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In this study, the swelling behaviors of recycled and neutralized soils derived from surplus construction materials, commonly referred to as construction sludge in Japan were investigated. Investigations are performed on the properties of recycled soils treated with cement and paper sludge ash for stabilization and sometimes neutralized with carbon dioxide to reduce their high alkalinity. Although the quality control during the manufacturing process confirms that both recycled soil and neutralized-recycled soil (recycled soil neutralized with CO2) meet Japanese soil environmental standards and strength requirements, their use is often avoided owing to concerns regarding their expansive nature. However, the expansive nature of recycled soil and neutralized recycled soil has not been investigated in detail. Therefore, the focus of this study is on understanding how the treatments in the manufacturing of recycled soil and neutralized recycled soil affect its potential to swell when immersed in water, which is critical for ensuring the suitability of the material for construction applications. The swelling potentials of recycled and neutralized recycled soils after compaction were evaluated using a series of soaking tests. Furthermore, the underlying mechanisms were investigated through cone index tests and microstructural and crystallographic analyses. These findings indicate that recycled soils exhibit a reduced tendency to swell compared to untreated clayey soils, which is attributed to their enhanced structural integrity. In contrast, neutralized soils, especially those treated early with carbon dioxide, exhibit a higher propensity for swelling, which is linked to reduced cohesion and changes in the hydrate composition. This study underscores the importance of controlling the manufacturing and compaction processes of recycled soils, particularly the CO2 curing time and degree of saturation during compaction, to mitigate swelling risks. Although secondary-treated soils demonstrate high strength, they also exhibit swelling under certain conditions, thus highlighting the need for the careful consideration of the hydration and compaction processes. These insights contribute to a broader understanding of recycled soil behavior, thus advocating for informed construction practices and further research to facilitate the effective and sustainable use of recycled construction materials.

To utilize industrial by-products in construction projects, soil stabilization using paper sludge ash-based stabilizers (PSASs) has recently been developed. PSASs can be manufactured by insolubilizing the heavy metals in original paper sludge (PS) ash, which is a waste product emitted from paper mills. This study assessed the durability of clays treated with PSASs in wet-dry or dry-wet environments through various evaluation tests. Initially, the particle size distributions (PSDs) in the clays that had undergone various pretreatments were assessed. Then, unconfined compression tests were performed on the treated clays that had undergone dry and wet curing cycles using demolded specimens. Finally, cone index tests were carried out on the treated clays that had undergone dry and wet curing cycles while the samples were constrained in molds. Based on the test results, the durability assessment of the clays treated with PSASs was discussed, considering the specifics of each evaluation test and contrasting the findings with those obtained for clays treated with cement. The generated PSDs were found to contain more fine particles as the time spent washing the samples prior to sieving was increased. The findings indicated that PSAS-treated clays will eventually become muddy, even though it is unlikely that these treated clays will ever be subjected to washing with water while being stirred after construction. The unconfined compression test results also demonstrated that, after several dry-wet cycles, the strength of the PSAS-treated specimens had decreased. It was revealed that clays treated with PSASs might be less resistant to dry-wet curing cycles than those treated with cement. However, PSAS-treated samples using unconfined compression test specimens are still being investigated and are a topic of debate because the strength development mechanisms of PSASs and cement are not similar. To address this issue, a series of cone index tests were conducted on samples treated with PSASs to examine the change in strength caused by the dry-wet curing process. The results of the cone index tests were different from those of the unconfined compression tests. The cone index test results revealed that the samples must be constrained during the assessment tests in order to assess the durability of PSAS-treated soils subjected to dry-wet curing. In addition, it was proposed in this study that the idea of maturity would be applicable to PSAS-treated soils with a range of curing temperatures and curing times as long as the soils were constrained during the assessment tests.

Liquefied soil stabilization methods have been applied in a wide range of construction projects especially in Japan for the effective geotechnical reuse of construction-generated soil. However, after the placement of the liquefied stabilized soil is complete, water often rises to the surface and causes the bleeding phenomenon. Chromium oxide, which is produced in the manufacturing process of iron ore and steel scrap, is sometimes added to the cement solidifier (OPC solidifier) that is mixed into the liquefied stabilized soil. The environmental conditions under which the cement solidifier and construction-generated soil encounter each other change the transformation of the chromium oxides, i.e., they leach as hexavalent chromium. Hexavalent chromium is more likely to be leached when the pH is low or the moisture content is high. These are some of the environmental issues related to the application of liquefied stabilized soil. In this study, the addition of a thickener for liquefied stabilized soil is proposed; it is used to prevent segregation while maintaining fluidity. Specifically, the authors conduct various laboratory experiments, and then perform several physical analyses using the specimens after the experiments to investigate how the addition of the thickener has changed the properties of the liquefied stabilized soil. The authors also examine the properties of each type of liquefied stabilized soil by comparing it to normal liquefied stabilized soil when the amount of OPC solidifier in the liquefied stabilized soil is reduced, and instead, a solidifier mixing with waste glass powder and blast furnace slag is used to make the liquefied stabilized soil.

