A Novel 16S rRNA PCR-Restriction Fragment Length Polymorphism Assay to Accurately Distinguish Zoonotic Capnocytophaga canimorsus and C. cynodegmi.

The zoonotic bacteria Capnocytophaga canimorsus and C. cynodegmi, the predominant Capnocytophaga species in the canine oral biota, can cause human local wound infections or lethal sepsis, usually transmitted through dog bites. Molecular surveying of these Capnocytophaga species using conventional 16S rRNA-based PCR is not always accurate due to their high genetic homogeneity. In this study, we isolated Capnocytophaga spp. from the canine oral cavity and identified them using 16S rRNA and phylogenetic analysis. A novel 16S rRNA PCR-restriction fragment length polymorphism (RFLP) method was designed based on our isolates and validated using published C. canimorsus and C. cynodegmi 16S rRNA sequences. The results showed that 51% of dogs carried Capnocytophaga spp. Among these, C. cynodegmi (47/98, 48%) was the predominant isolated species along with one strain of C. canimorsus (1/98, 1%). Alignment analysis of 16S rRNA sequences revealed specific site nucleotide diversity in 23% (11/47) of the C. cynodegmi isolates, which were misidentified as C. canimorsus using previously reported species-specific PCR. Four RFLP types could be classified from all the isolated Capnocytophaga strains. The proposed method demonstrates superior resolution in distinguishing C. cynodegmi (with site-specific polymorphism) from C. canimorsus and especially in distinguishing C. canimorsus from other Capnocytophaga species. After in silico validation, this method was revealed to have an overall detection accuracy of 84%; notably, accuracy reached 100% in C. canimorsus strains isolated from human patients. Overall, the proposed method is a useful molecular tool for the epidemiological study of Capnocytophaga in small animals and for the rapid diagnosis of human C. canimorsus infections. IMPORTANCE With the increased number of small animal breeding populations, zoonotic infections associated with small animals need to be taken more seriously. Capnocytophaga canimorsus and C. cynodegmi are part of common biota in the mouths of small animals and can cause human infections through bites or scratches. In this study, C. cynodegmi with site-specific 16S rRNA sequence polymorphisms was erroneously identified as C. canimorsus during the investigation of canine Capnocytophaga by conventional PCR. Consequently, the prevalence of C. canimorsus is incorrectly overestimated in epidemiological studies in small animals. We designed a new 16S rRNA PCR-RFLP method to accurately distinguish zoonotic C. canimorsus from C. cynodegmi. After validation against published Capnocytophaga strains, this novel molecular method had high accuracy and could detect 100% of C. canimorsus-strain infections in humans. This novel method can be used for epidemiological studies and the diagnosis of human Capnocytophaga infection following exposure to small animals.

Comparative electrocardiographic study of the Asian freshwater box turtle Cuora flavomarginata and the Asian yellow pond turtle Mauremys mutica using non-invasive methods.

Background: Evaluation of cardiac function is an integral part of clinical examination of chelonians. However, information about electrocardiography (ECG) in turtles and tortoises is limited and fragmentary. Its application is limited due to the lack of ECG reference values. This study aimed to compare specific ECG parameters using non-invasive methods in the Asian box turtle Cuora flavomarginata (CF) and the Asian yellow pond turtle Mauremys mutica (MM). Methods: We included 116 clinically healthy and conscious turtles. Two non-invasive methods, using adhesive patches or crocodile clips, for ECG were applied where possible. The ambient temperature was within the preferred optimum temperature zone of both species. We used specific digital ECG monitoring equipment to record the ECG data and analysed the data using specific software. Results: The MM group showed better ECG quality and lower heart rate than the CF group. Comparing both methods, the adhesive patches method yielded higher ECG quality in the CF group, while the crocodile clips method yielded higher ECG quality in the MM group. Conclusions: The study population was selected as presumed healthy turtles; the presence of systemic or cardiac disease could not be excluded completely due to limited investigation. Both ECG methods were clinically potentially useful for obtaining ECG parameters; the ECG quality was influenced by the method used.

