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The overexpression of the multidrug resistance (mdr1) gene and its product, P-glycoprotein (P-gp), is thought to limit the successful chemotherapy of human tumors. Recent studies demonstrate that SN-38, a metabolite of the camptothecin (CPT) derivative CPT-11, has antitumor effects on several tumors, but the mechanisms responsible for its cytotoxicity remain unclear. We therefore determined whether SN-38 has cytotoxic effects on MDR human glioblastoma GB-1 cells and non-MDR human glioblastoma U87-MG cells. Furthermore, we determined what role SN-38 plays in the induction of cytotoxicity in these tumor cells. In this study, we demonstrated that SN-38 had significantly stronger antitumor effects on GB-1 and U-87MG cells than did CPT (P < 0.01 and P < 0.05, respectively). In addition, findings obtained using a DNA fragmentation assay, Hoechst 33258 staining, in situ end-labeling and cell cycle analysis demonstrated that SN-38 induced apoptosis in these tumors. Our results suggest that SN-38 has a stronger antitumor effect on malignant glioma cells regardless of MDR expression than does CPT, and therefore can be considered a new chemotherapeutic agent potentially effective in the treatment of human primary or recurrent malignant gliomas resistant to chemotherapy. [ABSTRACT FROM AUTHOR]

β-Glycyrrhetinic acid (BGA) is a natural antibacterial agent. Previous studies reported that BGA has antibacterial effects against several bacteria. This study evaluated the effects of BGA on the regulation of supragingival plaque bacteria. First, the minimum inhibitory concentrations (MICs) of BGA against oral bacteria were measured. Next, the minimum concentrations for inhibition of biofilm formation were evaluated against Streptococcus mutans and Streptococcus sobrinus, possessing insoluble glucan synthesis abilities. The MICs of biofilm formation by these bacteria ranged from 1/8 to 2× MIC. Furthermore, the inhibition effects of BGA against the coaggregation of Porphyromonas gingivalis and Streptococcus gordonii were evaluated. BGA at 32 or 64 μg/mL inhibited the coaggregation of these bacteria after a 30 min incubation. Lastly, the inhibition effects of BGA against human supragingival plaque bacteria were evaluated. Human supragingival plaque samples were obtained from 12 healthy donors. The inhibition effects of BGA against biofilm formation by these plaque bacteria were evaluated. Of 12 samples, the biofilm formation by 11 was significantly attenuated by 128-256 μg/mL of BGA. The number of colony forming units in these biofilms was also significantly attenuated. In conclusion, it was revealed that BGA inhibits the growth and biofilm formation of bacteria, furthermore, the same effect was confirmed with supragingival plaque bacteria. BGA is a good candidate for a natural agent that prevents the outbreak and progression of periodontal disease because it suppresses not only the growth and biofilm formation of bacteria, but also the coaggregation of P. gingivalis with plaque bacteria., (© 2021 The Societies and John Wiley & Sons Australia, Ltd.)

Nontarget screening studies have recently revealed the accumulation of typically unmonitored organohalogen compounds (OHCs) in various marine animals, but information for terrestrial food chains is still lacking. This study investigated the accumulation profiles of known and unknown OHCs in the liver of representative wild bird specimens from Osaka, Japan using nontarget analysis based on two-dimensional gas chromatography-time-of-flight mass spectrometry. A large number of unmonitored OHCs were identified, including anthropogenic contaminants and marine halogenated natural products (HNPs), and their accumulation profiles were considered to be influenced by terrestrial and brackish water-based diets. Anthropogenic OHCs were highly accumulated in terrestrial predator species (peregrine falcon, hawks, and black kite), and some unmonitored highly chlorinated contaminants reached the levels of microgram per gram lipid in the liver, i.e., C 10 -/C 15 -based chlordane related compounds (CHLs) and their epoxides, dichlorodiphenyldichloroethylene (DDE) homologues, and polychlorinated terphenyls (PCTs). In contrast, HNPs were accumulated at higher levels in piscivorous birds (gray heron and common cormorant). Considering the enrichment of the unmonitored C 10 -/C 15 -based CHLs, PCTs, and DDE homologues relative to structurally similar persistent organic pollutants (POPs) in high trophic-level species such as raptors, further studies are needed to elucidate their environmental levels, behavior in terrestrial food chains, and ecotoxicological impacts.

