogens is crucial in order to monitor changing patterns of susceptibility and to be able to update treatment recommendations on a regular basis., Journal of Infection and Chemotherapy, 21(6), pp.410-420; 2015

Background/Aims: In the present study, we examined whether DNA methylation of the brain-derived neurotrophic factor (BDNF) promoter is associated with the manifestation and clinical presentation of Alzheimer's disease (AD). Methods: Of 20 patients with AD and 20 age-matched normal controls (NCs), the DNA methylation of the BDNF promoter (measured using peripheral blood samples) was completely analyzed in 12 patients with AD and 6 NCs. The resulting methylation levels were compared statistically. Next, we investigated the correlation between the DNA methylation levels and the clinical presentation of AD. Results: The total methylation ratio (in %) of the 20 CpG sites was significantly higher in the AD patients (5.08 ± 5.52%) than in the NCs (2.09 ± 0.81%; p < 0.05). Of the 20 CpG sites, the methylation level at the CpG4 site was significantly higher in the AD subjects than in the NCs (p < 0.05). Moreover, the methylation level was significantly and negatively correlated with some neuropsychological test subscores (registration, recall, and prehension behavior scores; p < 0.05). Conclusion: These results suggest that the DNA methylation of the BDNF promoter may significantly influence the manifestation of AD and might be associated with its neurocognitive presentation.

Background and Aims Influenza virus infections are serious public health concerns worldwide that cause considerable mortality and morbidity. Moreover, the emergence of resistance to anti-influenza viral agents underscores the need to develop new anti-influenza viral agents and novel treatment strategies. Recently, we identified anti-influenza viral activity of manuka honey. Therefore, we hypothesized that methylglyoxal (MGO), a key component of manuka honey, may impart anti-influenza viral activity. The aim of this study was to evaluate the anti-influenza viral activity of MGO and its potential in combination treatments with neuraminidase (NA) inhibitors. Methods MDCK cells were used to evaluate anti-influenza viral activity. To evaluate the mechanism of MGO, plaque inhibition assays were performed. The synergistic effects of MGO and viral NA inhibitors were tested. Results MGO inhibited influenza virus A/WSN/33 replication 50% inhibitory concentration = 240 ± 190 μM; 50% cytotoxic concentration = 1.4 ± 0.4 mM; selective index (SI) = 5.8, which is related to its virucidal effects. Moreover, we found that MGO showed promising activity against various influenza strains. A synergistic effect was observed by a marked increase in SI of NA inhibitors at ∼1/100 th of their single usage. A synergistic effect of MGO and oseltamivir was also observed against oseltamivir-resistant virus. Conclusions Our results showed that MGO has potent inhibitory activity against influenza viruses and also enhanced the effect of NA inhibitors. Thus, the co-administration of MGO and NA inhibitors should be considered for treatment of influenza virus infections.

We investigated the relationship between tuberculosis recurrence and Mycobacterium tuberculosis antigen-stimulated interferon-gamma (IFN-γ) responses during treatment.Plasma IFN-γ levels in active pulmonary tuberculosis patients (n = 407) were analyzed using QuantiFERON-TB Gold In-Tube™ (QFT-IT) at 0, 2, and 7 months of the 8-month treatment received from 2007 to 2009 and the patients were followed up for another 16 months after treatment. Risk factors for recurrence were assessed using the log-rank test and Cox proportional hazard models. Random coefficient models were used to compare longitudinal patterns of IFN-γ levels between groups.QFT-IT showed positive results in 95.6%, 86.2%, and 83.5% at 0, 2, and 7 months, respectively. The antigen-stimulated IFN-γ responses varied significantly during the treatment course (P0.0001). Unexpectedly, positive-to-negative conversion of QFT-IT results between 0 and 2 months was significantly associated with earlier recurrence (adjusted hazard ratio, 5.57; 95% confidence interval, 2.28-13.57). Time-dependent changes in IFN-γ levels were significantly different between the recurrence and nonrecurrence groups (P0.0001).Although the IGRA response varies individually, early response during the treatment course may provide an insight into host immune responses underlying tuberculosis recurrence.