Risk Factors for Antimicrobial Resistance of Staphylococcus Species Isolated from Dogs with Superficial Pyoderma and Their Owners.

The microbial communities on the skin of dogs include several species of bacteria, which contribute to skin health and disease. Staphylococcus pseudintermedius, cultured at high frequency from the skin of dogs, is an opportunistic pathogen causing superficial pyoderma. Effective treatment against S. pseudintermedius infections is an important issue in veterinary medicine. However, multiple antibiotic-resistant mechanisms gradually developed by bacteria make treatment more challenging nowadays. Drug-resistant genes may have the chance to be transferred from infected dogs to other staphylococci in humans. The objective of this survey is to investigate the bacterial species that cause canine superficial pyoderma and characterize the antibiotic-resistant profiles and drug-resistant genes of isolated S. pseudintermedius. In addition, the possible risk factors causing S. pseudintermedius colonizing owners were also evaluated by a questionnaire survey. Sixty-five bacteria were isolated from dogs with superficial pyoderma, which included 47 S. pseudintermedius (72.3%), 12 other staphylococci (18.5%), 4 other Gram-positive bacteria (6.2%) and 2 Gram-negative bacteria (3.1%). Strains containing mecA and blaZ genes showed multiple-drug resistance characteristics. Dogs that received antimicrobial treatment within a recent month were at significantly higher risk of MRSP infections. Only five S. pseudintermedius strains (8.33%) were isolated from 60 samples of owners. Risk factor analysis indicated there was no significant association between S. pseudintermedius isolated from dogs and owners, but the "Keeping three or more dogs" and "Dogs can lick the owner's face" have high odds ratios of 3.503 and 5.712, respectively. MRSP isolates belonged to three different dru types, including dt11y (29.41%), dt11a (47.06%) and dt10cp (23.53%). In conclusion, the major pathogen of canine superficial pyoderma is found to be S. pseudintermedius in Taiwan, and isolates which are mecA- or blaZ-positive are generally more resistant to commonly used antibiotics. Although S. pseudintermedius isolated from the owners might be transferred from their dogs, definite risk factors should be examined in the future study.

Cobalt protoporphyrin promotes human keratinocyte migration under hyperglycemic conditions.

BACKGROUND: Complete healing of diabetic wounds continues to be a clinically unmet need. Although robust therapies such as stem cell therapy and growth factor treatment are clinically applied, these treatments are costly for most diabetic wound patients. Therefore, a cheaper alternative is needed. Cobalt protoporphyrin (CoPP) has recently been demonstrated to promote tissue regeneration. In this study, the therapeutic benefits of CoPP in diabetic wound healing were examined. METHODS: An in vitro wound healing model that mimics re-epithelialization was established to examine the effect of CoPP on the migratory capability of human keratinocytes (HaCaT) in either normal glucose (NG) or high glucose (HG) media, as well as in the presence of either H2O2 or lipopolysaccharide (LPS). At the end of the migration assays, cells were collected and subjected to Western blotting analysis and immunostaining. RESULTS: HaCaT were found to migrate significantly more slowly in the HG media compared to the NG media. CoPP treatment was found to enhance cell migration in HG media, but was found to decrease cell migration and proliferation when HaCaT were cultured in NG media. CoPP treatment induced high levels of expression of Nrf-2/HO-1 and FoxO1 in HaCaT cultured in either glucose concentration, although the FoxO1 expression was found to be significantly higher in HaCaT that underwent the migration assay in NG media compared to those in HG media. The higher level of FoxO1 expression seen in CoPP-treated HaCaT cultured in NG media resulted in upregulation of CCL20 and downregulation of TGFß1. In contrast, HaCaT migrated in HG media were found to have high levels of expression of TGFß1, and low levels of expression of CCL20. Interestingly, in the presence of H2O2, CoPP-pretreated HaCaT cultured in either NG or HG media had similar expression level of Nrf-2/HO-1 and FoxO1 to each other. Moreover, the anti-apoptotic effect of CoPP pretreatment was noticed in HaCaT cultured in either glucose concentration. Additionally, CoPP pretreatment was shown to promote tight junction formation in HaCaT suffering from LPS-induced damage. CONCLUSIONS: CoPP enhances cell migratory capacity under hyperglycemic conditions, and protects cells from oxidative and LPS-induced cellular damage in HG media containing either H2O2 or LPS.

