Development of a DNA microarray-based multiplex assay of avian influenza virus subtypes H5, H7, H9, N1, and N2.

Outbreaks of highly pathogenic avian influenza have caused considerable economic losses in the poultry industry and have also resulted in human deaths since 2004. Rapid subtyping of highly pathogenic avian influenza viruses(HPAIVs) in clinical specimens is a prerequisite of prompt control of disease and prevention of its spreading. In this study, we describe development of a DNA microarray-based detection and subtyping of HPAIVs in field samples. DNA copies of matrix (M) protein genes for the H5, H7, and H9 subtypes of hemagglutinin (HA) and the N1 and N2 subtypes of neuraminidase (NA) were prepared by RT-PCR and specific primers and then spotted onto aldehyde slides to form DNA microarrays. The HPAIV samples to be tested were subjected to total RNA isolation, RT-PCR with universal primers and Cy3 labeling, and the obtained double-stranded DNAs (targets) were finally hybridized with DNA microarrays (probes). A fluorescent spot on the microarray, detected by scanning indicated positive hybridization, i.e. the involved subtype. The assay was specific as various heterologous viruses or HPAIVs of other subtypes tested were negative. No cross-hybridization among different subtypes could be detected. The assay was more sensitive than RT-PCR and chicken embryo inoculation and could be also used for field samples. Summing up, the assay has proved useful for simultaneous detection and differentiation of main epidemic HPAIV subtypes.

Whole-genome resequencing reveals genetic structure and introgression in Pudong White pigs.

Pudong White (PDW) pigs, historically originating from Shanghai, are the only Chinese indigenous pigs characterised by their completely white coats, with the exception of Rongchang pigs. However, there is limited information concerning their overall genetic structure or relationship with other breeds, especially the East Chinese (ECN) and European pigs. To uncover the genetic structure, selection signatures, and potential exotic introgression in PDW pigs, we sampled 15 PDW pigs using whole-genome sequencing (~20×). We then conducted in-depth population genetic analyses in 320 pigs from 27 global pig groups, namely, European wild boars, Chinese wild boars, and outgroup. Neighbour-joining tree and principal component analysis confirmed that PDW pigs belonged to the ecotype of ECN pigs. Both f3, D-statistics, and structure analysis showed that PDW pigs shared apparent alleles with Large White (LW) pigs. Three statistics, rIBD, a haplotype heat map and copy number variation, further indicated that PDW pigs shared apparent alleles with LW pigs at the KIT Proto-Oncogene, Receptor Tyrosine Kinase (KIT) and PARG-MARCHF8 loci, suggesting that the lineage of European pigs in PDW originated from LW pigs. After further detecting the KIT mutations in different pig breeds, PDW was confirmed to have the same duplication region 1, duplication region 2, and the splicing mutation on intron 17 of KIT as LW pigs that determine the white coat colour phenotype in European white pigs. We hypothesised that LW pigs were imported to China ∼110-160 years ago according to the admixture time estimate and then crossed with ECN pigs, resulting in the introgression of the KIT alleles that produce the white coat colour phenotype in the PDW pig breed. To our knowledge, this study presents the first thorough description of the genetic structure of PDW pigs via whole-genome resequencing data; moreover, the results provide a basis for the national project for the conservation of this unique Chinese local population.

Development and application of isothermal amplification methods for rapid detection of F4 fimbriae producing Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the causative agent of a wide range of diseases, which are the important cause of illness and mortality in piglets. ETEC strains expressing F4 fimbriae are frequently associated with post-weaning diarrhea (PWD) and lead to great economic losses in swine production industry worldwide. The aim of this study was to establish a rapid and effective isothermal amplification method for detection of F4 fimbriae. Loop-mediated isothermal amplification (LAMP), Polymerase spiral reaction (PSR) and cross-priming ampli- fication (CPA) were used to develop and optimize the detection method first time. Subsequently, the specificity and sensitivity of these methods were evaluated, and the clinical samples were detected with these methods. All the F4-positive samples could produce ladder-like amplifica- tions products and lead the chromogenic substrate SYBR Green I produce green fluorescence, while in blank control and negative samples lack of this pattern or remained orange. The sensi- tivity of LAMP and CPA were 10 times higher than PSR method. Meanwhile, these three methods were validated with clinical samples, 7 were found positive, while 125 samples were negative, the testing results were consisted with the real-time PCR method. These findings suggested that the isothermal amplification based on the F4 fimbriae is a rapid, effective and sensitive method under resource constrains.

Clinical therapeutic effects of human umbilical cord-derived mesenchymal stem cells transplantation in the treatment of end-stage liver disease.

We aimed to evaluate clinical therapeutic effects of human umbilical cord-derived mesenchymal stem cell (UCMSC) transplantation in the treatment of end-stage liver diseases. The human UCMSCs were cultured and prepared, and then transplanted into the hepatic tissues of 50 patients with decompensated cirrhosis. The liver function, thrombin function, Model for End-Stage Liver Disease (MELD) score, and hemodynamic index value were detected during a 24-week follow-up period, with the addition of hepatoprotective, antiviral, and other conventional treatments. No complications or serious side effects were observed. In the first 2-3 weeks after surgery, symptoms including abdominal distension, oliguria, edema, and others decreased significantly, with increased appetite compared with before surgery. In the 24-week follow-up period, the levels of serum albumin and prealbumin increased significantly compared with the preoperative levels; the decrease of coagulation indicators was not significant. The MELD scores were also markedly increased. Alpha-fetoprotein levels increased without significance after treatment. There was no significant difference in the hemodynamic changes in the portal and splenic veins according to ultrasound. Moreover, no significant differences in the liver and thrombin functions between the hepatitis B virus group and the other-etiology group were observed.