Ebola virus disease is a severe disease, often fatal, with a mortality rate of up to 90%. Presently, effective treatment and safe prevention options for Ebola virus disease are not available. Therefore, there is an urgent need to develop control measures to prevent or limit future Ebola virus outbreaks. Ebola virus protein-based virus-like particle (VLP) and inactivated whole virion vaccines have demonstrated efficacy in animal models, and the addition of appropriate adjuvants may provide additional benefits to these vaccines, including enhanced immune responses. In this study, we screened 24 compounds from injectable excipients approved for human use in Japan and identified six compounds that significantly enhanced the humoral response to Ebola VLP vaccine in a murine model. Our novel adjuvant candidates for Ebola VLP vaccine have already been demonstrated to be safe when administered intramuscularly or subcutaneously, and therefore, they are closer to clinical trials than adjuvants whose safety profiles are unknown., Competing Interests: Y.K. has received speaker’s honoraria from Toyama Chemical and Astellas; grant support from Chugai Pharmaceuticals, Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Otsuka Pharmaceutical, and Kyoritsu Seiyaku; and is a founder of FluGen. All of the other authors declare that they have no conflicts of interest.

To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in Madin-Darby canine kidney subclone II cells and detected two mammal-adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased the virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney subclone II, and human lung adenocarcinoma [Calu-3] cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates, in addition to the lungs. Bat influenza viruses have not been tested for their virulence or organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferrets, respectively. A mutant virus possessing both the NA-F144C and NA-T342A mutations was isolated from both the lung and the trachea, suggesting that it has a broader organ tropism than the wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that the propagation of bat influenza H18N11 virus in mammalian cells can result in mammal-adapting mutations that may increase the replicative ability and/or organ tropism of the virus; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets. IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes have been identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammal-adapting mutations that may increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate to high titers in all infected animals., (Copyright © 2020 American Society for Microbiology.)

Previous studies have suggested a possible relationship between age-related changes to human gingival hemodynamics and periodontal disease. However, firmly establishing this has been difficult because of a lack of suitable tools. Our study investigated whether a non-invasive laser speckle flowgraphy (LSFG)-based 2-dimensional technique could be used to assess maxillary anterior gingival blood flow under resting conditions. In total, 124 healthy male volunteers aged between 22 and 69 years were included in the study and delineated into young (Y; 22-37 years, n = 45), middle-aged (M; 38-53 years, n = 43), and elderly groups (E; 54-69 years, n = 36). The differences in gingival hemodynamics were compared among age groups and pulse waveform analysis performed to calculate blood flow indices, mean blur rate (MBR), gingival vascular conductance (MBR/mean blood pressure [MBP]), and three pulse waveform parameters (acceleration time index [ATI], falling rate, and blowout time [BOT]). Although no statistically significant differences were observed in the MBR of the three age groups, vascular conductance (MBR/MBP) was lower in groups M and E compared to group Y and correlated negatively with age. ATI and falling rates were also significantly higher in group E relative to group Y, whereas average BOT was significantly lower. All of the assessed parameters correlated with age. These data suggest that there are age-related decreases in the ability to maintain blood flow in the human maxillary anterior gingiva under resting conditions which may impact the likelihood of periodontal disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Influenza A and B viruses have eight-segmented, single-stranded, negative-sense RNA genomes, whereas influenza C and D viruses have seven-segmented genomes. Each genomic RNA segment exists in the form of a ribonucleoprotein complex (RNP) in association with nucleoproteins and an RNA-dependent RNA polymerase in virions. Influenza D virus was recently isolated from swine and cattle, but its morphology is not fully studied. Here, we examined the morphological characteristics of D/bovine/Yamagata/10710/2016 (D/Yamagata) and C/Ann Arbor/50 (C/AA), focusing on RNPs packaged within the virions. By scanning transmission electron microscopic tomography, we found that more than 70% of D/Yamagata and C/AA virions packaged eight RNPs arranged in the "1+7" pattern as observed in influenza A and B viruses, even though type C and D virus genomes are segmented into only seven segments. These results imply that influenza viruses generally package eight RNPs arranged in the "1+7" pattern regardless of the number of RNA segments in their genome. IMPORTANCE The genomes of influenza A and B viruses are segmented into eight segments of negative-sense RNA, and those of influenza C and D viruses are segmented into seven segments. For progeny virions to be infectious, each virion needs to package all of their genomic segments. Several studies support the conclusion that influenza A and B viruses selectively package eight distinct genomic RNA segments; however, the packaging of influenza C and D viruses, which possess seven segmented genomes, is less understood. By using electron microscopy, we showed that influenza C and D viruses package eight RNA segments just as influenza A and B viruses do. These results suggest that influenza viruses prefer to package eight RNA segments within virions independent of the number of genome segments., (Copyright © 2018 American Society for Microbiology.)