HPLC/ESI-MS and NMR Analysis of Chemical Constitutes in Bioactive Extract from the Root Nodule of Vaccinium emarginatum.

Vaccinium emarginatum Hayata is a medicinal plant that has been historically used in ethnopharmacy to treat diseases in Taiwan. The objective of this study is to evaluate the anti-cancer and anti-bacterial constitutes from the root nodule extract of V. emarginatum. The chemical composition of V. emarginatum fractions was analyzed by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and the chemical constitutes were isolated and structurally identified by nuclear magnetic resonance (NMR) spectroscopy. Bioassay-guided chromatography showed that the ethyl acetate (EA) fraction was bioactive on the hepatocellular carcinoma (HepG2). By LC-ESI-MS/MS analysis, twenty peaks of EA fraction were partially identified and the phytochemical investigation of the fractions led to the isolation and identification of protocatuchuic acid (1), epicatechin (2), catechin (3), procyanidin B3 (4), procyanidin A1 (5), hyperin (6), isoquercetin (7), quercetin (8), lupeol (9), beta-amyrin (10), and alpha-amyrin (11). Both procyanidin B3 and A1 exhibited anti-proliferative activity against HepG2 and gastric adenocarcinoma (AGS) cells at IC50 values between 38.4 and 41.1 µM and 79.4 and 83.8 µM, respectively. In addition, isoquercetin displayed the strongest anti-proliferative activity against the HepG2, lung carcinoma (A549), and AGS cell at 18.7, 24.6 and 68.5 µM, respectively. Among the triterpenoids, only lupeol showed the inhibitory activity against all tested tumor cell lines at IC50 values between 72.9 and 146.8 µM. Furthermore, procyanidins B3, A1 and isoquercetin displayed moderate anti-bacterial activity against Staphylococcus aureus. In conclusion, this study provides background information on the exploitation of V. emarginatum as a potential natural anti-cancer and anti-bacterial agent in pharmaceutical research.

GluN2B/CaMKII mediates CFA-induced hyperalgesia via HDAC4-modified spinal COX2 transcription.

Histone deacetylase 4 (HDAC4), which actively shuttles between the nucleus and cytoplasm, is an attractive candidate for a repressor mechanism in epigenetic modification. However, the potential role of HDAC4-dependent epigenetics in the neural plasticity underlying the development of inflammatory pain has not been well established. By injecting complete Freund's adjuvant (CFA) into the hind-paw of Sprague-Dawley rats (200-250 g), we found animals displayed behavioral hyperalgesia was accompanied with HDAC4 phosphorylation and cytoplasmic redistribution in the dorsal horn neurons. Cytoplasmic HDAC4 retention led to its uncoupling with the COX2 promoter, hence prompting spinal COX2 transcription and expression in the dorsal horn. Moreover, the GluN2B-bearing N-methyl-d-aspartate receptor (GluN2B-NMDAR)/calmodulin-dependent protein kinase II (CaMKII) acted as an upstream cascade to facilitate HDAC4 phosphorylation/redistribution-associated spinal COX2 expression after inflammatory insults. The results of this pilot study demonstrated that the development and/or maintenance of inflammatory pain involved the spinal HDAC4-dependent epigenetic mechanisms. Our findings open up a new avenue for the development of a novel medical strategy for the relief of inflammatory pain.

Investigation of gastrointestinal parasites of dairy cattle around Taiwan.