Background Omalizumab, a humanized anti-IgE monoclonal antibody, is the first molecularly targeted drug for severe asthmatics. However, responses to omalizumab vary widely among patients. Objectives This study aimed to assess the potential of baseline serum cytokine levels as predictors of responsiveness to omalizumab. Methods Thirty-one patients with severe, persistent asthma were enrolled in this study and administered omalizumab for at least 1 year. Response to omalizumab was assessed based on the physician's global evaluation of treatment effectiveness (GETE) at 48 weeks of treatment. Blood samples were collected at baseline and 16 and 32 weeks after starting omalizumab and measured for 30 cytokines by Luminex 200 and ELISA. Exhaled nitric oxide (FeNO) levels, peripheral blood eosinophil counts, pre-bronchodilator pulmonary functions and Asthma Quality of Life Questionnaire scores were determined at baseline and 16, 32 and 48 weeks after starting omalizumab. The numbers of clinically significant asthma exacerbations in the previous year and during 48 weeks of treatment with omalizumab were assessed. Results GETE assessment showed 19 responders (61.3%) and 12 non-responders (38.7%). Responders showed significantly higher levels of CXCL10 and IL-12 at baseline compared to non-responders (CXCL10: responders, 1530.0 ± 315.2 pg/ml vs. non-responders, 1066.0 ± 396.8 pg/ml, P = 0.001; IL-12: responders, 60.2 ± 39.2 pg/ml vs. non-responders, 32.2 ± 26.3 pg/ml, P = 0.04). ROC curves to distinguish responders from non-responders using the baseline serum CXCL10 level showed a good AUC of 0.83. At 32 weeks of omalizumab therapy, serum CXCL10 tended to be increased (1350 ± 412.3 pg/ml at baseline vs. 1529 ± 637.6 pg/ml at 32 weeks, P = 0.16) and serum IL-12 tended to be decreased (49.4 ± 37.0 pg/ml at baseline vs. 43.9 ± 30.9 pg/ml at 32 weeks, P = 0.05). On the other hand, serum IL-5 and PDGF were significantly decreased (IL-5: 54.2 ± 13.8 pg/ml at baseline vs. 49.1 ± 12.5 pg/ml at 32 weeks, P = 0.008; PDGF: 4821 ± 2458 pg/ml at baseline vs. 4219 ± 1951 pg/ml at 32 weeks, P = 0.048). Conclusions High baseline serum CXCL10 and IL-12 levels may be useful in predicting a good omalizumab response in severe asthmatics.

Objectives Prediabetes is a precursor of diabetes and increases the risk for cardiovascular disease. A high C-reactive protein (CRP) level is a risk factor for diabetes, and individuals with prediabetes have higher CRP levels than those with normal glucose tolerance. In addition, systemic inflammation may play a role in the early-phase deterioration of glucose metabolism. We examined the association between serum CRP levels and prediabetes. Methods Overall, 4,101 subjects without diabetes underwent oral glucose tolerance tests. Levels of serum CRP were divided into quartiles; the lowest quartile was used as the reference when calculating odds ratios (ORs) and confidence intervals. Isolated fasting glucose, isolated glucose tolerance and elevated glycated hemoglobin levels (i.e. between 42 and 47 mmol/mol [6.0% to 6.4%]) were indicative of prediabetes. Results In the multiple logistic regression analysis, the ORs (95% confidence intervals) for impaired glucose tolerance, impaired fasting glucose and elevated glycated hemoglobin levels corresponding to the highest quartile of CRP levels were 1.67 (1.31 to 2.14); 1.62 (1.15 to 2.28); and 1.47 (1.14 to 1.90), respectively. In the stratified analysis, the ORs for impaired glucose tolerance were consistently higher in the uppermost quartile than in the reference quartile in both the presence and absence of hypertension or dyslipidemia. In contrast, the ORs for impaired fasting glucose in the uppermost quartile were higher only in the presence of hypertension and dyslipidemia, and the OR for elevated glycated hemoglobin levels in the uppermost quartile was higher only in the presence of hypertension. Conclusions Elevated serum CRP levels are associated with prediabetes, particularly impaired glucose tolerance.

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be "sub-microscopic" blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.

