Changes in the gut microbiome of the Chinese mitten crab (Eriocheir sinensis) in response to White spot syndrome virus (WSSV) infection.

Intestinal microorganisms play important roles in maintaining host health, but their functions in aquatic animal hosts have yet to be fully elucidated. The Chinese mitten crab, Eriocheir sinensis, is one such example. We attempted to identify the shift of gut microbiota that occurred in response to infection of white spot syndrome virus (WSSV), an emerging viral pathogen in the crab aquaculture industry. The microbiota may exert some control over aspects of the viral pathogenesis. We investigated the changes in composition and structure of the crab gut microbiome during various WSSV infection stages of 6 h post-infection (hpi) and 48 hpi, using a 16S rRNA approach on the MiSeq Illumina sequencing platform. Four phyla (Firmicutes, Proteobacteria, Tenericutes and Bacteroidetes) were most dominant in the gut of E. sinensis regardless of the WSSV infection stages. However, further analysis revealed that over 12 bacterial phyla, 44 orders and 68 families were significantly different in abundance at various states of WSSV infection. Several intriguing aspects of E. sinensis gut bacteria that had not been previously reported were also uncovered, such as class Mollicutes was dominant here, but absent in crabs from Yangtze River estuary and Chongming Islands. Overall, this study provided the first evidence that changes in gut microbiome were closely associated with the severity of WSSV infection and that indicator taxa could be used to evaluate the crab health status.

Occurrence, Distribution, and Molecular Characterization of Citrus yellow vein clearing virus in China.

In 2009, a new citrus viral disease caused by Citrus yellow vein clearing virus (CYVCV) was discovered in China. To more effectively monitor the presence of CYVCV, a survey was conducted in 166 citrus orchards from 11 major citrus-growing provinces in China from May 2014 to April 2016. In all, 458 of a total of 2,350 citrus samples tested positive for CYVCV, demonstrating that the virus is widely distributed in China. In this study, the complete genome sequences of 19 CYVCV isolates from different provinces and hosts were sequenced and characterized. Comparisons of the whole-genome sequences of these 19 CYVCV isolates as well as 4 isolates previously reported from around the world revealed that the sequence identity ranged from 97.1 to 99.8%, indicating that there is a very low level of sequence heterogeneity among CYVCV isolates of different geographic origins and hosts. Phylogenetic analysis of these 23 genomic sequences suggested that all of the isolates from China were clustered into the same clade, clearly apart from the CYVCV isolates from Turkey and Pakistan. To our knowledge, this is the first extensive survey conducted in China for CYVCV incidence.

Chinese community-dwelling elders' needs: promoting ageing in place.

BACKGROUND: 'Ageing in place' is widely promoted as a response to global ageing and increased demand for services, but little evidence is available about what older people think they need in terms of services and supports to remain at home. AIM: To investigate older people's needs when ageing in place in order to provide evidence to inform policies and strategies promoting the option of ageing in place. METHODS: A total of 568 elderly persons in Hangzhou, China, were surveyed in 2009-2011 using a modified questionnaire validated in the USA and China. RESULTS: Overall, 88.9% of older adults were satisfied with the community in which they live; 97.2% were satisfied with life quality. Health problems and healthcare access difficulty increased with age. House repairs and housework were the most troubling. Respondents identified high need for social and health promotion services and this varied across age groups. LIMITATIONS: Cultural adaptation and validation of the questionnaire could have been influenced by differences in socioeconomic and cultural factors. The sample excluded older adults with disabilities, bed-ridden and/or unable to communicate thus limiting the scope of relevance. CONCLUSION: A majority of older adult respondents ageing at home lived a relatively healthy life; however, they required more comprehensive health insurance to cover costs of long-term health problems and access to home care support. IMPLICATIONS FOR NURSING AND HEALTH POLICY: The needs of community-dwelling older Chinese people in the Xiacheng District are not being fully met and much remains be done to increase community and regional capacity before ageing in place can be promoted as a policy strategy. More generally, nursing and health policies geared to enhance the self-sufficiency of older people residing in their communities must draw upon evidence of assessed needs and client perspectives of their requirements before services can be designed and delivered.

First Report of Citrus bark cracking viroid and Citrus viroid V Infecting Citrus in China.