BACKGROUND: Parasitic nematodes are one of the most important causes of production losses in most cattle-producing countries of the world. The aim of the present study is to make a through estimate of helminth and protozoan infection prevalence in dairy cattle around Taiwan. METHODS: Coprological techniques, including direct fecal smear, simple flotation, and simple sedimentation, were used to detect gastrointestinal helminths and protozoan in dairy cattle. A total of 1259 rectal fecal samples were collected from Holstein dairy cattle at 94 farms in 13 counties in Taiwan. RESULTS: The overall prevalence of gastrointestinal parasitic infection was 86.9%. The infection rates of protozoa, nematodes, trematodes, and cestodes were 81.3%, 7.9%, 1.6%, and 0.6%, respectively. Among all parasites, Buxtonella sulcata (61.7%) was the most predominant one, followed with Cryptosporidium spp. (32.6%) and Eimeria spp. (11.8%). There were significant differences in the prevalence of protozoa and nematodes between different age groups and distributional area groups. CONCLUSION: The present study demonstrated that gastrointestinal parasitic infections occur frequently in dairy cattle around Taiwan, especially protozoan infections. The results indicated that a superior management system and regular anthelmintic treatment should be used for the control of parasitic infections in dairy cattle farms.

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach.

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.

Study of the endoparasitic fauna of commensal rats and shrews caught in traditional wet markets in Taichung City, Taiwan.

BACKGROUND: Rats live in close proximity to human populations. Feral rodents are known to transmit diseases and act as reservoir hosts to many zoonotic parasites that pose health risks to humans. The aim of this study is to investigate endoparasitic infections in commensal rats and shrews caught in traditional wet markets in Taichung City, Taiwan. METHODS: A total of 51 commensal wild rodents and shrews were caught in traditional wet markets in Taichung City, including 32 Rattus norvegicus, 11 R. rattus, and eight Suncus murinus. All tissues, organs, and intestinal contents were carefully examined after euthanasia for the detection of parasites. RESULTS: The overall prevalence of infection was 94.1%, and the infection rates in R. norvegicus, R. rattus, and S. murinus were 93.8%, 90.9%, and 100.0%, respectively. Four cestodes (Taenia taeniaeformis, Hymenolepis diminuta, H. nana, and Raillietina celebensis), seven nematodes (Angiostrongylus cantonensis, Capillaria hepatica, Heterakis spumosa, Nippostrongylus brasiliensis, Strongyloides ratti, Syphacia muris, and Trichosomoides crassicauda), and one protozoan (Sarcocystis spp.) were detected. CONCLUSION: Our findings indicate that commensal rodents and shrews found in the traditional wet markets of Taichung City are hosts to various zoonotic parasites and, therefore, pose a serious health risk to humans and domestic animals in Taiwan.

Proteomic approaches to study epigallocatechin gallate-provoked apoptosis of TSGH-8301 human urinary bladder carcinoma cells: roles of AKT and heat shock protein 27-modulated intrinsic apoptotic pathways.

Epigallocatechin-3-gallate (EGCG), a polyphenol constituent present in green tea, has been shown to inhibit the growth of cancer cells in vitro and in vivo. However, studies regarding human bladder carcinoma cells are limited and not well investigated. Hence, our study focused on the evaluation of EGCG-triggered apoptosis in TSGH-8301 human urinary bladder carcinoma cells in vivo and in vitro as well as its related molecular mechanisms. In an in vivo study, EGCG inhibited xenograft tumor size of TSGH-8301 cells in a nude mouse model. Based on an in vitro study, EGCG resulted in morphological changes and increased growth inhibition in a dose- and time-dependent manner in TSGH-8301 cells. Furthermore, sub-G1 populations were shown and caspase-9 and -3 activities were stimulated in EGCG-treated TSGH-8301 cells. Moreover, a caspase-9 inhibitor (Z-LEHD-FMK) and a caspase-3 inhibitor (Z-DEVD-FMK) were able to reduce EGCG-stimulated caspase-9 and -3 activities, respectively. Loss of mitochondrial membrane potential (∆Ψm) resulted in an increase of protein levels of cytochrome c, Apaf-1, caspase-9 and -3 in TSGH-8301 cells following exposure to EGCG. Proteomic analysis revealed that EGCG affected the expression levels of various proteins, including HSP27, porin, tropomyosin 3 isoform 2, prohibitin and keratin 5, 14, 17 in TSGH-8301 cells. EGCG also suppressed AKT kinase activity and protein levels and also altered the expression levels of Bcl-2 family-related proteins such as Bcl-2, Bax, BAD and p-BAD. Based on the above findings, this study suggests that EGCG-provoked apoptotic death in TSGH-8301 cells is mediated through targeting AKT and HSP27 and modulating p-BAD, leading to activation of the intrinsic apoptotic pathway.