Recently, a technology was developed in Japan to accelerate the carbonation of alkaline construction sludge and reduce its alkalinity. Alkaline construction sludge is treated in advance using a paper sludge ash-based stabilizer (PSAS) so that it becomes granular and well aerated. Subsequently, CO2 gas is injected from the bottom to the top of the treated sludge layer in the underground pit. This technology can reduce the pH of alkaline sludge within short periods; however, the completion period of pH neutralization has often been predicted empirically. This may be due to the combined effects of density, water content, particle size, PSAS amount in the sludge, and CO2 concentration in the accelerated carbonation on the pH neutralization process. However, there have been few studies on how these factors affect the pH neutralization and CO2 capture of PSAS-treated alkaline sludges subjected to accelerated carbonation. Therefore, in this study, the effects of several accelerated carbonation factors on neutralization and CO2 capture from alkaline sludge were experimentally investigated. PSAS-treated alkaline sludges with different densities, water contents, particle sizes, and amounts of PSAS were cured in incubators with controlled CO2 concentrations. Based on the pH measurements, the pH neutralization completion period (tN) was evaluated for each alkaline sludge sample. The test results revealed that tN increased with increasing dry density and particle size and decreasing CO2 concentration. The results also suggest that tN increases with increasing amounts of PSAS. However, the water content had a more significant effect on tN than the PSAS amount. There may be an optimum water content for pH neutralization if conditions other than the water content are the same. From the results of the carbonate content evaluation, the amounts of CO2 captured in each specimen (mCO2) and mCO2 at tN were determined for each alkaline sludge sample. The results revealed that the density, water content, particle size of the alkaline sludge, and CO2 concentration in the incubators did not significantly affect the amount of CO2 captured by the sludge (mCO2) upon the completion of pH neutralization. However, the amount of PSAS significantly affects the captured CO2 content. A higher amount of PSAS caused mCO2 to increase at tN.

In this study, a novel testing method was developed to evaluate the water absorption and retention of waste-based stabilizers using suction filtration. Suction filtration was seen to remove most of the unabsorbed and unretained water from the waste-based stabilizer without losing any waste-based stabilizer particles. Therefore, oven-drying of the wet stabilizer, which can deteriorate the stabilizer, was not necessary. In addition, a formula was introduced to estimate the residual free water in the stabilizer after suction filtration based on the particle size. This estimate was used as a correction to determine the water absorption and retention rate (Wab) of the stabilizer. Subsequently, newly developed tests were conducted on fly ash (FA), a paper sludge ash-based stabilizer (PSAS), biomass ash (BMA), and the PSAS–BMA hybrid to evaluate Wab. The results show that the PSAS exhibited the highest Wab, followed by the BMA and FA. This is attributed to the CaO content of each stabilizer, which may have initiated hydration reactions. The Wab of the PSAS–BMA hybrid was similar to that of the PSAS, which is attributed to the synergistic effects of the components of each type of waste. Finally, the strength development of the FA, PSAS, BMA, and PSAS–BMA hybrid-treated clays was investigated via cone index tests. In addition to the stabilizer addition ratio (A), the Wab of the stabilizers is suggested to be an essential indicator for predicting the strength development of clays treated with waste-based stabilizers. Parameter β, which is the product of Wab and A, governs the cone index qc of the treated clays instead of A and Wab. Thus, the modified water content, w*, which considers the absorbed and retained water as a solid instead of a liquid, was applied to the cone index test results. The results show that the qc of the treated clays is more correlated to w* than to the measured water content. An empirical relationship for predicting cone index qc using w* was also proposed.

Recently, the capture of carbon dioxide (CO2) using alkaline waste and byproducts has garnered considerable interest. Construction sludge may be categorized as alkaline waste, as it often exhibits high alkalinity during its generation or intermediate treatment. Hence, researchers have attempted to accelerate pH neutralization and recycle alkaline construction sludge by curing it under a high CO2 concentration. By exposing concentrated CO2 gas to an alkaline sludge, cement hydrates such as calcium hydroxide and calcium–silicate–hydrate gels form calcium carbonate (CaCO3). Subsequently, the generation of CaCO3 is expected to reduce the pH of the sludge. However, the amount of CO2 captured in sludge has not been investigated extensively, unlike those of other alkaline wastes. Therefore, the amount of CO2 captured in alkaline sludge that is associated with pH neutralization is evaluated in this study. It is demonstrated that accelerated carbonation tests using a CO2 incubator and carbonate content evaluation tests based on the gas pressure method successfully reveal the amount of CO2 captured in the alkaline sludge that is associated with pH neutralization. Additionally, the test results show that the amount of mCO2 (i.e., the amount of CO2 captured per 1 g of dry mass of alkaline sludge) increases with ΔpH (ΔpH is the difference between the initial pH and the pH after the alkaline sludge is neutralized). A maximum of 0.021 g of CO2 is captured per 1 g of dry mass of alkaline sludge when the addition ratio of quicklime AQL = 3% and 0.040 g when AQL = 6%. The CO2 capture ratio mCO2/mCO2max, which represents the ratio of CO2 captured in the sludge to the maximum capturing capacity, increases with ΔpH. CO2 capture ratios of up to 90.0% and 84.9% are recorded when AQL = 3% and AQL = 6%, respectively. It is discovered that a higher AQL results in a higher mCO2. Moreover, the test results indicate that a higher AQL causes a more significant change in the CO2 capture ratio, even when the pH decreases slightly.