One new dihydrochalcone diglycoside has been isolated from the EtOAc fraction of the Egyptian seagrass Thalassodendrin ciliatum (Forsk.) den Hartog, and was identified as 6′-O-rhamnosyl-(1? → 6″)-glucopyranosyl asebogenin for which a trivial name Thalassodendrone was established. Furthermore, five known phenolics were isolated and identified as asebotin, quercetin 3,7-diglucoside, protocatechuic acid, ferulic acid and p-hydroxybenzoic acid. The structures of all the isolated compounds were established based on 1D and 2D NMR spectroscopy and high-resolution-mass spectrometer. High-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained using a JEOL JMS-T100TD spectrometer (JEOL Ltd., Tokyo, Japan). The anti-influenza A virus activity of the isolated new compound and asebotin was evaluated, and the obtained results revealed that the inhibition dose concentration of asebotin was more than that of Thalassodendrone with IC 50 = 2.00 and 1.96 μg/mL, respectively, and with cytotoxic concentration (CC50) of 3.36 and 3.14 μg/mL, respectively., Natural Product Research, 28(6), pp.377-382; 2014

Highlights • To elucidate vRNP export, we investigated the interactions among Hsc70, M1, and NS2. • Hsc70 interacts with M1 more strongly than with NS2. • Hsc70 competes with NS2 for M1 binding. • These results suggest the involvement of Hsc70 in the nuclear export of vRNP., The influenza virus replicates in the host cell nucleus, and the progeny viral ribonucleoprotein complex (vRNP) is exported to the cytoplasm prior to maturation. NS2 has a nuclear export signal that mediates the nuclear export of vRNP by the vRNP–M1–NS2 complex. We previously reported that the heat shock cognate 70 (Hsc70) protein binds to M1 protein and mediates vRNP export. However, the interactions among M1, NS2, and Hsc70 are poorly understood. In the present study, we demonstrate that Hsc70 interacts with M1 more strongly than with NS2 and competes with NS2 for M1 binding, suggesting an important role of Hsc70 in the nuclear export of vRNP.

Background: We explored catalytic antibodies applicable to patients. Results: A human light chain (22F6) possessing both amidase and nuclease activities and preventing infection of influenza virus was obtained. Conclusion: The 22F6 light chain holds a huge potential as a new drug for patient therapies in the future. Significance: The procedure developed in this study is highly noteworthy for developing new drugs., Human antibody light chains belonging to subgroup II of germ line genes were amplified by a seminested PCR technique using B-lymphocytes taken from a human adult infected with influenza virus. Each gene of the human light chains was transferred into the Escherichia coli system. The recovered light chain was highly purified using a two-step purification system. Light chain 22F6 showed interesting catalytic features. The light chain cleaved a peptide bond of synthetic peptidyl-4-methyl-coumaryl-7-amide (MCA) substrates, such as QAR-MCA and EAR-MCA, indicating amidase activity. It also hydrolyzed a phosphodiester bond of both DNA and RNA. From the analysis of amino acid sequences and molecular modeling, the 22F6 light chain possesses two kinds of active sites as amidase and nuclease in close distances. The 22F6 catalytic light chain could suppress the infection of influenza virus type A (H1N1) of Madin-Darby canine kidney cells in an in vitro assay. In addition, the catalytic light chain clearly inhibited the infection of the influenza virus of BALB/c mice via nasal administration in an in vivo assay. In the experiment, the titer in the serum of the mice coinfected with the 22F6 light chain and H1N1 virus became considerably lowered compared with that of 22F6-non-coinfected mice. Note that the catalytic light chain was prepared from human peripheral lymphocyte and plays an important role in preventing infection by influenza virus. Considering the fact that the human light chain did not show any acute toxicity for mice, our procedure developed in this study must be unique and noteworthy for developing new drugs.

Cervical cancer, the third most common cancer that affects women worldwide, is caused by the human papillomavirus (HPV) and is treatable when detected at an early stage. To date, more than 100 different HPV types have been described, and the development of simple, low-cost, and accurate methods to distinguish HPV genotypes is highly warranted. In this study, an HPV genotyping assay based on polymerase chain reaction (PCR) was evaluated. This method involved the use of MY09/11 primers followed by restriction fragment length polymorphism (RFLP) analysis with the restriction enzymes HpyCH4V and NlaIII. Cervical specimens preserved using CytoRich Blue fluid were collected from 1,134 female volunteers for HPV detection, and 1,111 valid samples were amplified using PCR. The PCR method was sensitive enough to detect 25 copies of HPV18, and three copies of HPV16. Out of 202 PCR-positive samples, HPV genotypes were determined in 189 samples (93.6%) by this RFLP method. Results were then evaluated further by capillary sequencing method. Concordant results between the two tests were as high as 96.0%. Thirteen samples, which tested negative with RFLP, were verified as non-specific amplifications with PCR. In conclusion, this PCR-RFLP method using restriction enzymes HpyCH4V and NlaIII is simple, non-labor intensive, and is applicable for the inexpensive determination of HPV genotypes in clinical samples.