Effects of high-voltage electrostatic fields on the quality of tilapia meat during refrigeration.

Fresh fish is typically brought to market refrigerated at approximately 4 °C, R-storage. A storage method has been devised that combines refrigeration with a high-voltage electrostatic field (100 kV/m; E-storage). It was developed to improve the quality and prolong the shelf life of foods. This study investigated changes in the freshness of tilapia meat under E-storage conditions. The total viable count of tilapia reached 107 CFU/g on the 7th d of refrigeration in R-storage. By the 6th d, K-value had increased from 20% to 61.7% for E-storage and to 94.7% for R-storage. Volatile basic nitrogen had increased from 12.54 mg/100 g to about 24.34 and 25.03 mg/100 g for R- and E-storage (on the 7th and 10th d), respectively. The sensory assessment also indicated that E-storage yielded an improvement in quality over that of R-storage. Practical application of the study model has the potential to prolong the freshness of fish.

Allyl isothiocyanate triggers G2/M phase arrest and apoptosis in human brain malignant glioma GBM 8401 cells through a mitochondria-dependent pathway.

Isothiocyanates (ITCs) are present as glucosinolates in various cruciferous vegetables. Allyl isothiocyanate (AITC) is one of the common naturally occurring isothiocyanates. Recent studies have shown that AITC significantly inhibited survival of leukemia HL-60, bladder cancer UM-UC-3 and colon cancer HT-29 cells in vitro. In this study, we demonstrate that AITC significantly decreased proliferation and viability of human brain malignant glioma GBM 8401 cells in a dose-dependent manner with IC50 9.25+/-0.69 microM for 24 h-treatment. The analysis of cell cycle distribution also showed that AITC induced significantly G2/M arrest and sub-G1 phase (apoptotic population) in GBM 8401 cells. AITC markedly reduced the CDK1/cyclin B activity and protein levels by CDK1 activity assay and Western blot analysis. AITC-induced apoptotic cell death and this evidence was confirmed by morphological assessment and DAPI staining. Pretreatment with specific inhibitors of caspase-3 (Z-DEVE-FMK) and -9 (Z-LEHD-FMK) significantly reduced caspase-3 and -9 activity in GBM 8401 cells. Western blot analysis and colorimetric assays also displayed that AITC caused a time-dependent increase in cytosolic cytochrome c, pro-caspase-9, Apaf-1, AIF, Endo G and the stimulated caspase-9 and -3 activity. Our results suggest that AITC is a potent anti-human brain malignant glioma drug and it shows a remarkable action on cell cycle arrest before commitment for apoptosis is reached.

Effect of thawing and cold storage on frozen chicken thigh meat quality by high-voltage electrostatic field.

One of the most popular issues in electrostatic biology is the effects of a high-voltage electrostatic field (HVEF) on the thawing of chicken thigh meat. In this study, chicken thigh meat was treated with HVEF (E-group), and compared to samples stored in a common refrigerator (R-group), to investigate how HVEF affects chicken thigh meat quality after thawing at low temperature storage (-3 and 4 degrees C). The results showed that there were no significant differences in biochemical and microorganism indices at -3 degrees C. However, the HVEF can significantly shorten thawing time for frozen chicken thigh meat at -3 degrees C. After thawing chicken thigh meat and storing at 4 degrees C, the total viable counts reached the Intl. Commission on Microbiological Specification for Foods limit of 10(7) CFU/g on the 6 and 8 d for the R- and E-group, respectively. On the 8th d, the volatile basic nitrogen had increased from 11.24 mg/100 g to 21.9 mg/100 g for the E-group and 39.9 mg/100 g for the R-group, respectively. The biochemical and microorganism indices also indicated that the E-group treatment yielded better results on thawing than the R-group treatment. The application of this model has the potential to keep products fresh.

Develop a novel method for removing fusel alcohols from rice spirits using nanofiltration.