Construction sludge generated from underground and pile construction works frequently appears in a liquid state. For its utilization as a construction geomaterial, it can be improved by mixing in cement or lime. The target strength can be achieved relatively easily by adjusting the amount of cement or lime added to the sludge. However, this type of chemical treatment is associated with high alkalinity, which causes environmental concerns associated with alkali leaching. In this study, therefore, the accelerated carbonation of alkaline construction sludge through treatment with a paper-sludge ash-based stabilizer (PSAS) was experimentally attempted to neutralize the pH. The alkaline sludge treated with the PSAS was exposed to pure CO2 gas to accelerate the carbonation. The effects of granulation via crumbling prior to the accelerated carbonation on the pH neutralization period were analyzed from the test results. It was observed that crumbling the sludge prior to the CO2 gas exposure accelerated the pH neutralization. These results suggest that a short pH neutralization period can be achieved by reducing the particle size. Therefore, the effect of the particle size of the PSAS-treated sludge on the pH neutralization period was also investigated. The results showed that the pH neutralization period decreased with an increasing particle size. However, it was also observed that, when the addition ratio of the PSAS was increased, the difference in the pH neutralization period induced by the change in particle size became less significant.

Soil stabilization using paper sludge ash-based stabilizers (PSASs) has been developed as a technique for using the sustainable materials generated from industrial processes in construction projects. PSASs can be produced by insolubilizing the heavy metals in original paper sludge (PS) ash particles, i.e., the waste generated by the incineration of the PS discharged from paper mills. The aim of this study was to investigate the effects of the primary curing conditions and subsequent crumbling on the physical, compaction, and strength characteristics of PSAS-treated soils, using two types of PSASs with different water absorption and retention performances. For comparison, the same investigation was conducted on soils treated with blast furnace cement type B (BFCB). The experimental results revealed that, after crumbling, the PSAS-treated samples produced sand and gravel-like granules, regardless of the sealed or air primary curing conditions. In addition, the lower the water content at crumbling, the smaller the particle size. This was also true for the BFCB-treated samples. Consequently, the compaction test results demonstrated that, for one of the two types of PSASs, the dry densities of the PSAS-treated samples with sealed primary curing were almost the same as those without primary curing. The same trend was observed for the BFCB-treated samples. However, for the other type of PSAS, the dry densities of the PSAS-treated samples with sealed primary curing were lower than those without primary curing. This could be due to the difference in the degrees of disturbance caused by crumbling, depending on the type of PSAS. The rapid formation of hydrates in one type of PSAS may have significantly disturbed the treated samples owing to crumbling, resulting in a decrease in dry density. Finally, after secondary curing in a soaked environment, cone index tests were conducted on the PSAS- and BFCB-treated samples. The results indicated that the cone indices of the PSAS-treated samples with primary curing were higher or lower than those of the samples without primary curing, depending on the primary curing environment, number of curing days, and type of PSAS. The different trends, depending on the conditions, were considered to be caused by the combined effects of the “strength reduction owing to crumbling,” and “strength increase owing to water content reduction at compaction.” These mechanisms suggest that, for PSAS-treated soils with early strength development, the strength reduction caused by crumbling must be considered. However, for PSAS-treated soils with slow strength development, adjusting the water content of the treated soils through primary curing before compaction is an effective approach. Moreover, it is suggested that the curing conditions used for the laboratory mixtures be designed and set to reflect the field conditions and to minimize any discrepancies between the field and laboratory observations for the PSAS treatment.

In an attempt to reduce environmental impact, paper sludge ash (PS ash) has recently been studied for its complementary reuse with cement for soil stabilization. In order to establish the optimal mixture design for combining PS ash and cement in soils, a detailed investigation into the stabilizing mechanism is required. To assess the combined effects of PS ash and cement on the strength development of stabilized clay soil, referred to as PS ash–cement-treated clay, a new critical parameter, the unabsorbed and unretained clay-water/cement ratio W*/C, was proposed. To determine W*/C, a new testing method for evaluating the water absorption and retention performance of PS ash was developed. It was revealed that the water absorption and retention rate Wab of PS ash increased with curing time. Unconfined compression tests conducted on the PS ash–cement-treated clay with various water-cement–PS ash mixture proportions and different curing times affirmed that the strength development was fundamentally governed by the parameter W*/C. This suggests that the water absorption and retention rate Wab obtained by the developed method is an essential material parameter in the mixture design for the PS ash–cement-treated clay. It was also found that the effect of the hybrid treatment method, which uses both cement and PS ash, was better than that of the method which uses cement alone, particularly under high W*/C conditions. This indicates that the water absorption and retention performance of PS ash can be fully utilized when the mixture has sufficient unabsorbed and unretained water for cement hydration.