The influenza A virus genome is composed of eight single-stranded negative-sense RNAs. Eight distinct viral RNA segments (vRNAs) are selectively packaged into progeny virions, with eight vRNAs in ribonucleoprotein complexes (RNPs) arranged in a specific "1+7" pattern, that is, one central RNP surrounded by seven RNPs. Here we report the genome packaging of an artificially generated seven-segment virus that lacks the hemagglutinin (HA) vRNA. Electron microscopy shows that, even in the presence of only seven vRNAs, the virions efficiently package eight RNPs arranged in the same "1+7" pattern as wild-type virions. Next-generation sequencing reveals that the virions specifically incorporate host-derived 18S and 28S ribosomal RNAs (rRNAs) seemingly as the eighth RNP in place of the HA vRNA. These findings highlight the importance of the assembly of eight RNPs into a specific "1+7" configuration for genome packaging in progeny virions and suggest a potential role for cellular RNAs in viral genome packaging.

During December 2016-February 2017, influenza A viruses of the H7N2 subtype infected ≈500 cats in animal shelters in New York, NY, USA, indicating virus transmission among cats. A veterinarian who treated the animals also became infected with feline influenza A(H7N2) virus and experienced respiratory symptoms. To understand the pathogenicity and transmissibility of these feline H7N2 viruses in mammals, we characterized them in vitro and in vivo. Feline H7N2 subtype viruses replicated in the respiratory organs of mice, ferrets, and cats without causing severe lesions. Direct contact transmission of feline H7N2 subtype viruses was detected in ferrets and cats; in cats, exposed animals were also infected via respiratory droplet transmission. These results suggest that the feline H7N2 subtype viruses could spread among cats and also infect humans. Outbreaks of the feline H7N2 viruses could, therefore, pose a risk to public health.

Present study determined concentrations and residue patterns of bromophenols (BPhs) in whole blood samples of pet cats and pet dogs collected from veterinary hospitals in Japan. BPhs concentrations were higher in cat blood than in dog blood, with statistically insignificant differences (p = 0.07). Among the congeners, 2,4,6-tribromophenol (TBPh) constituted the majority of BPhs (>90%) detected in both species. Analysis of commercial pet food to estimate exposure routes showed that the most abundant congener in all pet food samples was 2,4,6-TBPh, accounting for >99% of total BPhs. This profile is quite similar to the blood samples of the pets, suggesting that diet might be an important exposure route for BPhs in pets. After incubation in polybrominated diphenyl ether (PBDE) mixtures (BDE-47, BDE-99 and BDE-209), 2,4,5-TBPh was found in dog liver microsomes but not in cat liver microsomes, implying species-specific metabolic capacities for PBDEs. Formation of 2,4,5-TBPh occurred by hydroxylation at the 1' carbon atom of the ether bond of BDE-99 is similar to human study reported previously. Hydroxylated PBDEs were not detected in cats or dogs; therefore, diphenyl ether bond cleavage of PBDEs can also be an important metabolic pathway for BPhs formation in cats and dogs.

The adverse effects of elevated polybrominated diphenyl ether (PBDE) levels, reported in the blood of domestic dogs and cats, are considered to be of great concern. However, the tissue distribution of PBDEs and their derivatives in these animals is poorly understood. This study determined the concentrations and profiles of PBDEs, hydroxylated PBDEs (OH-PBDEs), methoxylated PBDEs (MeO-PBDEs), and 2,4,6-tribromophenol (2,4,6-tri-BPh) in the blood, livers, bile, and brains of dogs and cats in Japan. Higher tissue concentrations of PBDEs were found in cats, with the dominant congener being BDE209. BDE207 was also predominant in cat tissues, indicating that BDE207 was formed via BDE209 debromination. BDE47 was the dominant congener in dog bile, implying a species-specific excretory capacity of the liver. OH-PBDE and MeO-PBDE concentrations were several orders of magnitude higher in cat tissues, with the dominant congener being 6OH-BDE47, possibly owing to their intake of naturally occurring MeO-PBDEs in food, MeO-PBDE demethylation in the liver, and lack of UDP-glucuronosyltransferase, UGT1A6. Relatively high concentrations of BDE209, BDE207, 6OH-BDE47, 2'MeO-BDE68, and 2,4,6-tri-BPh were found in cat brains, suggesting a passage through the blood-brain barrier. Thus, cats in Japan might be at a high risk from PBDEs and their derivatives, particularly BDE209 and 6OH-BDE47.