The removal effect on excessive fusel alcohols from rice spirits were investigated using nanofiltration (NF) and ultrafiltration (UF). Compared to UF (GE and GH membranes), NF (DK and DL membranes) showed 10 times greater effect for fusel alcohols rejection due to molecular weight cut-off. On operating pressures, 488.95 kPa was suitable with a rejection rate attaining 44.2% for DK membrane. Only slight changes in physicochemical indices including ethanol concentration, flavor, total acidity, pH value, and soluble solid content were observed for rice-spirits after NF treatment. Moreover, rice spirits treated with the DK membrane achieved a higher score in sensory evaluation. We anticipated a practical application of the nonheat processes in rice spirits production.

Endogenous ephrinB2 mediates colon-urethra cross-organ sensitization via Src kinase-dependent tyrosine phosphorylation of NR2B.

Recently, the role of EphB receptor (EphBR) tyrosine kinase and their ephrinB ligands in spinal pain-related neural plasticity has been identified. To test whether Src-family non-receptor tyrosine kinase-dependent glutamatergic N-methyl-d-aspartate receptor (NMDAR) NR2B subunit phosphorylation underlies lumbosacral spinal EphBR activation to mediate cross-organ sensitization between the colon and the urethra, external urethra sphincter electromyogram activity evoked by pelvic nerve stimulation and protein expression in the lumbosacral (L6-S2) dorsal horn were studied before and after intracolonic mustard oil (MO) instillation. We found MO instillation produced colon-urethra reflex sensitization along with an upregulation of endogenous ephrinB2 expression as well as phosphorylation of EphB 1/2, Src-family kinase, and NR2B tyrosine residues. Intrathecal immunoglobulin fusion protein of EphB1 and EphB2 as well as PP2 reversed the reflex sensitization and NR2B phosphorylation caused by MO. All these results suggest that EphBR-ephrinB interactions, which provoke Src-family kinase-dependent NMDAR NR2B phosphorylation at the lumbosacral spinal cord level, are involved in cross-organ sensitization, contributing to the development of viscero-visceral referred pain between the bowel and the urethra.

Estrogen-dependent facilitation on spinal reflex potentiation involves the Cdk5/ERK1/2/NR2B cascade in anesthetized rats.

Cyclin-dependent kinase-5 (Cdk5), a proline-directed serine/threonine kinase, may alter pain-related neuronal plasticity by regulating extracellular signal-related kinase-1/2 (ERK1/2) activation. This study investigated whether Cdk5-dependent ERK activation underlies the estrogen-elicited facilitation on the repetitive stimulation-induced spinal reflex potentiaton (SRP) that is presumed to be involved in postinflammatory/neuropathic hyperalgesia and allodynia. Reflex activity of the external urethra sphincter electromyogram evoked by pelvic afferent nerve test stimulation (TS; 1 stimulation/30 s for 10 min) and repetitive stimulation (RS; 1 stimulation/1 s for 10 min) was recorded in anesthetized rats. TS evoked a baseline reflex activity, whereas RS produced SRP. Intrathecal (it) beta-estradiol facilitated the repetitive stimulation-induced SRP that was reversed by pretreatment with the estrogen receptor anatogonist ICI 182,780 (10 nM, 10 microl it), Cdk5 inhibitor roscovitine (100 nM, 10 microl it), ERK inhibitor (U-0126; 100 microM, 10 microl it) and N-methyl-D-aspartate (NMDA) NR2B subunit antagonist (Co-101244; 100 nM, 10 microl it). Moreover, ERalpha (propylpyrazoletriol; 100 nM, 10 microl it) and ERbeta (diarylpropionitrile; 100 microM, 10 microl it) agonists both facilitated the SRP, similar to results with a beta-estradiol injection. In association with the facilitated RS-induced SRP, an intrathecal beta-estradiol injection elicited ERK1/2 and NR2B subunit phosphorylation that were both reversed by intrathecal roscovitine and U-0126. These results indicated that the Cdk/ERK cascade, which is activated by ERalpha and ERbeta, may subsequently phosphorylate the NR2B subunit to develop NMDA-dependent postinflammatory hyperalgesia and allodynia to maintain the protective mechanisms of the body.