High-temperature stress during grain filling hastens the growth rate of endosperm and causes grain chalkiness. Scanning microscopy of chalky areas reveals loosely packed, rounded starch granules with occasional small pits. Intensive investigation of the transcriptome, proteome, and metabolome in developing caryopses under high-temperature stress revealed the downregulation of starch synthesis enzymes and the upregulation of α-amylases. High-temperature ripening may unbalance the synthesis and degradation of starch in the developing endosperm cells. In addition to starches, storage proteins are synthesized, assembled, and stored in developing seeds. Several lines of evidence suggest that redox regulation affects seed maturation, including the accumulation of storage starches and proteins, and thus grain quality. A heat-tolerant cultivar of rice shows a characteristic high expression of superoxide dismutase (SOD). H2O2 produced by SOD under high-temperature stress possibly acts as a signal that rapidly can promote the expression of stress-response proteins. Herein, we will discuss the possible molecular physiology of grain chalking under high-temperature stress.

Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic α-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional α-amylase genes, identification of the α-amylase(s) that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify α-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight α-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most α-amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of α-amylase genes, except for Amy3E, in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E-overexpressing grains contained enhanced α-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by α-amylases. Moreover, the chalky grains contained increased amounts of soluble sugars including maltooligosaccharides at the expense of starch. The integrated analyses proposed that expression of Amy1A, Amy3C, and Amy3D at the specific regions of the developing endosperm could generate the chalkiness. This finding provides the fundamental knowledge to narrow down the targets for the development of high temperature-tolerant premium rice.

Jasmonic acid (JA) transiently accumulated during temperature-dependent synchronous necrotic lesion formation in Tobacco mosaic virus-infected tobacco leaves. The accumulation of JA was preceded by activation of a tobacco mitogen-activated protein kinase, WIPK, which functions upstream of JA in wound signal transduction pathways.

In Tobacco mosaic virus (TMV)-infected tobacco plants carrying the N resistance gene, a hypersensitive reaction or response (HR) occurs to enclose the virus in the infected tissue. Although a contribution of peroxidases to the resistance has been proposed, no evidence has been presented that tobacco peroxidase genes respond to HR. Here, we describe the HR-induced expression of a tobacco peroxidase gene (tpoxC1) whose induction kinetics were slightly different from those of acidic and basic tobacco pathogenesis-related (PR) protein genes. Interestingly, tpoxC1 was insensitive to the inducers of PR genes such as salicylic acid, methyl jasmonate, and ethephon. Spermine activated tpoxC1 gene expression at a low level and both acidic and basic PR gene expression at a considerably higher level. These results indicate that the induced expression of tpoxC1 is regulated differently from that of classical tobacco PR genes in the N gene-mediated self-defense system in tobacco plants.

Abstract Background Understanding swine influenza virus (SIV) ecology has become more and more important from both the pig industry and public health points of views. However, the mechanism whereby SIV occurs in pig farms is not well understood. The purpose of this study was to develop a proper strategy for SIV surveillance. Findings We conducted longitudinal monitoring in 6 farrow-to-finish farms in the central region of Thailand from 2008 to 2009. Nasal swabs and serum samples were collected periodically from clinically healthy pigs consisting of sows, fattening pigs, weaned piglets and pigs transferred from other farms. A total of 731 nasal swabs were subjected to virus isolation and 641 serum samples were subjected to detection of SIV antibodies against H1 and H3 subtypes using the hemagglutination inhibition test and ELISA. Twelve SIVs were isolated in this study and eleven were from piglets aged 4 and 8 weeks. Phylogenetical analysis revealed that SIVs isolated from different farms shared a common ancestor. Antibodies against SIVs were detected in fattening pigs on farms with no SIV isolation in the respective periods studied. These observations suggested that piglets aged 8 weeks or younger could be a main target for SIV isolation. Farm-to-farm transmission was suggested for farms where pigs from other farms are introduced periodically. In addition, antibodies against SIVs detected in fattening pigs could be a marker for SIV infection in a farm. Conclusions The present study provided important information on SIV surveillance that will enable better understanding of SIV ecology in farrow-to-finish farms.

A comprehensive and quantitative evaluation of multiple intracellular structures or proteins is a promising approach to provide a deeper understanding of and new insights into cellular polarity. In this study, we developed an image analysis pipeline to obtain intensity profiles of fluorescent probes along the apical-basal axis in elongating Arabidopsis thaliana zygotes based on two-photon live-cell imaging data. This technique showed the intracellular distribution of actin filaments, mitochondria, microtubules, and vacuolar membranes along the apical-basal axis in elongating zygotes from the onset of cell elongation to just before asymmetric cell division. Hierarchical cluster analysis of the quantitative data on intracellular distribution revealed that the zygote may be compartmentalized into two parts, with a boundary located 43.6% from the cell tip, immediately after fertilization. To explore the biological significance of this compartmentalization, we examined the positions of the asymmetric cell divisions from the dataset used in this distribution analysis. We found that the cell division plane was reproducibly inserted 20.5% from the cell tip. This position corresponded well with the midpoint of the compartmentalized apical region, suggesting a potential relationship between the zygote compartmentalization, which begins with cell elongation, and the position of the asymmetric cell division., (© 2023. The Author(s).)