Citrus is the most cultivated and highest value crop in the 15 southern provinces and municipalities in South China. Trifoliate orange (Poncirus trifoliata) is the main rootstock for citrus cultivars and is known to be susceptible to citrus viroids. Surveys conducted from 1995 to 2007 revealed 42 symptomatic samples from 33 cultivars (21 from sweet oranges (Citrus sinensis), 6 from mandarins (C. reticulata), 2 from satauma mandarins (C. unshiu), 6 from lemons (C. jambhiri), and 7 from mandarin hybrids). Symptoms included stunting, bark scaling, and cracking on the Trifoliate orange rootstock collected from citrus orchards in the Chongqing municipality, Sichuan, Zhejiang, Jiangxi, Hunan, and Yunnan provinces. Of the 42 samples, 27 were cultivars imported from abroad and 15 were local cultivars. Budwood from infected trees were grafted onto Arizona 861-S1 'Etrog citron' (C. medica) on rough lemon (C. jambhiri) rootstock. After more than 12 months, 39 of 42 samples revealed typical viroid symptoms of stunting, epinasty. and leaf rolling on the Etrog indicator plants. In September 2009, total RNA was extracted with TRIZOL Reagent and a one-step multiplex reverse transcription (RT)-PCR assay (3) was used to detect simultaneously Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), and Citrus dwarfing viroid (CDVd). Also, a one-step RT-PCR protocol using two primer pairs targeting the complete genome sequences was used to detect Citrus bark cracking viroid (CBCVd) (1) and Citrus viroid V (CVd-V) (2). Of the 42 samples, 37 and 35 were positive for HSVd and CDVd, respectively. CEVd and CBLVd were found, respectively, in 14 and 13 of 42 samples. CBCVd was detected in cv. Meishan No. 9 (C. sinensis) from Sichuan Province and cvs. Akemi (C. reticulata) and Nishirokaori (C. reticulata) from Zhejiang Province. CVd-V was detected in cvs. Nishirokaori, Haruka (C. tamuranua), and Kiyomi (C. unshiu × C. sinensis) from Zhejiang, Hunan, and Chongqing Province, respectively. Only Meishan No. 9 is a local cultivar, whereas Akemi, Nishirokaori, Haruka, and Kiyomi are cultivars imported from Japan. Of 42 samples, 3 without typical symptoms on Etrog citrons were infected with HSVd only. Of 42 infected citrus plants, 36 harbored more than one viroid species. RT-PCR products of CBCVd and CVd-V were cloned by standard methods. Eight clones for CBCVd (one from Meishan No. 9 [Accession No. HM042742, 284 bp], three from Akemi [Accession Nos. HM042743-HM042745, 283 to 284 bp], and four from Nishirokaori [Accession Nos. HM042746-HM042749, 286 bp]) and six clones for CVd-V (four from Nishirokaori [Accession Nos. HM042750-HM042753, 294 bp], one from Kiyomi [Accession No. HM042754, 294 bp], and one from Haruka [Accession No. HM042755, 294 bp]) were sequenced and deposited in GenBank. BLAST analysis of the CBCVd (Accession No. HM042742) and CVd-V (Accession No. HM042751) sequences revealed highest nucleotide sequence identity (100 and 96%) to a CBCVd isolate from Cuba (Accession No. AJ630360) and a CVd-V isolate from Spain (Accession No. EF617306), respectively. To our knowledge, this is the first report of CBCVd and CVd-V in China. Our finding emphasizes the need for CBCVd and CVd-V indexing in production and distribution of pathogen-free citrus plants in China. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (2) P. Serra et al. Phytopathology 98:1199, 2008. (3) X. F. Wang et al. Eur. J. Plant. Pathol. 124:175, 2009.

First Report of Citrus bent leaf viroid and Citrus dwarfing viroid from Citrus in Punjab, Pakistan.