Many broadly reactive human monoclonal antibodies against the hemagglutinin (HA) stem of influenza A virus have been developed for therapeutic applications. These antibodies typically inhibit viral entry steps, especially the HA conformational change that is required for membrane fusion. To better understand the mechanisms by which such antibodies inhibit viral replication, we established broadly reactive human anti-HA stem antibodies and determined the properties of these antibodies by examining their reactivity with 18 subtypes of HA, evaluating their in vivo protective efficacy, identifying their epitopes, and characterizing their inhibitory mechanisms. Among the eight human monoclonal antibodies we generated, which recognized at least 3 subtypes of the soluble HA antigens tested, clone S9-1-10/5-1 reacted with 18 subtypes of HA and protected mice from lethal infection with H1N1pdm09, H3N2, H5N1, and H7N9 viruses. This antibody recognized the HA2 helix A in the HA stem, and inhibited virus particle release from infected cells but did not block viral entry completely. These results show that broadly reactive human anti-HA stem antibodies can exhibit protective efficacy by inhibiting virus particle release. These findings expand our knowledge of the mechanisms by which broadly reactive stem-targeting antibodies inhibit viral replication and provide valuable information for universal vaccine development., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Unlabelled: The genomes of influenza A and B viruses comprise eight segmented, single-stranded, negative-sense viral RNAs (vRNAs). Although segmentation of the virus genome complicates the packaging of infectious progeny into virions, it provides an evolutionary benefit in that it allows viruses to exchange vRNAs with other strains. Influenza A viruses are believed to package their eight different vRNAs in a specific manner. However, several studies have shown that many viruses are noninfectious and fail to package at least one vRNA. Therefore, the genome-packaging mechanism is not fully understood. In this study, we used electron microscopy to count the number of ribonucleoproteins (RNPs) inside the virions of different influenza A and B virus strains. All eight strains examined displayed eight RNPs arranged in a "7+1" configuration in which a central RNP was surrounded by seven RNPs. Three-dimensional analysis of the virions showed that at least 80% of the virions packaged all eight RNPs; however, some virions packaged only five to seven RNPs, with the exact proportion depending on the strain examined. These results directly demonstrate that most viruses package eight RNPs, but some do indeed package fewer. Our findings support the selective genome-packaging model and demonstrate the variability in the number of RNPs incorporated by virions, suggesting that the genome-packaging mechanism of influenza viruses is more flexible than previously thought., Importance: The genomes of influenza A and B viruses contain segmented RNAs, which complicates genome packaging but provides the evolutionary advantage of allowing the exchange of individual genome segments with those of other strains. Some studies have shown that influenza A viruses package all eight genome segments in a specific manner, whereas others have shown that many virions are noninfectious and fail to package at least one genome segment. However, such viruses have never been directly observed. Here, we used electron microscopy to provide the first direct visual evidence of virions packaging an incomplete set of ribonucleoproteins. The percentage of these noninfectious virions varied from 0 to 20, depending on the virus strain, indicating that most virions package all eight genome segments. These results extend our knowledge about how infectious and noninfectious virions coordinate for successful virus infection., (Copyright © 2016 Nakatsu et al.)

Background: Corynebacterium ulcerans is a pathogen causing diphtheria-like illness to humans. In contrast to diphtheria by Corynebacterium diphtheriae circulating mostly among humans, C. ulcerans infection is zoonotic. The present study aimed to clarify how a zoonotic pathogen C. ulcerans circulates among wild birds and animals., Results: By screening 380 birds, a single strain of toxigenic C. ulcerans was isolated from a carnivorous bird, ural owl (Strix uralensis). The bacterium was also isolated from two individuals of Japanese shrew-mole (Urotrichus talpoides), a food preference of the owl. Analysis by ribotyping showed that the owl and mole isolates were classified in a group, suggesting that C. ulcerans can be transmissible among wild birds and their prey animals. Moreover, our isolates were found to belong to a group of previously reported C. ulcerans isolates from dogs and a cat, which are known to serve as sources for human infection., Conclusion: The findings suggest that the shrew-mole may be a potential reservoir of a zoonotic pathogen C. ulcerans.

There are growing concerns about the increase in hyperthyroidism in pet cats due to exposure to organohalogen contaminants and their hydroxylated metabolites. This study investigated the blood contaminants polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) and their hydroxylated and methoxylated derivatives (OH-PCBs, OH-PBDEs, and MeO-PBDEs), in pet dogs and cats. We also measured the residue levels of these compounds in commercially available pet foods. Chemical analyses of PCBs and OH-PCBs showed that the OH-PCB levels were 1 to 2 orders of magnitude lower in cat and dog food products than in their blood, suggesting that the origin of OH-PCBs in pet dogs and cats is PCBs ingested with their food. The major congeners of OH-/MeO-PBDEs identified in both pet food products and blood were natural products (6OH-/MeO-BDE47 and 2'OH-/MeO-BDE68) from marine organisms. In particular, higher concentrations of 6OH-BDE47 than 2'OH-BDE68 and two MeO-PBDE congeners were observed in the cat blood, although MeO-BDEs were dominant in cat foods, suggesting the efficient biotransformation of 6OH-BDE47 from 6MeO-BDE47 in cats. We performed in vitro demethylation experiments to confirm the biotransformation of MeO-PBDEs to OH-PBDEs using liver microsomes. The results showed that 6MeO-BDE47 and 2'MeO-BDE68 were demethylated to 6OH-BDE47 and 2'OH-BDE68 in both animals, whereas no hydroxylated metabolite from BDE47 was detected. The present study suggests that pet cats are exposed to MeO-PBDEs through cat food products containing fish flavors and that the OH-PBDEs in cat blood are derived from the CYP-dependent demethylation of naturally occurring MeO-PBDE congeners, not from the hydroxylation of PBDEs.