Mammographic and clinicopathological features of triple-negative breast cancer.

OBJECTIVE: Triple-negative breast cancer (TNBC) lacks effective treatment and has a poor prognosis. This study assessed mammographic findings and clinicopathological features of TNBC by comparing with non-TNBC in order to improve clinical diagnosis of TNBC. METHODS: A total of 426 patients with pathologically confirmed breast cancer were retrospectively assigned into two groups, TNBC (n = 54) and non-TNBC (n = 372), and then analysed. RESULTS: TNBC frequently showed a high histological grade, presented with a mass (79.6%) and was less frequently associated with focal asymmetric density (11.1%), microcalcifications (5.6%) and distortion (3.7%) on mammography. TNBC mammographic masses were most frequently round/oval (58.1%) or lobular (30.2%) in shape and were less frequently irregular in shape (11.6%). Masses with circumscribed margins were the most frequent (37.2%), with microlobulated (25.6%) and obscured (16.3%) margins being commonly observed, but masses with spiculated margins were rare (9.3%). CONCLUSION: TNBC could have distinct mammographic and clinicopathological features compared with non-TNBC, and thus mammography may be useful in the diagnosis of TNBC. ADVANCES IN KNOWLEDGE: This study demonstrated distinct mammographic and clinicopathological features to help in diagnosis of Chinese patients with TNBC.

Vitrification of boer goat morulae and early blastocysts by straw and open-pulled straw method.

The aim of this study was to investigate the effects of different vitrification solutions [EFS30 or EFS40 contains 30% (v/v) ethylene glycol (EG), 40% (v/v) EG; EDFS30 or EDFS40 contains 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) EG and 20% (v/v) DMSO], equilibrium time during vitrification (0.5-2.5 min) and vitrification protocols [one-step straw, two-step straw and open-pulled straw (OPS)] on in vivo development of vitrified Boer goat morulae and blastocysts after embryo transfer. In the one-step straw method, the lambing rates of vitrified embryos in EFS30 (37.5%), EFS40 (40.5%) or EDFS30 (38.2%) group were similar to that of fresh embryos (57.5%) and conventional freezing method (46.7%) when the equilibrium time was 2 min. In the two-step straw method, the highest lambing rate was obtained when embryos were pretreated with 10% EG for 5 min and then exposed to EFS40 for 2 min (51.4%), showing similar lambing rates compared with fresh embryos (56.1%) or the embryos cryopreserved by conventional freezing method (45.2%). In the OPS method, the lambing rate in EFS40, EDFS30 or EDFS40 groups were similar to that (57.1%) of fresh embryos, or to that (46.0%) of embryos cryopreserved by conventional freezing method. The highest lambing rate (51.4%) of the group of OPS was obtained when the embryos were vitrified with EDFS30. In conclusion, either the two-step straw method in which embryos were pretreated in 10% EG for 5 min and then exposed to EFS40 for 2 min, or the OPS method in which embryos were pretreated in 10% EG + 10% DMSO for 30 s and then exposed to EDFS30 for 25 s was a simple and efficient method for the vitrification of Boer goat morulae and blastocysts.

Computers against disease.

In order to improve the efficiency of the collection, compilation and dissemination of information on 35 communicable diseases in China's Sichuan Province, a computerized network has been established. In 1987 a microcomputer link was set up between the Provincial Health and Anti-epidemic Centre at Chengdu and the Health Information Centre in Beijing, and in 1990 a microcomputer network was established between Chengdu and the 21 prefectures in the province. Substantial progress in disease control is already apparent as a consequence of these developments.

PIP: In China's Sichuan Province, home to more than 107 million people, 503,900 cases of the 35 communicable diseases which require notification to health authorities occurred in 1990. Clinics and hospitals make daily reports to one of the 235 local country or municipal health and anti-epidemic centers, which have the main responsibility for controlling infectious diseases. The data from all 213 counties are analyzed, and monthly reports are sent to the 21 prefectural centers, the provincial center, and the Ministry of Public Health. Data on each patient include name, sex, age, race. address, occupation, school or work unit, kind of exposure, and history of attacks as well as dates of onset, hospitalization, diagnosis, cure, death, or other developments. The manual processing of these data was so slow it often reached the Ministry of Health too late for the Ministry to be of help in implementing strategies and control measures. In 1987, therefore, a microcomputer link was established between the Provincial Health and Anti-epidemic center at Chengdu and the Health Information Center in Beijing. By 1990, a computer network was set up between Chengdu and the 21 prefectures in Sichuan. This allows the rapid deployment of funds, medical teams and medicines in urgent situations; it saves time and manpower, improves reliability and analysis of data, and facilities information transfer among health agencies. Other developing countries may find computers as helpful in controlling infectious disease.