Background: Host effector mechanism against Mycobacterium tuberculosis (Mtb) infection is dependent on innate immune response by macrophages and neutrophils and the alterations in balanced adaptive immunity. Coordinated release of cytolytic effector molecules from NK cells and effector T cells and the subsequent granule-associated killing of infected cells have been documented; however, their role in clinical tuberculosis (TB) is still controversy. Objective: To investigate whether circulating granulysin and other effector molecules are associated with the number of NK cells, iNKT cells, Vγ9+Vδ2+ T cells, CD4+ T cells and CD8+ T cells, and such association influences the clinical outcome of the disease in patients with pulmonary TB and HIV/TB coinfection. Methods: Circulating granulysin, perforin, granzyme-B and IFN-γ levels were determined by ELISA. The isoforms of granulysin were analyzed by Western blot analysis. The effector cells were analyzed by flow cytometry. Results: Circulating granulysin and perforin levels in TB patients were lower than healthy controls, whereas the granulysin levels in HIV/TB coinfection were much higher than in any other groups, TB and HIV with or without receiving HAART, which corresponded to the number of CD8+ T cells which kept high, but not with NK cells and other possible cellular sources of granulysin. In addition, the 17kDa, 15kDa and 9kDa isoforms of granulysin were recognized in plasma of HIV/TB coinfection. Increased granulysin and decreased IFN-γ levels in HIV/TB coinfection and TB after completion of anti-TB therapy were observed. Conclusion: The results suggested that the alteration of circulating granulysin has potential function in host immune response against TB and HIV/TB coinfection. This is the first demonstration so far of granulysin in HIV/TB coinfection.

From the standpoint of early interventions for dementia, a convenient method of diagnosis using biomarkers is required for Alzheimer’s disease (AD) in the early stage as well as amnesic mild cognitive impairment (aMCI). Focusing on differences in DNA methylation due to AD and aMCI, in the present study, we first conducted genome-wide screening, measuring blood DNA methylation levels by the Illumina Infinium HD Methylation Assay in 3 small age-and gender-matched groups consisting of 4 subjects each: normal controls (NC), aMCI and AD. The genome-wide analysis produced 11 DNA methylation loci that distinguished the 3 groups. For confirmation, we increased group sizes and examined samples by pyrosequencing which revealed that DNA methylation in the NCAPH2/LMF2 promoter region was significantly decreased in the AD (n = 30) and aMCI (n = 28) groups as compared to the NC group (n = 30) (P < 0.0001, ANCOVA). No association was found between methylation levels and APOE genotype. NCAPH2/LMF2 methylation levels were considered to potentially be a convenient and useful biomarker for diagnosis of AD and aMCI.

We investigated whether neurotrophin (NT)-3 polymorphisms influenced the executive function of patients with 2 separate disease stages with similar dementia conditions: amnestic mild cognitive impairment (A-MCI) or mild Alzheimer disease (AD).Among 215 outpatients with dementia and MCI, 155 with mild AD (n = 108) or A-MCI (n = 47) were recruited and divided into three genotypic groups based on the representative NT-3 functional polymorphisms rs6332 and rs6489630. Next, we compared the frontal assessment battery (FAB) total and subtest scores between the three genotypic groups.The total FAB score was not significantly associated with the rs6332 and rs6489630 genotypes; however, the conflicting instructions score among the 6 subtests was significantly associated with the rs6332 genotype (p0.05). Moreover, in patients with mild AD, the conflicting instructions score differed significantly among the three genotypic groups of rs6332 (p0.05) (G/GA/A: p = 0.042 and G/AA/A: p = 0.041). No significant differences in any other demographic variables were observed among the three genotypes of rs6332 and rs6489630.These results suggested that an NT-3 polymorphism, rs6332, may significantly influence executive function, reflecting interference performances among patients with mild-stage AD.