DNA topoisomerase II (TOP2) is an enzyme that resolves DNA topological problems and plays critical roles in various nuclear processes. Recently, a heterozygous H58Y substitution in the ATPase domain of human TOP2B was identified from patients with autism spectrum disorder, but its biological significance remains unclear. In this study, we analyzed the nuclear dynamics of TOP2B with H58Y (TOP2B H58Y). Although wild-type TOP2B was highly mobile in the nucleus of a living cell, the nuclear mobility of TOP2B H58Y was markedly reduced, suggesting that the impact of H58Y manifests as low protein mobility. We found that TOP2B H58Y is insensitive to ICRF-187, a TOP2 inhibitor that halts TOP2 as a closed clamp on DNA. When the ATPase activity of TOP2B was compromised, the nuclear mobility of TOP2B H58Y was restored to wild-type levels, indicating the contribution of the ATPase activity to the low nuclear mobility. Analysis of genome-edited cells harboring TOP2B H58Y showed that TOP2B H58Y retains sensitivity to the TOP2 poison etoposide, implying that TOP2B H58Y can undergo at least a part of its catalytic reactions. Collectively, TOP2 H58Y represents a unique example of the relationship between a disease-associated mutation and perturbed protein dynamics., (© 2022. The Author(s).)

Pathogenic FLNA variants can be identified in patients with seizures accompanied by periventricular nodular heterotopia (PVNH). It is unusual to find FLNA aberrations in epileptic patients without PVNH on brain imaging. We report a boy with cryptogenic West syndrome followed by refractory seizures and psychomotor delay. We performed whole-exome sequencing and identified a de novo missense variant in FLNA. It is noteworthy that this patient showed no PVNH. As no other pathogenic variants were found in epilepsy-related genes, this FLNA variant likely caused West syndrome but with no PVNH.

Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.

A total of 300 nasal swabs were collected from 5 pig farms in two provinces in the Eastern part of Thailand in February 2011 and were subjected to viral isolation of influenza A viruses. Two H3N2 and 6 H1N1 influenza A viruses were isolated from swabs collected from clinically healthy weaning pigs on farms in Chonburi and Chachoengsao provinces, respectively. The H3N2 isolates consisted of the hemagglutinin (HA) and neuraminidase (NA) genes closely related to Thai SIVs and derived from a cluster of human seasonal H3N2 strains circulating around 1996-1997. The remaining gene segments of the isolates originated from the Pandemic (H1N1) 2009 (A (H1N1) pdm09) virus. Antigenicity of the H3N2 isolates was distinguishable from a human seasonal vaccine strain in the 1996-1998 seasons that represented antigenicity of the seasonal strains around 1996-1998. Nasal swabs from a Chachoengsao farm yielded A (H1N1) pdm09 viruses in chicken embryonated eggs and MDCK cells. A (H1N1) pdm09 viruses isolated in this study grew poorly in MDCK cells. Deduced amino acid sequences of the HA1 region of the HA protein of egg isolated viruses were identical to the sequences directly amplified from original swab samples. Our result demonstrated that the A (H1N1) pdm09 virus has been established in the Thai pig population and this has resulted in genetic reassortment with Thai SIV that previously circulated among pigs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Surveillance of swine influenza viruses (SIVs) in 31 pig farms in northern and southern parts of Vietnam was conducted. Six H3N2 influenza A viruses were isolated from a pig farm in southern Vietnam. They were novel genetic reassortants between a triple-reassortant SIV and a human seasonal H3N2 virus. Their hemagglutinin and neuraminidase genes were derived from a human virus circulating around 2004-2006 and the remaining genes from a triple-reassortant SIV that originated in North America. This is the first report describing the isolation of a novel triple-reassortant SIV in Vietnam., (© 2011 Blackwell Publishing Ltd.)

In Thailand, highly pathogenic avian influenza (HPAI) viruses of subtype H5N1 had been isolated from various wild birds during the HPAI outbreak in poultries. In this study, we examined the pathogenicity of two wild bird isolates (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) in mice. They showed similar replication in several organs and lethal outcome. However, on day 3 post-infection, Pigeon04 induced mRNA expression of proinflammatory cytokines (IL6 and TNFα) and MIP-2, neutrophil chemoattractant, in the lungs, resulting in severe pneumonia that was accompanied by neutrophil infiltration. In contrast, on day 7 post-infection, T.sparrow05 induced the expression of several cytokines to a greater extent than Pigeon04; it also potently induced mRNA expression of several cytokines in brains of the infected mice that triggered frequent inflammatory events. In sum, our study demonstrated that two HPAI viruses induced different host responses, despite having similar replications, resulting in lethal outcome in mice., (Copyright © 2010 Elsevier Inc. All rights reserved.)