Objective: We evaluated the diagnostic performance of computed tomography (CT) as an initial radiologic test for assessing the optimal timing of colonoscopy in patients with acute lower gastrointestinal bleeding (LGIB) and investigated the effectiveness of contrast-enhanced (CE) CT for detecting colonic diverticular bleeding., Methods: This was a retrospective study of 1,604 consecutive patients who visited or were referred to St. Marianna University Hospital due to acute LGIB and underwent colonoscopy within three months after presentation between September 2004 and December 2012. The clinicopathological data of the subjects were obtained from their medical records., Results: Among the 1,604 patients presenting with LGIB, 879 (55%) underwent a CT scan. Elective colonoscopy was considered in cases in which typical colonic wall thickening was observed on CT, suggesting colonic inflammation or malignancy (239 patients; 27%). The diagnoses in the elective cases included ischemic colitis (38%), infectious colitis (8%), inflammatory bowel disease (8%) and malignancy (5%). Urgent colonoscopy was performed after the CT examination in 640 cases (73%). The most common presumptive CT diagnosis was diverticulum (402/640; 63%). Of the 638 patients who underwent CE-CT, diverticula were observed in 346 cases, including 104 cases of extravasation indicating ongoing diverticular bleeding. Among these 104 patients, the site of bleeding was identified in 71 subjects (68%) during colonoscopy. The rate of detection of the bleeding source on colonoscopy was significantly higher in the patients with extravasation on CE-CT than in those without extravasation on CE-CT (68% vs. 20%, respectively; p<0.001)., Conclusion: Urgent CT is useful for determining the optimal timing of colonoscopy in cases of acute LGIB. CE-CT may be used to depict the presence and location of active hemorrhage and provides useful information for subsequent colonoscopy, especially in patients with diverticular bleeding.

Unlabelled: Freeze-thaw impregnation is a technique used for the rapid impregnation of substances into foodstuffs. Freeze-thaw impregnation with macerating enzymes has been applied to soften foodstuffs, while retaining their original shapes and flavors. In this study, we found that co-impregnation with citrate ions and macerating enzymes significantly facilitated the softening of root crops. When burdock roots were processed by the impregnating solution at pH 4.0-5.0, co-impregnated burdock roots exhibited 1/6-1/3 firmness values compared with burdock roots impregnated with only enzymes. The impregnation with citrate ions alone at pH 4.0 to 5.0 did not soften burdock roots. The firmness of burdock roots was positively correlated with the amount of water-insoluble calcium in the samples. The results suggested that the degradation of pectins by pectinolytic activities could promote contact with citrate to bridging-calcium ions interacting with the pectin chains. Therefore, the softening by the synergistic effect of citrate ions and macerating enzymes was related to the amount of pectins contained in root crops. That is, the synergistic effect was significant with burdock roots and carrots (from which 50% of polysaccharides are pectins) unlike with lotus rhizomes and bamboo shoots (from which 30% and 10% of polysaccharides are pectins, respectively)., Practical Application: Freeze-thaw impregnation with macerating enzymes and citrate ions can be applied for the production of care foods which can be eaten without chewing. The softened products induce the pleasure of eating for consumers because their original shapes and flavors are retained., (© 2014 Institute of Food Technologists®)

Purpose: This report describes an attempt to reduce the expression level of Hanganutziu-Deicher (H-D) antigens by small interfering RNA (siRNA) for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (pCMAH)., Methods: A pig endothelial cell (PEC) line, and PEC and fibroblasts from an α1,3galactosyltransferase knockout (GalT-KO) piglet were used. Real-time PCR was used to evaluate the degradation of mRNA by siRNA. The H-D antigen was stained, and then the cells were incubated with human serum for the FACS analysis. The extent of lysis in human serum was next calculated using an LDH assay., Results: Suppression of the mRNA of pCMAH by each siRNA was first determined. The mixture of siRNAs for pCMAH reduced the expressions of the H-D antigen on the PEC and fibroblasts to a considerable extent. The further reduction in the xenoantigenicity for human serum of the GalT-KO cells was then confirmed. In addition, the PEC line showed a significant downregulation in complement-dependent cytotoxicity by the siRNAs, thus indicating that the anti-H-D antigen in human serum is capable of causing lysis of the pig cells., Conclusion: pCMAH silencing by siRNA reduced the expression of the H-D antigen and its antigenicity, thus confirming that the H-D antigen is one of the major non-Gal antigens in this situation.