Many pathogenic viruses, such as the influenza virus and the Human Immunodeficiency Virus (HIV)-1, are a threat to humans, thus leading to thousands of deaths annually. The development of antiviral drugs is urgent, and it is an essential strategy for the suppression of these infectious diseases. However, regardless of the rapid emergence of many infectious diseases, the development of novel antiviral drugs has been slow, except for the case of the AIDS. In addition, several viruses can easily mutate and escape the inhibitory activity of anti-viral drugs. It was already well-established that HIV escapes from anti-viral drug effects because of the lack of proofreading activity in its reverse transcriptase. It is known that the influenza virus, which is resistant to Tamiflu, is already spread all over the world. Viruses utilize the host cell environment and cellular factors to propagate. Therefore, the development of novel drugs which inhibit viral protein-host protein interactions or cellular functions appear to be good candidates. The influenza virus is unique in replicating in host nuclei, and we therefore focused on the nuclear export processes for the development of anti-influenza viral drugs. We previously reported that leptomycin B (LMB), which inhibited the nuclear export processes via the nuclear export signal (NES) inhibited the nuclear export of influenza viral RNP (vRNP), and resulted in the inhibition of influenza viral propagation. We herein examined novel CRM1 inhibitors, valtrate from Valerianae Radix, and 1'-acetoxychavicol acetate (ACA) from Alpinia galanga as potent inhibitors for the influenza virus replication.

The influenza virus genome replicates in the host cell nucleus, and the progeny viral ribonucleoproteins (vRNPs) are exported to the cytoplasm prior to maturation. The influenza virus NS2 protein has a nuclear export signal (NES) and binds to M1. It is therefore postulated that vRNP is exported from the nucleus by binding to NS2 through M1. However, the significance of the association between NS2 and M1 for the nuclear export of vRNP is still poorly understood. We herein demonstrate that the C-terminal domain of NS2 (residues 81–100) is essential for M1 binding and the nuclear export of progeny vRNPs.Structured summaryMINT-8057301, MINT-8057317: NS2 (uniprotkb:P03508) binds (MI:0407) to M1 (uniprotkb:P03485) by pull down (MI:0096)

A highly selective and facile assay of human immunodeficiency virus protease (HIV-PR) has been required for the screening of medicinal inhibitors and also for classifying the subtypes of HIV in the therapeutic treatment of acquired immune deficiency syndrome (AIDS). This article describes a novel assay method of HIV-PR based on the selective fluorogenic reaction of peptides. A peptide fragment generated from a substrate by the enzymatic digestion with HIV-PR could be selectively quantified by the spectrofluorometric detection after the fluorogenic reaction with catechol in the presence of sodium periodate and sodium borate (pH 7.0). This assay system uses an N-terminal acetyl peptide as the substrate and crude extracts from Escherichia coli expressing recombinant HIV-PR. The activity obtained by the proposed assay correlated with that obtained by a conventional HIV-PR assay based on fluorescence resonance energy transfer detection.

In 1997, an outbreak of alveolar echinococcosis in Japanese monkeys (Macaca fuscata) occurred in a zoo in Hokkaido, Japan. Twelve infected monkeys from a colony (n = 57) were diagnosed serologically by Western blotting, and ultrasonography showed the presence of tumor-like tissue in the livers of nine monkeys. The 12 infected monkeys have been treated with albendazole for 10 years without surgical resection. Ten of these monkeys have died so far; diagnoses were confirmed histopathologically through autopsy. Two of these monkeys are still alive. Recently, a significant difference between the two living monkeys was recognized. A difference in curative effect was demonstrated between the two living monkeys by radiography, contrast enhanced computed tomography, and contrast ultrasound. One showed metastasis to various organs, and the other appeared to be almost cured, as demonstrated by size reduction and calcification of the lesion after albendazole treatment for 10 years. This time, serological reexamination was performed to corroborate this apparent difference. The serological tests supported the preliminary imaging findings. In addition, the presence of albendazole metabolites in sera was confirmed by high-performance liquid chromatography. In this study, it was demonstrated that tests which have been used in human cases were also effective for diagnosing alveolar echinococcosis and for assessing curative effects in nonhuman primates such as M. fuscata.