Surveillance of endoparasitic infections and the first report of Physaloptera sp. and Sarcocystis spp. in farm rodents and shrews in central Taiwan.

A total of 95 rodents and shrews including 82 Rattus norvegicus, 7 Rattus rattus, and 6 Suncus murinus were trapped from different localities of Taichung, Taiwan. The overall prevalence of parasites was 93.7%. The infection rates for R. norvegicus, R. rattus, and S. murinus were 93.9%, 85.7%, and 100%, respectively. The rats were infected with four cestodes, Taenia taeniaeformis (48.4%), Hymenolepis diminuta (38.9%), Hymenolepis nana (5.3%), and Raillietina celebensis (45.3%); ten nematodes, Angiostrongylus cantonensis (16.8%), Capillaria hepatica (49.5%), Gongylonema neoplasticum (1.1%), Heterakis spumosa (35.8%), Nippostrongylus brasiliensis (57.9%), Physaloptera sp. (1.1%), Strongyloides ratti (81.1%), Syphacia muris (2.1%), Trichosomoides crassicauda (29.5%), and Trichurus sp. (1.1%), and one protozoan, Sarcocystis spp. (33.7%). Physaloptera sp. from S. murinus and Sarcocystis spp. from both R. norvegicus and R. rattus were reported for the first time in Taiwan. The importances of zoonotic species were discussed.

TRPV1 mediates the uterine capsaicin-induced NMDA NR2B-dependent cross-organ reflex sensitization in anesthetized rats.

Spinal cord-mediated cross-organ sensitization between the uterus and the lower urinary tract may underlie the high concurrence of obstetrical/gynecological inflammation and chronic pelvic pain syndrome characterized by urogenital pain. However, the neural pathway and the neurotransmitters involved are still unknown. We tested the hypothesis that the excitation of capsaicin-sensitive primary afferent fibers arising from the uterus through the stimulation of transient receptor potential vanilloid 1 (TRPV1) induces cross-organ sensitization on the pelvic-urethra reflex activity. Capsaicin (1-1,000 microM, 0.05 ml) was instilled into the uterus to induce cross-organ reflex sensitization. Activation of capsaicin-sensitive primary afferent fibers by capsaicin instillation into the uterine horn sensitized the pelvic-urethra reflex activity that was reversed by an intrauterine pretreatment with capsaizepine, a TRPV1-selective antagonist. Intrathecal injection of AP5, a glutamatergic N-methyl-D-aspartate (NMDA) antagonist, and Co-101244, an NMDA NR2B-selective antagonist, both abolished the cross-organ reflex sensitization caused by capsaicin instillation. These results demonstrated that TRPV1 plays a crucial role in contributing to the capsaicin-sensitive primary afferent fibers mediating the glutamatergic NMDA-dependent cross-organ sensitization between the uterus and the lower urinary tract when there is a tissue injury.

Alteration of extracellular collagen matrix in the myocardium of canines infected with Dirofilaria immitis.

The heart consists of cardiocytes and the interstitial extracellular matrix (ECM), which is made up mainly of collagens. The ECM has been suggested to be important in maintaining the structure and function of the heart. This investigation attempted to elucidate the changes in the ECM collagens in the hearts of canines with dirofilariasis. The ECM collagen fibrils of the heart are grouped into endomysial struts, epimysial weaves, and perimysial coils. In the present study, we used the modified silver impregnation technique to stain paraffin-embedded sections to demonstrate three types of ECM. The results revealed that the ECM content of the heart was significantly reduced in heartworm-infected dogs, and became fragmented and dissociated. In addition, the amounts of collagen in the septum (Sep), RVs and LVs in canines with dirofilariasis (Sep=11.55+/-0.65, RV=12.07+/-0.59, LV=11.72+/-0.62 microg/mg, n=24) were significantly lower (p<0.01) than that in the normal canines (Sep=15.09+/-0.72, RV=15.16+/-0.83, LV=14.91+/-0.89 microg/mg, n=8). These results indicated that heartworm infection induced the remodeling of the extracellular matrix, thus markedly altering the architecture and function of the heart.