Pakistan is among the top 10 citrus-producing countries of the world and the leader in Kinnow mandarin production with production concentrated in the province of Punjab, which produces more than 96% of the total citrus crop. To evaluate the presence and distribution of citrus viroids in this area, 34 samples were collected in September 2008 from citrus orchards in the Sargodha, Bhalwal, and Faisalabad areas of Punjab, including 15 'Mosambi' and two 'Bloodred' sweet oranges (Citrus sinensis), eight 'Kinnow' and four 'Feutrell Early' mandarins (C. reticulata), three 'Jatti Khatti' rough lemon (C. jambhiri), and two grapefruit (C. paradisi), which showed stunting, bark scaling, and cracking symptoms on the rootstock which was either citrange (Poncirus trifoliata × C. sinensis) or sweet lime (C. limetta). Infected budwood from these trees was grafted onto indicator plants of Arizona 861-S-1 'Etrog citron' (C. medica) budded on rough lemon rootstock, and after 3 months, the citron showed typical viroid symptoms of mild epinasty and leaf roll with 23 of the 34 samples. A one-step multiplex reverse transcription (RT)-PCR assay (3) was used to detect simultaneously Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd). On the basis of amplification of the appropriately sized DNA, CEVd, CBLVd, HSVd, and CDVd were detected in 12, 8, 31, and 17 samples, respectively, whereas CBCVd was not detected. Twenty-three of 34 infected samples harbored more than one viroid species and one had four viroids. Budwood from 11 trees did not induce viroid symptoms on Etrog citron. Two of these trees were infected with CBLVd only and nine with HSVd only. Four primer pairs were used to amplify the full sequences of CEVd, CBLVd, HSVd, and CDVd by RT-PCR (2), which were cloned by standard methods. Sequences of three cDNA clones each of CEVd (Nos. FJ773253, FJ773254, and FJ773255), CBLVd (Nos. FJ773262, FJ773263, and FJ773267), HSVd (Nos. FJ773268, FJ773269, and FJ773271), and CDVd (Nos. FJ773274, FJ773275, and FJ773276) were deposited in GenBank. BLAST analysis showed that these nucleotide sequences had greater than 97% nucleotide identity to the most similar genome sequences in GenBank. One of the HSVd sequences, FJ773271, presented the cachexia determinants. To our knowledge, this is the first report of CBLVd and CDVd in Pakistan (1). These results indicate the need for proper indexing of mother trees and a virus-free propagation scheme to create healthy budwood sources in Pakistan. References: (1) M. Arif et al. Pak. J. Bot. 37:407, 2005. (2) L. Bernard and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (3) X. F. Wang et al. Eur. J. Plant. Pathol, 124:175, 2009.

Identification of early tolerance regulator genes induced by allochimeric therapy using microarray-based genomewide scan.

We have demonstrated that peri- or postoperative delivery of allochimeric [a1h(u)]-RT1.A(a) class I major histocompatibility complex molecules with donor-type (RT1A(u)) immunogenic epitopes presented in recipient-type (RT1A(a)) sequences induced donor-specific tolerance in ACI (RT1a) recipients of WF (RT1u) heart allografts. A genomic scan during the early posttransplant period was performed to elucidate the underlying operative mechanisms. A rat genome study after transplantation was carefully designed using Affymetrix Rat Genome 230 2.0 Array. The allochimeric treatment group is 3-day cyclosporine (CsA)-treated ACI recipients that accepted Wistar Furth RT1u cardiac allografts with postoperative dosage of allochimeric molecules, while the control is 3-day CsA-treated ACI recipients of WF cardiac allografts. All the samples were harvested 5 days after heart transplant as the early stage of tolerance detection. Following array data normalization and modeling, we compared the above two treatment groups and identified a total of 250 tolerance regulator genes induced by allochimeric molecules only.

Purification of a novel myofibril-bound serine proteinase inhibitor (MBSPI) from the skeletal muscle of lizard fish.

A novel myofibril-bound serine proteinase inhibitor (MBSPI) was purified to homogeneity from the skeletal muscle of lizard fish (Saurida wanieso). Purification was carried out by ammonium sulfate fractionation, followed by column chromatographies on DEAE-Sephacel, SP-Sepharose and Sephadex G-150. MBSPI was purified 7.7-fold starting from the DEAE-Sephacel fraction, with a yield of 0.2%. It is a monomeric protein with the molecular mass of 50 kDa as estimated by SDS-PAGE and gel filtration. MBSPI reveals high inhibition specificity toward a myofibril-bound serine proteinase (MBSP) purified from lizard fish muscle. No inhibition is detected toward bovine trypsin, bovine chymotrypsin, two trypsins from carp hepatopancreas and a serine proteinase isolated from the sarcoplasmic fraction of white croaker muscle. It does not exert any inhibitory activity toward a myofibril-bound serine proteinase from carp muscle.