From February to March 2009, six strains of H7N6 subtype avian influenza virus were isolated from quails in three farms in Aichi prefecture in Japan. The isolates were shown to be low pathogenic for chicken by the examination performed using the "Manual of Standards for Diagnostic Tests and Vaccines" by World organisation for Animal Health (OIE). The deduced amino acid sequence at the cleavage site was PE (I/Q/L) PKRR (nucleotide sequences were cct gaa (a/c) (t/a) a cc (a/g) aaa aga aga), suggesting persistence in domestic poultry for some time. The direct putative ancestor strain could not be elucidated by phylogenetic analysis of all genome segments of the quail isolates. Diverged date from a putative common ancestor in a non-rooted phylogenetic tree among quail viruses was estimated between March 2002 and July 2004. Three putative N-linked glycosylation sites resided in the vicinity of the receptor binding pocket of HA1 region. They are considered to decrease the reactivity of neutralizing antibody against the virus. Experiments for the infectivity and pathogenicity of a quail strain to poultry indicated that the quail isolate had higher infectivity to quails than chickens and ducks. Direct and dust-borne and/or droplet-borne transmissions among quail were proven in quails with and without direct contact with experimentally infected quails. The virus is seldom transmitted among chickens either directly or indirectly, and indirect transmission from infected quails to chickens was not observed. The pathogenicity of the quail strain for mammalian, pig and mouse was low, although it could replicate in those animals., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Highly pathogenic avian influenza virus (HPAIV) of the H5N1 subtype has been reported to infect pigeons asymptomatically or induce mild symptoms. However, host immune responses of pigeons inoculated with HPAIVs have not been well documented. To assess host responses of pigeons against HPAIV infection, we compared lethality, viral distribution and mRNA expression of immune related genes of pigeons infected with two HPAIVs (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) isolated from wild birds in Thailand. The survival experiment showed that 25% of pigeons died within 2 weeks after the inoculation of two HPAIVs or medium only, suggesting that these viruses did not cause lethal infection in pigeons. Pigeon04 replicated in the lungs more efficiently than T.sparrow05 and spread to multiple extrapulmonary organs such as the brain, spleen, liver, kidney and rectum on days 2, 5 and 9 post infection. No severe lesion was observed in the lungs infected with Pigeon04 as well as T.sparrow05 throughout the collection periods. Encephalitis was occasionally observed in Pigeon04- or T.sparrow05-infected brain, the severity, however was mostly mild. To analyze the expression of immune-related genes in the infected pigeons, we established a quantitative real-time PCR analysis for 14 genes of pigeons. On day 2 post infection, Pigeon04 induced mRNA expression of Mx1, PKR and OAS to a greater extent than T.sparrow05 in the lungs, however their expressions were not up-regulated concomitantly on day 5 post infection when the peak viral replication was observed. Expressions of TLR3, IFNα, IL6, IL8 and CCL5 in the lungs following infection with the two HPAIVs were low. In sum, Pigeon04 exhibited efficient replication in the lungs compared to T.sparrow05, but did not induce excessive host cytokine expressions. Our study has provided the first insight into host immune responses of pigeons against HPAIV infection.

Since the Pandemic H1N1 2009 (H1N1pdm) influenza virus emerged in human in 2009, H1N1pdm, classical swine H1, Eurasian avian-like H1, human-like H1 and human-like H3 swine influenza viruses have circulated in pig populations, and avian H9N2 viruses have been isolated in pigs as well. In this study, TaqMan single-step real-time reverse transcription-PCR (rtRT-PCR) assays targeting the hemagglutinin gene were developed to differentiate H1N1pdm from other genetic lineages of the H1 subtype and other subtypes of influenza viruses circulating in human and pig populations for veterinary use. H1N1pdm rtRT-PCR detected H1N1pdm RNA and did not cross-react with classical swine H1, Eurasian avian-like H1, human-like H1, human-like H3 swine and avian H9 influenza viruses RNA. Classical swine H1, Eurasian avian-like H1, human-like H1 and H3 and avian H9 rtRT-PCR were reacted exclusively with viral RNA of their respective lineages and subtypes. The results demonstrate that these assays are useful for the diagnosis of the H1N1pdm virus in both human- and animal-health-related fields., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The etiology of nocturia in 70 patients with benign prostatic hyperplasia (BPH) who had nocturia of two or more times were examined based on frequency volume charts (FVC). Nocturia was classified into four groups: nocturnal polyuria, low capacity, combined nocturia, and no evidence of abnormality. Nearly half of the cases had nocturnal polyuria only. A little under 70% of patients had associated nocturnal polyuria (nocturnal polyuria+combined nocturia). Naftopidil was administered for three months to the patients with BPH who had nocturia with a urinary frequency of two or more times. Clinical efficacy was evaluated in 32 patients based on FVC and naftopidil was shown to improve nocturia. The improvement in nocturia was determined by the increment in voided volume.

Avian influenza A H5N1 viruses were isolated from humans for the first time in Hong Kong in 1997. The virulence of A/Hong Kong/156/97 (HK156) strain in mice was found to change significantly depending on the passage history of the virus. Madin-Darby canine kidney (MDCK) cell-grown parental virus and three of its clones derived from mouse brain showed high pathogenicity in mice after intranasal or intracerebral infection. In contrast, the egg-derived parental virus HK156-E3 and its cloned viruses were markedly less pathogenic in mice. It appeared that differences in pathogenicity among viruses derived from MDCK cells and eggs were due to their ability or inability to disseminate from the lungs to the brain. Sequence analysis of the entire protein coding regions of all eight RNA genome segments revealed a total of six conserved amino acid differences in the HA1 domain (residue 211) of the HA protein, as well as the PB1 (residues 456 and 712), PA (residue 631), NP (residue 127), and NS1 (residue 101) proteins that correlated with observed changes in virulence and neurovirulence of HK156 virus in mice. Thus it was evident that the passaging of HK156 in embryonated eggs led to the adaptation and selection of variants demonstrating markedly decreased pathogenicity and neurovirulence in mice that appeared to be attributable to specific amino acid changes in the HA and internal proteins., (Copyright 2000 Academic Press.)