Host genetic susceptibility to adult pulmonary Mycobacterium avium complex disease remains unknown. To identify genetic loci for the disease, we prepared 3 sets of pooled DNA samples from 300 patients and 300 sex- matched control subjects and genotyped 19,651 microsatellite markers in a case-control manner. D6S00091― located in the MICA (major histocompatibility complex class I chain-related A) gene, which encodes a ligand of the NKG2D receptor―had the lowest P value in pooled and individual DNA typing. The A6 allele of the micro- satellite was significantly associated with female patients (P

We investigated whether exogenously supplied precursors of bergapten, namely umbelliferone, psoralen and bergaptol, could be utilized to produce bergapten without elicitation in Glehnia littoralis cell suspension cultures. The levels of added psoralen and bergaptol in the medium soon decreased, and this was followed by the detection of bergapten in both culture fluid and cells. Umbelliferone was also incorporated but in this case no bergapten was produced; instead, skimmin, umbelliferone monoglucoside, was detected. To determine whether conversion of psoralen to bergapten was due to enzyme induction by precursor feeding, the transcript accumulations and enzyme activities of bergaptol O-methyltransferase (BMT, EC 2.1.1.69), which catalyzes the last step of bergapten synthesis, and of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), which catalyzes the initial step of the phenylpropanoid biosynthetic pathway and is known as a marker enzyme of elicitation, were examined. The results showed that both the expression and the activity of BMT were always detected in all cells, including control cells. Since PAL was slightly induced in the cells supplied with/without precursors, phenylethyl alcohol (PEA, a competitive inhibitor of PAL) was applied to suspension cells prior to the addition of psoralen. PAL activity was effectively inhibited by PEA at 1-5 mM concentrations. Under these conditions, PEA did not affect bergapten production by cell cultures fed with psoralen at all. These results demonstrate that BMT is constitutively expressed in G. littoralis cell cultures., Plant Cell Reports, 28(2), pp.257-265; 2009

We investigated the role of apoptosis in relation to proliferative activity in hepatocellular carcinoma (HCC) using in situ DNA nick end labeling (ISNEL) and immunostaining for the Ki-67 antigen in 35 patients with HCC. We also performed immunostaining for Fas and Fas ligand (Fas L) to determine the relationship between the Fas system and apoptosis. The ratio of the ISNEL labeling index (LI) to the Ki-67 LI was significantly lower in HCC than in surrounding nontumorous liver tissue (p

Betanodavirus, a bipartite RNA virus of fishes and a member of Nodaviridae family, targets nervous tissues and is the causative agent of viral nervous necrosis in marine farmed fish. Betanodavirus is thought to be propagated only in fish cells because betanodavirus has only been isolated in fish and it is not well propagated in mammalian culture cells. However, the host specificity of betanodavirus has not yet been well analyzed. To analyze the host specificity of betanodavirus, various mammalian cells were screened for their permissiveness to betanodavirus. As a result, redspotted grouper nervous necrosis virus can be propagated efficiently in the murine astrocytoma cell line, DBT. The level of viral production in DBT was 10-fold-higher than in the fish cell line, E-11. This result is the first to demonstrate the efficient propagation of betanodavirus in mammalian cells and may help to elucidate the mechanism of the host specificity of betanodavirus.

Summary The determination of the host ranges of viruses is important because of the possible emergence of infectious agents, which may result from the zoonotic transmission of animal viruses to humans. The family Nodaviridae, whose members are non-enveloped, positive-stranded bipartite RNA viruses, is comprised of the genera Alphanodavirus and Betanodavirus, whose members predominantly infect insects and fish, respectively. The alphanodaviruses can also infect suckling mice and suckling hamsters, resulting in paralysis and death. Pigs near the site of isolation of the Nodamura virus (NoV), an alphanodavirus, have been reported to have high levels of NoV neutralizing antibody, suggesting that they may be part of the natural host range of this virus. Betanodaviruses are the causative agents of viral nervous necrosis, which occurs in several species of fish. However, little is known regarding the mechanism of infection of these viruses. Whether betanodaviruses can infect hosts other than fish remains unclear. In this study, we examined the possibility that a betanodavirus, redspotted grouper nervous necrosis virus (RGNNV), can infect human cell lines and showed that this virus can attach to the cells but cannot penetrate them, although human cells can support the replication of the betanodavirus when viral RNAs are transfected. The betanodavirus in its present form cannot infect human cells.