Background: Besides α-Gal expression, the differences of glycosylation and antigenicity between adult pig islets (APIs) and neonatal porcine islet-like cell clusters (NPCCs) are altogether unclear. In this study, lectin microarray analyses of NPCCs were performed and the results compared with the corresponding values for wild-type APIs and NPCCs from α-Gal transferase knockout (GalT-KO) pig., Methods: NPCCs were isolated from 1-3-d-old neonatal wild-type pigs and cultured for 1 d, 5 d, and 9 d, using a previously described technique. Alternatively, the isoration of APIs were isolated based on the method for human islets., Results: In a comparison between NPCCs and APIs, all of the NPCCs showed higher signals for Sambucus nigra, Sambucus sieboldiana, and Trichosanthes japonica I and the binding of α2,6 sialc acid, whereas the APIs showed stronger signals for Lotus tetragonolobus, Aleuria aurantia, Narcissus pseudonarcissus, and Galanthus nivalis, suggesting that APIs contain high levels of high-mannose forms. Among the NPCCs, NPCC (day1) appeared to be richer than the others in Lotus tetragonolobus, Narcissus pseudonarcissus, Galanthus nivalis, and Urtica dioica, implying the presence of high-mannose forms. However, as a whole, the signals for many lectins for NPCCs were very similar. The NPCCs from a GalT-KO pig indicated not only the downregulation of α-Gal expression but α-GalNAc as well, and α2-6 sialic acid was upregulated., Conclusions: The results reported herein contain useful information for the future production of immunomodified pigs with less antigenicity than GalT-KO pigs toward clinical applications of NPCCs., (Copyright © 2013. Published by Elsevier Inc.)

Residue levels and patterns of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), their hydroxylated metabolites (OH-PCBs, OH-PBDEs), and methoxylated PBDEs (MeO-PBDEs) in the blood of various terrestrial mammals in Japan, including cats, raccoon dogs, dogs, masked palm civets, foxes, raccoons, badgers, and mongooses were determined. Tri- through penta-chlorinated OH-PCBs were predominant in cat blood, whereas hexa- through octa-chlorinated OH-PCBs were found in other species. High proportion of BDE209 was found in all species, suggesting exposure to municipal waste and soil containing higher levels of deca-BDE products. 6OH-/MeO-BDE47 and 2'OH-/MeO-BDE68 were dominant in all terrestrial mammals. This is first report on the detection of OH-/MeO-PBDEs in the blood of terrestrial mammals. High concentrations of OH-/MeO-PBDEs were found in cats, suggesting the intake of these compounds from seafood. Cats exhibited higher accumulation and specific patterns of OH-PCBs, OH-PBDEs, and MeO-PBDEs, they may be at a high risk from these metabolites., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aim: To elucidate the colonoscopic features of serrated lesions of the colorectum using magnifying colonoscopy., Methods: Broad division of serrated lesions of the colorectum into hyperplastic polyps (HPs), traditional serrated adenomas (TSAs), and sessile serrated adenomas/polyps (SSA/Ps) has been proposed on the basis of recent molecular biological studies. However, few reports have examined the colonoscopic features of these divisions, including magnified colonoscopic findings. This study examined 118 lesions excised in our hospital as suspected serrated lesions after magnified observation between January 2008 and September 2011. Patient characteristics (sex, age), conventional colonoscopic findings (location, size, morphology, color, mucin) and magnified colonoscopic findings (pit pattern diagnosis) were interpreted by five colonoscopists with experience in over 1000 colonoscopies, and were compared with histopathological diagnoses. The pit patterns were categorized according to Kudo's classification, but a more detailed investigation was also performed using the subclassification [type II-Open (type II-O), type II-Long (type II-L), or type IV-Serrated (type IV-S)] proposed by Kimura T and Yamano H., Results: Lesions comprised 23 HPs (23/118: 19.5%), 39 TSAs (39/118: 33.1%: with cancer in one case), 50 SSA/Ps (50/118: 42.4%: complicated with cancer in three cases), and six others (6/118: 5.1%). We excluded six others, including three regular adenomas, one hamartoma, one inflammatory polyp, and one juvenile polyp for further analysis. Conventional colonoscopy showed that SSA/Ps were characterized as larger in diameter than TSAs and HPs (SSA/P vs HP, 13.62 ± 8.62 mm vs 7.74 ± 3.24 mm, P < 0.001; SSA/Ps vs TSA, 13.62 ± 8.62 mm vs 9.89 ± 5.73 mm, P < 0.01); common in the right side of the colon [HPs, 30.4% (7/23): TSAs, 20.5% (8/39): SSA/P, 84.0% (42/50), P < 0.001]; flat-elevated lesion [HPs, 30.4% (7/23): TSAs, 5.1% (2/39): SSA/Ps, 90.0% (45/50), P < 0.001]; normal-colored or pale imucosa [HPs, 34.8% (8/23): TSAs, 10.3% (4/39): SSA/Ps, 80% (40/50), P < 0.001]; and with large amounts of mucin [HPs, 21.7% (5/23): TSAs, 17.9% (7/39): SSA/Ps, 72.0% (36/50), P < 0.001]. In magnified colonoscopic findings, 17 lesions showed either type II pit pattern alone or partial type II pit pattern as the basic architecture, with 14 HPs (14/17, 70.0%) and 3 SSA/Ps. Magnified colonoscopy showed the type II-O pit pattern as characteristic of SSA/Ps [sensitivity 83.7% (41/49), specificity 85.7% (54/63)]. Cancer was also present in three lesions, in all of which a type VI pit pattern was also present within the same lesion. There were four HPs and four TSAs each. The type IV-S pit pattern was characteristic of TSAs [sensitivity 96.7% (30/31), specificity 89.9% (72/81)]. Cancer was present in one lesion, in which a type VI pit pattern was also present within the same lesion. In our study, serrated lesions of the colorectum also possessed the features described in previous reports of conventional colonoscopic findings. The pit pattern diagnosis using magnifying colonoscopy, particularly magnified colonoscopic findings using subclassifications of surface architecture, reflected the pathological characteristics of SSA/Ps and TSAs, and will be useful for colonoscopic diagnosis., Conclusion: We suggest that this system could be a good diagnostic tool for SSA/Ps using magnifying colonoscopy.