The entire nucleotide sequences of all six internal genes of six human H5N1 influenza A viruses isolated in Hong Kong in 1997 were analysed in detail from a phylogenetic point of view and compared with the evolutionary patterns of the haemagglutinin and neuraminidase genes. Despite being isolated within a single year in the same geographical location, human H5N1 viruses were characterized by a variety of amino acid substitutions in the ribonucleoprotein complex [PB2, PB1, PA and nucleoprotein (NP)] as well as the matrix (M) proteins 1 and 2 and nonstructural (NS) proteins 1 and 2. The presence of previously reported amino acid sequences specific for human strains was confirmed in the PB2, PA, NP and M2 proteins. Nucleotide and amino acid sequence identities of the six internal genes of H5N1 viruses examined here were separated into at least two variant groups. In agreement with the above result, phylogenetic trees of the six internal genes of human H5N1 viruses were generally composed of two minor clades. Additionally, variable dendrogram topologies suggested that reassortment among viruses contributed further to the genetic variability of these viruses. As a result, it became clear that human H5N1 viruses are characterized by divergent gene constellations, suggesting the possible occurrence of genetic reassortment between viruses of the two evolutionary lineages.

Phylogenetic patterns of the three polymerase (PB2, PB1 and PA) genes of a total of 20 influenza B viruses isolated during a 58 year period, 1940-1998, were analysed in detail in a parallel manner. All three polymerase genes consistently showed evolutionary divergence into two major distinct lineages and their amino acid profiles demonstrated conserved lineage-specific substitutions. Dendrogram topologies of the PB2 and PB1 genes were very similar and contrasted with that of the PA gene. It was of particular interest to reveal that even though the PA gene evolved into two major lineages, that of three recent Asian Victoria/1/87-like strains formed a branch cluster located in the same lineage as that of recent Yamagata/16/88-like isolates. Differences in the phylogenetic pathways of three polymerase genes were not only a reflection of genetic reassortment between co-circulating influenza B viruses, but also an indication that the polymerase genes were not co-evolving as a unit. As a result, comparison of the phylogenetic patterns of the three polymerase genes with previously determined patterns of the HA, NP, M and NS genes of 18 viruses defined the existence of eight distinct genome constellations. Also, similar phylogenetic profiles among the PA, NP and M genes, as well as between the PB2 and PB1 genes, were observed, suggesting possible functional interactions among these proteins. Completion of evolutionary analysis of the six internal genes and the HA gene of influenza B viruses revealed frequent genetic reassortment among co-circulating variable strains and suggested co-dependent evolution of genes.

Phylogenetic profiles of the genes coding for the hemagglutinin (HA) protein, nucleoprotein (NP), matrix (M) protein, and nonstructural (NS) proteins of influenza B viruses isolated from 1940 to 1998 were analyzed in a parallel manner in order to understand the evolutionary mechanisms of these viruses. Unlike human influenza A (H3N2) viruses, the evolutionary pathways of all four genes of recent influenza B viruses revealed similar patterns of genetic divergence into two major lineages. Although evolutionary rates of the HA, NP, M, and NS genes of influenza B viruses were estimated to be generally lower than those of human influenza A viruses, genes of influenza B viruses demonstrated complex phylogenetic patterns, indicating alternative mechanisms for generation of virus variability. Topologies of the evolutionary trees of each gene were determined to be quite distinct from one another, showing that these genes were evolving in an independent manner. Furthermore, variable topologies were apparently the result of frequent genetic exchange among cocirculating epidemic viruses. Evolutionary analysis done in the present study provided further evidence for cocirculation of multiple lineages as well as sequestering and reemergence of phylogenetic lineages of the internal genes. In addition, comparison of deduced amino acid sequences revealed a novel amino acid deletion in the HA1 domain of the HA protein of recent isolates from 1998 belonging to the B/Yamagata/16/88-like lineage. It thus became apparent that, despite lower evolutionary rates, influenza B viruses were able to generate genetic diversity among circulating viruses through a combination of evolutionary mechanisms involving cocirculating lineages and genetic reassortment by which new variants with distinct gene constellations emerged.

This study was initiated with the isolation of influenza A and B viruses from clinical throat swabs in both fertile chicken eggs (egg) and MDCK cells, which were used in subsequent vaccine production in the above two hosts. On the basis of haemagglutination-inhibiting (HI) tests, immune mouse sera from mice vaccinated with MDCK cell-derived vaccines revealed antigenic similarities among H3N2 or B viruses isolated in MDCK cells or eggs. Similarly, antiserum prepared by immunization with egg-derived H3N2 vaccine showed equivalent antigenicity between homologous and heterologous (MDCK cell-derived) viruses. In contrast, antigenicity of egg-derived B vaccines was differed somewhat from that of MDCK cell-derived vaccines, suggesting the occurrence of antigenic change due to passaging in eggs. The time-course of immune responses based on HI titres indicated that MDCK cell-derived vaccines elicited extremely high antibody levels. Also, it was evident that antibody production by MDCK cell-grown H3N2 vaccine was very similar to that of vaccine prepared from egg-grown viruses. These results were comparable to those of plaque neutralization tests, although antigenic differences between egg- and MDCK cell-derived challenge viruses were confirmed in the test with antiserum to MDCK cell-derived vaccine. Consistent with HI-antibody production, the immunogenicity of MDCK cell-derived B vaccine appeared to be low by plaque neutralization test, while immune responses in mice which received egg-derived vaccines were significantly higher than that of the former. Furthermore, immune responses confirmed in mice immunized with B virus vaccines prepared in eggs revealed slight antigenic differences between two viruses derived from their respective hosts. Nevertheless, through evaluation of immune responses, MDCK cell-derived influenza vaccines may be useful when weak immunogenicity of B virus vaccine is improved.