Summary Betanodaviruses, members of the family Nodaviridae, have small positive-stranded bipartite RNA genomes and are the causal agent of viral nervous necrosis (VNN) in many species of marine farmed fish. In the aquaculture industry, outbreaks of betanodavirus infection and spread in larval and juvenile fish result in devastating damage and heavy economic loss. Although an urgent need exists to develop drugs that inhibit betanodavirus infection, there have been no reports about anti-betanodavirus drugs. Recently, it was reported that betanodaviruses were detected in the endosomes of infected cells, suggesting that betanodaviruses enter fish cells by endocytosis. This finding prompted us to examine whether blocking this endosomal pathway could provide a target for antiviral drug development. In this study, we examined the inhibitory effect of several lysosomotropic agents against betanodavirus infection in fish E-11 cells. The presence of 1 mM NH4Cl or 1 µM chloroquine in the medium inhibited the entry of betanodaviruses into cells and inhibited viral infection. The lysosomotropic agents bafilomycin A1 and monensin also inhibited virus-induced cytopathology and virus production. Our data demonstrate that inhibitors of endosomal acidification are candidates as antiviral agents against betanodavirus.

The influence of the topographical structure of natural coastal marine environments on the composition of culturable microbial communities in marine sediments was analyzed by comparing the composition of the culturable fraction of sedimental bacterial communities from five topographically disparate marine ecosystems. The sampling areas included open, enclosed and semi-closed coastal seas near Nagasaki, Japan. Bacterial identification and phylogenetic analyses based on 16S rDNA gene sequencing showed that 84.0% of the isolated strains belonged to known species. The marine indigenous γ-Proteobacteria were significantly higher (P < 0.05) in abundance among the strains isolated from the open coastal seas, but were less abundant in the enclosed and the semi-closed coastal seas. Conversely, the abundance of the low G + C gram-positive bacteria, mainly soil-derived Bacillus species, was significantly higher (P < 0.001) in the enclosed sea than in the open coastal seas. It was found that the composition of the culturable fraction of coastal sediment bacterial communities might be influenced by topographic factors and could be utilized as a microbiological tool for the assessment of the deterioration of environmental conditions in enclosed coastal seas.

In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii., Author Summary Malaria infections are very often composed of multiple strains of malaria parasites. It is thought that these strains may compete for resources such as space and nutrients within the host. Here we show that such “within-host competition” has repercussions for the virulence of the malaria infection, so that infections composed of multiple strains are more virulent in terms of disease severity, than single strain infections containing the constituent parasites. Following from this, it has been proposed that as such competition would favour those parasites with faster growth rates, then these parasites would be selected in nature when within-host competition is common. We show, however, that this is not necessarily the case, as parasites with faster growth rates in the mammalian host were no more successful at transmitting to mosquitoes than parasites with slower growth rates. These results show that a reassessment of our current understanding of the role of within-host competition in the selection of virulence in malaria parasites is required.

The aim of the present study was to determine whether expression of molecules associated with cell cycle regulation and apoptosis might reflect tumor grade and patients' prognosis of gastrointestinal stromal tumor (GIST). Forty-nine cases of gastric GIST were divided into three grades; low, intermediate, and high risk. Ki-67, cyclin A, cyclin D1, cyclin E, p16(Ink4), p21(Waf1), p27(Kip1), cyclin-dependent kinase (cdk)2, cdk4 and single-strand DNA (ssDNA) were immunohistochemically stained and assessed. Ki-67, ssDNA, cyclin A and cdk2 had higher labeling indices (LI) in high-risk than in low-risk cases. Cyclin E expression was greater in the intermediate- than in the low-risk grade. On Kaplan-Meier analysis, tumor size, necrosis, cellularity, Ki-67, ssDNA, and cyclin A LI were significantly correlated with disease-free survival. Necrosis, cellularity, and Ki-67 LI were significant as prognostic factors on univariate, and Ki-67 LI on multivariate Cox hazard tests. Within the high-risk grade, high cellularity and low p27(Kip1) subgroups had the worst prognosis. The histological grade is related to cell turnover, assessed in terms of Ki-67, ssDNA, cyclin A, cyclin E, and cdk2 levels. Ki-67, ssDNA, and cyclin A are useful for prediction of prognosis, with cellularity and p27(Kip1) expression as further prognostic factors in high-risk cases.

Abnormal prion protein (PrP Sc ) plays a central role in the transmission of prion diseases, but the molecular basis of prion strains with distinct biological characteristics remains to be elucidated. We analyzed the characteristics of prion disease by using mice inoculated with the Chandler and Fukuoka-1 strains propagated in a cultured mouse neuronal cell line, GT1-7, which is highly permissive to replication of the infectious agents. Strain-specific biological characteristics, including clinical manifestations, incubation period as related to the infectious unit, and pathological profiles, remained unchanged after passages in the cell cultures. We noted some differences in the biochemical aspects of PrP Sc between brain tissues and GT1-7 cells which were unlikely to affect the strain phenotypes. On the other hand, the proteinase K-resistant PrP core fragments derived from Fukuoka-1-infected tissues and cells were slightly larger than those from Chandler-infected versions. Moreover, Fukuoka-1 infection, but not Chandler infection, gave an extra fragment with a low molecular weight, ∼13 kDa, in both brain tissues and GT1-7 cells. This cell culture model persistently infected with different strains will provide a new insight into the understanding of the molecular basis of prion diversity.