The soil-borne bacterial pathogen Ralstonia solanacearum invades a broad range of plants through their roots, resulting in wilting of the plant, but no effective protection against this disease has been developed. Two bacterial wilt disease-inhibiting compounds were biochemically isolated from tobacco and identified as sclareol and cis-abienol, labdane-type diterpenes. When exogenously applied to their roots, sclareol and cis-abienol inhibited wilt disease in tobacco, tomato and Arabidopsis plants without exhibiting any antibacterial activity. Microarray analysis identified many sclareol-responsive genes in Arabidopsis roots, including genes encoding or with a role in ATP-binding cassette (ABC) transporters, and biosynthesis and signaling of defense-related molecules and mitogen-activated protein kinase (MAPK) cascade components. Inhibition of wilt disease by sclareol was attenuated in Arabidopsis mutants defective in the ABC transporter AtPDR12, the MAPK MPK3, and ethylene and abscisic acid signaling pathways, and also in transgenic tobacco plants with reduced expression of NtPDR1, a tobacco homolog of AtPDR12. These results suggest that multiple host factors are involved in the inhibition of bacterial wilt disease by sclareol-related compounds.

Porcine endogenous retrovirus (PERV) is a major problem associated with successful clinical xenotransplantation. In our previous study, reducing the high mannose type of N-glycan content proved to be very effective in downregulating PERV infectivity. In this study, dolichyl-phosphate mannosyltransferase (D-P-M), an enzyme related to the early stages of N-linked sugar synthesis was studied. The pig cDNA of the encoding D-P-M was newly isolated. The RNA interference (siRNA) for the D-P-M was applied and transfected to PEC(Z)/PB cells, a pig endothelial cell line with the Lac Z gene and PERV-B, to reduce the levels of high mannose type N-glycans. Compared with the mock line, the temporary PEC(Z)/PB lines showed a decreased mRNA expression for pig D-P-M, and each line then showed a clear destruction of PERV infectivity to human cells in the Lac Z pseudotype assay. The PEC(Z)/PB was next transfected with pSXGH-siRNA, H1-RNA gene promoter. The established PEC(Z)/PB clones with pSXGH-siRNA clearly led to the downregulation of PERV infectivity, as evidenced by the decreased levels of the mRNA for pig D-P-M. Reducing D-P-M enzyme activity represents a potentially useful approach to address the problem of PERV infections in clinical xenotransplantations.

We evaluated the therapeutic effect of gemcitabine (GEM) therapy in 153 unresectable pancreatic cancer (UPC) patients, divided into younger patients (under 65), early-stage elderly patients (age 65-74) and advanced-stage elderly patients (age 75 and over). Among those patients who received only best supportive care (BSC), the most common reasons to be selected for BSC were family requests in the advanced-stage elderly patients, and poor general condition in the rest. Among the patients who received GEM therapy, there were no significant differences in response rate, or adverse events including the rate of dose reduction, postponement or cessation of GEM administration due to toxicity. Multivariate analysis using patient backgrounds and response to GEM therapy showed that CA 19-9 response and performance status did not change with age. GEM therapy for both early-stage and advanced-stage elderly UPC patients was as safe and useful as in younger patients.