Efficacy of porcine reproductive and respiratory syndrome virus vaccine and porcine interleukin-12.

The efficacy of porcine recombinant interleukin-12 (prIL-12) as an adjuvant to stimulate immunity was investigated in pigs vaccinated against porcine reproductive and respiratory syndrome virus (PRRSV). Porcine recombinant IL-12 was cloned and expressed in the insect baculovirus system. Thirty pigs were randomly allocated to five groups, and individual pigs were injected with PRRSV modified live virus with and without coadministration of prIL2; PRRSV killed virus with and without coadministration of prIL-12; or prIL-12 only (control). The effect of prIL-12 was evaluated through monitoring of humoral and cell-mediated immune (CMI) responses. Results indicated that the killed virus given in conjunction with prlL-12 induced humoral and CMI responses of similar magnitude to those of the modified live virus. However, without prIL-12, the immunologic response to vaccination with the killed virus vaccine was poor and similar to that of controls.

Purification of a novel serine proteinase inhibitor from the skeletal muscle of white croaker (Argyrosomus argentatus).

A novel serine proteinase inhibitor has been purified to homogeneity from the skeletal muscle of white croaker (Argyrosomus argentatus). The purification was carried out by ammonium sulfate fractionation, DEAE-Sephacel, heating treatment followed by column chromatographies on SP-Sepharose, Sephadex G-150 and gel-filtration high performance liquid chromatography. The molecular mass of the inhibitor was 55 kDa as estimated by SDS-PAGE and gel filtration. It specifically inhibited a myofibril-bound serine proteinase (MBSP) isolated from the skeletal muscle of lizard fish (Saurida wanieso). No inhibition, however, was detected toward other serine proteinases such as bovine trypsin, bovine chymotrypsin and a myofibril-bound serine proteinase from carp (Cyprinus carpio) muscle. Interestingly, the sequences of tryptic digested peptide fragments of MBSPI revealed high identity to that of porcine phosphoglucose isomerase (PGI) (76%) and other PGIs. Furthermore, purified MBSPI exhibits PGI activity, suggesting the inhibitor is a protein closely related to PGI. When rabbit muscle PGI was investigated, it also specifically suppressed the activity of MBSP. It thus strongly suggests that MBSPI is actually PGI and conversely, PGI is a specific inhibitor toward myofibril-bound serine proteinase(s).

Identification of a myofibril-bound serine proteinase (MBSP) in the skeletal muscle of lizard fish Saurida wanieso which specifically cleaves the arginine site.

A myofibril-bound serine proteinase (MBSP) from the skeletal muscle of lizard fish (Saurida wanieso) was purified to homogeneity by a heating treatment followed by a series of column chromatographies on DEAE-Sephacel, Sephacryl S-200, Q-Sepharose, Hydroxyapatite and Benzamidine-Sepharose 6B, and characterized enzymatically. On SDS-poly-acrylamide gel electrophoresis (SDS-PAGE), the purified enzyme showed a band with molecular mass of approximately 29 kDa under reducing conditions, while 60 kDa under non-reducing conditions. The optimum temperature of the enzyme was 50 degrees C using t-butyloxycarbonyl-Phe-Ser-Arg-4-methylcoumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) as a substrate. Substrate specificity analysis both using MCA-substrates and peptides showed that MBSP specifically cleaved at the carboxyl side of the arginine residue. Inhibitor susceptibility analysis revealed that MBSP was inhibited effectively by Pefabloc SC, soybean trypsin inhibitor (STI) and aprotinin, indicating the characteristic of a serine proteinase. When myofibril was incubated with the enzyme, it optically degraded myosin heavy chain at 55-60 degrees C, while alpha-actinin and actin were not at all hydrolyzed as detected by immunoblotting. The N-terminal amino acid sequence of MBSP was partially determined as IVGGAEXVPY- and was very homologous to other serine proteases.

Cleavage specificity of a myofibril-bound serine proteinase from carp (Cyprinus carpio) muscle.