Nucleotide sequences of all eight RNA segments of 10 human H3N2 influenza viruses isolated during a 5-year period from 1993 to 1997 were determined and analyzed phylogenetically in order to define the evolutionary pathways of all genes in a parallel fashion. It was evident that the hemagglutinin and neuraminidase genes of these viruses evolved essentially in a single lineage and that amino acid changes accumulated sequentially with respect to time. In contrast, amino acid differences in the internal proteins were erratic and did not accumulate over time. Parallel analysis of the phylogenetic patterns of all genes revealed that the evolutionary pathways of the six internal genes were not linked to the surface glycoproteins. Genes coding for the basic polymerase-1, nucleoprotein, and matrix proteins of 1997 isolates were closest phylogenetically to those of earlier isolates of 1993 and 1994. Furthermore, all six internal genes of four viruses isolated in the 1995 epidemic season consistently divided into two distinct branch clusters, and two 1995 isolates contained PB2 genes apparently originating from those of viruses before 1993. It was apparent that the lack of correlation between the topologies of the phylogenetic trees of the genes coding for the surface glycoproteins and internal proteins was a reflection of genetic reassortment among human H3N2 viruses. This is the first evidence demonstrating the occurrence of genetic reassortment involving the internal genes of human H3N2 viruses. Furthermore, internal protein variability coincided with marked increases in the activity of H3N2 viruses in 1995 and 1997.

We measured prostatic acid phosphatase (PAP), gamma-seminoprotein (gamma-Sm) and prostatic specific antigen (PSA) levels simultaneously in the serum of 52 patients with untreated prostatic cancer and 44 patients with benign prostatic hypertrophy to assess the clinical usefulness of these tumor markers. PAP and PSA were measured by radioimmunoassay and gamma-Sm by enzyme immunoassay. The positive rates of PAP, gamma-Sm and PSA in patients with prostatic cancer were 50.0, 61.5 and 69.2%, respectively, and those in patients with benign prostatic hypertrophy were 11.4, 13.6 and 13.6%, respectively. In patients with early stage prostatic cancer (stage A and B), the positive rates of PAP, gamma-Sm and PSA were 20.8, 41.7 and 54.2%. The efficiency of PSA was the highest among the three markers. The positive rate of the combination assay of PAP and PSA, that of gamma-Sm and PSA and that of PAP, gamma-Sm and PSA were slightly higher than that of the PSA assay alone. However, the efficiency of the PSA assay alone was higher than that of any combination. No significant correlation was found between histopathological grade and the level of each tumor marker. A significant correlation was found between PAP and gamma-Sm (r = 0.68, P less than 0.001), and between PAP and PSA (r = 0.61, P less than 0.001), but there was no correlation between gamma-Sm and PSA. These results suggest that PSA is the most useful marker and the combination assay of multiple markers is not so advantageous, at least for screening of prostatic cancer.

We reviewed the outcome of 300 cases without urinary tract infections to determine the relationship between leucocytosis and severe pain due to ureteral calculi. Leucocytosis above 9,000 cells per cmm was seen in 87.7% of the cases. Many patients (61%) had a leucocyte count between 9,000 and 12,000, but none had that exceeding 22,000. This leucocytosis can be attributed to a transient distributional alteration by means of leucopheresis to circulation sites from a storage pool such as the blood vessels in the spleen, liver, lung, etc. Since leucocytosis often accompanies ureteral calculi, caution should be exercised in distinguishing it from appendicitis.

The level of serum gamma-seminoprotein (gamma-Sm) was measured by enzyme immunoassay in 62 patients with untreated prostatic cancer and 89 patients with benign prostatic hypertrophy histologically diagnosed to assess the clinical usefulness as a tumor marker. The level of serum prostatic acid phosphatase (PAP) was also measured by radioimmunoassay in these patients simultaneously. Serum gamma-Sm levels in prostatic cancer were significantly higher than in benign prostatic hypertrophy. There was a tendency for serum gamma-Sm levels in prostatic cancer to increase with statistically significant difference as the stage progressed. A gamma-Sm level of over 5.0 ng/ml was considered to be positive. The positive rate of gamma-Sm was 56.5% in prostatic cancer (stage A.B: 32.3%, stage C: 75.0%, stage D: 90.9%) and 19.1% in benign prostatic hypertrophy. In stage A.B cases, the positive rate of gamma-Sm was higher than that of PAP. Therefore, the measurement of gamma-Sm is considered to be useful in the diagnosis of early prostatic cancer.