Background: Expression of complement regulatory proteins (CRP) on pig endothelial cells (PEC) is an effective means of avoiding induction of hyperacute rejection by human sera. However, pig endogenous retrovirus (PERV) from PEC transfected with CRP may acquire resistance to human sera. This study investigated a form of transfected CRP that is easily expressed on PERV particles., Methods: The PEC line was transfected with the Lac Z gene and PERV-B to investigate PERV infectivity using a Lac Z pseudo-type assay. The cDNAs of several modified DAF (CD55) were then transfected into the PEC(Lac Z)/P-B lines using lipofection. DAF expression was verified by FACS analysis. Complement-dependent PEC lysis was tested to verify the complement regulatory function of the expressed DAF. HEK293 cells were incubated with PEC culture supernatants with or without human sera. The inoculated 293 cells were histochemically stained and Lac Z-positive blue foci were counted. The rate of reduction in Lac Z-positive cells resulting from the addition of human serum was then calculated. In addition, to assess the localization of the expressed DAF, flotation sucrose density analysis was performed., Results: While PERV released from PEC expressing delta-short consensus repeat 2 (delta-SCR2) DAF (lacking CRP function) showed no change in resistance to human serum compared to control cells, PERV from cells expressing delta-SCR1 DAF (with CRP function) showed a significant increase in resistance. The DAF-blocking antibody assay indicated that PERV from the DAF transfectants expressed DAF molecules on the surface of the retrovirus. While delta-SCR1 DAF (PI-anchor form) significantly inhibited the reduction of Lac Z-positive cells by human serum, the reduction of Lac Z-positive cells by human serum was less inhibited in the case of transmembrane (TM)-types of DAF-HLA-G, modified influenza hemagglutinin (HA) and MCP (delta-CYT form). However, the reduction in each TM-type DAF was slightly less than that observed in naive and mock cells. The flotation sucrose density analysis of these transfectants indicated that the PI-anchor form of DAF is a raft-associated protein, and most TM-types of DAF are non-raft proteins., Conclusion: Induction of resistance to human serum in PERV, depends on the form of the CRP tail. The CRP/TM hybrid that does not associate with lipid rafts, is a suitable form of CRP for gene transduction.

The present study investigated concentrations and patterns of brominated flame retardants, such as polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs), and persistent organochlorines (OCs) in liver and adipose tissues of raccoon dogs (RD: Nyctereutes procyonoides) collected from two metropolises and a local prefecture in Japan during 2001-2006. Relatively high concentrations of PBDEs were found in RD livers, while HBCD levels were the lowest among the measured organohalogen compounds. Among PBDE congeners, BDE 209 was predominant in RDs from all the regions, indicating that pollution derived from the technical decaBDE product is extensive across Japan. On the other hand, concentrations of tetra- to nona-BDE congeners in RDs from a metropolis were significantly higher than those from the other two regions, implying that there were regional differences in the past usage of the technical tetraBDE and octaBDE products. Such a regional difference was also observed for HBCD levels. Lipid-normalized concentration ratios of liver to adipose tissue (L/A ratio) for tri to hepta-BDE congeners were lower than 1.0 in the investigated eight RDs, suggesting lipid-dependent accumulation. However, the LA ratios of BDE 209 exceeded 1.0 in all the specimens, suggesting hepatic retention of this compound. In addition, lipid-dependent accumulation of a-HBCD was observed, but the L/A ratios of gamma-HBCD were greater than 1.0 in some specimens. These results indicate that Japanese RDs have been recently exposed to BDE 209 and gamma-HBCD and accumulated both these compounds preferentially in blood-rich organs, probably due to their binding to proteins and/or rapid biotransformation, as reported in experimental rodents.

The activity of granzyme B, a main effector molecule of natural killer (NK) cells and cytotoxic T lymphocytes, is regulated by the intracellular serine proteinase inhibitor 9 (PI-9). Pig PI-9 was first cloned, and the sequences that encode pig PI-9, including the start codon and stop codon, were identified. The cDNA was inserted into the cloning site of pCXN2 (chicken beta actin promoter and cytomegalovirus enhancer), transfected into pig endothelial cells (PEC), and several stable PEC clones were established. An NK cell-mediated cytolysis test was next applied to the PEC clones, using YT cells (an NK-like cell line). The PEC transfectants with pig PI-9 had a significant inhibitory effect on NK cell-mediated PEC lysis. The overexpression of the anti-apoptotic molecule, pig PI-9, has the potential for use in protecting graft cells from human NK cells.