Previously, we reported the purification and characterization of a myofibril-bound serine proteinase (MBP) from carp muscle (Osatomi K, Sasai H, Cao M-J, Hara K, Ishihara T. Comp Biochem Physiol 1997;116B:159-66). In the present study, the N-terminal amino acid sequence of the enzyme was determined, which showed high identity with those of other trypsin-like serine proteases. The cleavage specificity of MBP for dibasic and monobasic residues was investigated using various fluorogenic substrates and peptides. Analyses of the cleaved peptide products showed that the enzyme hydrolyzed peptides both at monobasic and dibasic amino acid residues. Monobasic amino acid residues were hydrolyzed at the carboxyl side; dibasic residues were cleaved either at the carboxyl side of the pair or between the two basic residues and the enzyme showed a cleavage preference for the Arg-Arg pair. Unexpectedly, MBP hydrolyzed lysyl-bradykinin and methionyl-lysyl-bradykinin at the carboxyl side of Gly fairly specifically and efficiently displaying a unique cleavage. Because MBP also degraded protein substrates such as casein and myofibrillar proteins, the substrate specificity of MBP appeared not to be strictly specific.

[Experimental study of metallic bone and joint prostheses with plasma-sprayed ceramic coating]. / Experimentelles Studium über metallische Knochen- und Gelenkprothesen mit plasmagesprühtem Keramiküberzug.

In order to solve the loosening problem of endoprosthesis, a titanium-based alloy bone and joint prosthesis with plasma-sprayed ceramic coating was developed by the authors. By the fabrication powdered alumina or zirconium oxide were melted in an electric arc by a temperature of 10,000 K and sprayed onto the metallic prosthesis. The coating was 0.3 to 0.6 mm in thickness and had innumerable micropores. The pores were 50 to 200 microns in diameter. The metallic bone and joint prosthesis with plasma-sprayed ceramic coating exhibits characteristics of metal and ceramic, such as high mechanical strength, high resistance to corrosion and elution and good biocompatibility. According to our experimental study, the plasma-sprayed ceramic coating allows tissue ingrowth into its micropores and prevents the loosening of the prosthesis. Successful cases have been experienced through 10 years clinical application.

Prosthetic replacement using titanium-based alloy artificial total femur incorporating its total hip and total knee joints with plasma-sprayed ceramic coating.

The authors developed a titanium-based alloy artificial total femur incorporating its total hip and total knee joints with plasma-sprayed ceramic coating 0.3 to 0.6 mm in thickness and a myriad of micropores of 50-200 microns in diameter. On the basis of reliable data obtained in the animal experiments and physical testing, the prosthesis was applied clinically with satisfactory results. This paper reports the results of two patients with a follow-up of 42-70 months.

[Total replacement of the femur with simultaneous total hip and knee joint endoprostheses with plasma sprayed ceramic coating]. / Totalersatz des Femurs unter gleichzeitiger Verwendung von Hüft- und Kniegelenkstotalendoprothesen mit plasmagesprühtem Keramiküberzug.

A method of prosthetic replacement using total femur incorporating its total hip and knee joints is reported. On the surface of the titanium endoprosthesis was plasma-sprayed a ceramic coating to prevent loosening of the prosthesis. It can preserve the limb and restore the function in a short period. It can be used in certain non-malignant tumours and diseases of the bone with extensively destructive lesions of the entire femur. This method was used in 3 patients with good results. The operated patients were followed-up for 32-70 (mean 54) months. Types of procedures and advantages of the operation are presented.

[Total femur and hip knee joint replacement with plasma-sprayed ceramic coated prosthesis]. / Totalersatz des Femurs unter gleichzeitiger Verwendung von Hüft- und Kniegelenktotalendoprothesen mit plasmagesprühtem Keramiküberzug.

A method of prosthetic replacement using total femur with total hip and knee joints is reported. On the surface of the titanium endoprosthesis a ceramic coating was plasma-sprayed to prevent loosening of the prosthesis. By this method the limb can be preserved and the function restored within a short period. It can be used in certain cases of non-malignant tumors and diseases of the bone with extensive destructive lesions of the entire femur. This method was used on 3 patients with good results. The patients operated on were followed up for 32-70 (mean 54) months. The procedures and advantages of the operation are presented.