Previous studies demonstrated that IL-13Ralpha2 chain-overexpressing cancer cells were highly sensitive to IL-13 cytotoxin (IL13-PE38QQR) and could be targeted by cytotoxin treatment. However, the majority of human tumors do not express high levels of IL-13Ralpha2 chain. To expand the IL-13 cytotoxin-mediated cancer targeting therapy, we combined cytotoxin treatment with gene transfer of IL-13Ralpha2 chain. We constructed a recombinant adenoviral vector carrying the human IL-13Ralpha2 gene (Ad-IL-13Ralpha2), which expresses high levels of IL-13Ralpha2 chain on infected cells. Human cancer cell lines A549 and HOS, which originally show no IL-13Ralpha2 expression and little sensitivity to IL-13 cytotoxin, were effectively converted to become sensitive to this cytotoxin after Ad-IL-13Ralpha2 infection. The CC(50) of IL-13 cytotoxin for Ad-IL-13Ralpha2-infected A549 cells was10 ng/ml, whereas the CC(50) for uninfected or control vector-infected cells was500 ng/ml. We also examined the antitumor activity of IL-13 cytotoxin in an established xenograft model of cytotoxin-resistant human lung tumor. Only a single i.t. injection of Ad-IL-13Ralpha2 markedly enhanced the sensitivity of established tumors to IL-13 cytotoxin treatment; furthermore, this antitumor effect was significantly sustained for more than 1 month after the last treatment with IL-13 cytotoxin. Taken together, these results suggest the combination of adenoviral vector-mediated IL-13Ralpha2 gene transfer and IL-13 cytotoxin administration can be an effective targeting approach for several types of IL-13 cytotoxin-resistant cancers which show no or little expression of IL-13Ralpha2 chain.

Patients with anorexia nervosa (AN) seldom present with infectious illness, despite malnutrition-induced immunodeficiency. We described two young women who had a long-standing history of severe emaciation and pulmonary or lymph node tuberculosis discovered during the treatment of AN. Both patients reported a positive history of BCG vaccination. Contact tracing failed to reveal sources of infection, although the tuberculosis was considered transferred. Since the decline of notification rates for tuberculosis have been stagnant and outbreaks in schools or hospitals have been increasing in Japan, special attention must be given to the possibility of opportunistic infections in AN patients.

Legionella pneumophila is a major cause of life-threatening pneumonia, which is characterized by a high incidence of acute lung injury and resultant severe hypoxemia. Mechanical ventilation using high oxygen concentrations is often required in the treatment of patients with L. pneumophila pneumonia. Unfortunately, oxygen itself may propagate various forms of tissue damage, including acute lung injury. The effect of hyperoxia as a cofactor in the course of L. pneumophila pneumonia is poorly understood. In this study, we show that exposure to hyperoxic conditions during the evolution of pneumonia results in a marked increase in lethality in mice with Legionella pneumonia. The enhanced lethality was associated with an increase in lung permeability, but not changes in either lung bacterial burden or leukocyte accumulation. Interestingly, accelerated apoptosis as evidenced by assessment of histone-DNA fragments and caspase-3 activity were noted in the infected lungs of mice exposed to hyperoxia. TUNEL staining of infected lung sections demonstrated increased apoptosis in hyperoxic mice, predominantly in macrophages and alveolar epithelial cells. In vitro exposure of primary murine alveolar epithelial cells to Legionella in conjunction with hyperoxia accelerated apoptosis and loss of barrier function. Fas-deficient mice demonstrated partial resistance to the lethal effects of Legionella infection induced by hyperoxia, which was associated with attenuated apoptosis in the lung. These results demonstrate that hyperoxia serves as an important cofactor for the development of acute lung injury and lethality in L. pneumophila pneumonia. Exaggerated apoptosis, in part through Fas-mediated signaling, may accelerate hyperoxia-induced acute lung injury in Legionella pneumonia.