Detection of Skeletonema costatum based on loop-mediated isothermal amplification combined with lateral flow dipstick.

We developed a new assay method, which combines loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) for the rapid and special detection of the diatom Skeletonema costatum. Four groups of LAMP primers were derived from a conserved DNA sequence unique to S. costatum. The amplifications were carried out at 61, 63, and 65 °C for 60 min in various combinations by the quantitative PCR thermal cycler to confirm optimal primers and reaction temperature. The LAMP-LFD detection limit was 0.94 pg/µL of S. costatum genomic DNA and was 100 times more sensitive than conventional PCR. The LAMP-LFD method had high specificity and accurately identified S. costatum algal isolates, but not other algal isolates. The new LAMP-LFD assay can be used as a reliable and easy method to detect S. costatum.

Experience of copy number variation sequencing applied in spontaneous abortion.

PURPOSE: We evaluated the value of copy number variation sequencing (CNV-seq) and quantitative fluorescence (QF)-PCR for analyzing chromosomal abnormalities (CA) in spontaneous abortion specimens. METHODS: A total of 650 products of conception (POCs) were collected from spontaneous abortion between April 2018 and May 2020. CNV-seq and QF-PCR were performed to determine the characteristics and frequencies of copy number variants (CNVs) with clinical significance. The clinical features of the patients were recorded. RESULTS: Clinically significant chromosomal abnormalities were identified in 355 (54.6%) POCs, of which 217 (33.4%) were autosomal trisomies, 42(6.5%) were chromosomal monosomies and 40 (6.2%) were pathogenic CNVs (pCNVs). Chromosomal trisomy occurs mainly on chromosomes 15, 16, 18, 21and 22. Monosomy X was not associated with the maternal or gestational age. The frequency of chromosomal abnormalities in miscarriages from women with a normal live birth history was 55.3%; it was 54.4% from women without a normal live birth history (P > 0.05). There were no significant differences among women without, with 1, and with ≥ 2 previous miscarriages regarding the rate of chromosomal abnormalities (P > 0.05); CNVs were less frequently detected in women with advanced maternal age than in women aged ≤ 29 and 30-34 years (P < 0.05). CONCLUSION: Chromosomal abnormalities are the most common cause of pregnancy loss, and maternal and gestational ages are strongly associated with fetal autosomal trisomy aberrations. Embryo chromosomal examination is recommended regardless of the gestational age, modes of conception or previous abortion status.

A highly specific and ultrasensitive approach to detect Prymnesium parvum based on RPA-CRISPR-LbaCas12a-LFD system.

BACKGROUND: Harmful algal blooms (HABs), caused by the rapid proliferation or aggregation of microorganisms, are catastrophic for the environment. The Prymnesium parvum is a haptophyte algal species that is found worldwide and is responsible for extensive blooms and death of larval amphibians and bivalves, causing serious negative impacts on the ecological environment. For the prevention and management of environmental pollution, it is crucial to explore and develop early detection strategies for HABs on-site using simple methods. The major challenge related to early detection is the accurate and sensitive detection of algae present in low abundance. RESULTS: Herein, recombinase polymerase amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats and Cas12a protein (CRISPR-LbaCas12a) systems, and the lateral flow dipstick (LFD) was used for the first time for early detection of P. parvum. The internal transcribed spacer (ITS) of P. parvum was selected as the target sequence, and the concentration of single-strand DNA reporters, buffer liquid system, reaction time, and amount of gold particles were optimized. The RPA-CRISPR-LbaCas12a-LFD approach demonstrated highly specificity during experimental testing, with no cross-reaction against different microalgae used as controls. In addition, the lowest detection limit was 10,000 times better than the lowest detection limit of the standalone RPA approach. The feasibility and robustness of this approach were further verified by using the different environmental samples. It also observed that P. parvum are widely distributed in Chinese Sea, but the cell density of P. parvum is relatively low (<0.1 cells/mL). SIGNIFICANCE: The developed approach has an excellent specificity and offers 10,000 times better sensitivity than the standalone RPA approach. These advantages make this approach suitable for early warning detection and prevention of HAB events in environmental water. Also, the outcomes of this study could promote a shift from traditional laboratory-based detection to on-site monitoring, facilitating early warning against HABs.

Identification and bioinformatics analysis of genes associated with pyroptosis in spinal cord injury of rat and mouse.

The mechanism of spinal cord injury (SCI) is highly complex, and an increasing number of studies have indicated the involvement of pyroptosis in the physiological and pathological processes of secondary SCI. However, there is limited bioinformatics research on pyroptosis-related genes (PRGs) in SCI. This study aims to identify and validate differentially expressed PRGs in the GEO database, perform bioinformatics analysis, and construct regulatory networks to explore potential regulatory mechanisms and therapeutic targets for SCI. We obtained high-throughput sequencing datasets of SCI in rats and mice from the GEO database. Differential analysis was conducted using the "limma" package in R to identify differentially expressed genes (DEGs). These genes were then intersected with previously reported PRGs, resulting in a set of PRGs in SCI. GO and KEGG enrichment analyses, as well as correlation analysis, were performed on the PRGs in both rat and mouse models of SCI. Additionally, a protein-protein interaction (PPI) network was constructed using the STRING website to examine the relationships between proteins. Hub genes were identified using Cytoscape software, and the intersection of the top 5 hub genes in rats and mice were selected for subsequent experimentally validated. Furthermore, a competing endogenous RNA (ceRNA) network was constructed to explore potential regulatory mechanisms. The gene expression profiles of GSE93249, GSE133093, GSE138637, GSE174549, GSE45376, GSE171441_3d and GSE171441_35d were selected in this study. We identified 10 and 12 PRGs in rats and mice datasets respectively. Six common DEGs were identified in the intersection of rats and mice PRGs. Enrichment analysis of these DEGs indicated that GO analysis was mainly focused on inflammation-related factors, while KEGG analysis showed that the most genes were enriched on the NOD-like receptor signaling pathway. We constructed a ceRNA regulatory network that consisted of five important PRGs, as well as 24 miRNAs and 34 lncRNAs. This network revealed potential regulatory mechanisms. Additionally, the three hub genes obtained from the intersection were validated in the rat model, showing high expression of PRGs in SCI. Pyroptosis is involved in secondary SCI and may play a significant role in its pathogenesis. The regulatory mechanisms associated with pyroptosis deserve further in-depth research.

[Molecular epidemiological analysis of α- and ß-thalassemia in Fujian province].

OBJECTIVE: To investigate the gene prevalence and spectrum of alpha- and beta-thalassemia in Fujian province. METHODS: A total of 11 234 of neonatal cord blood samples were collected for a prevalence study of alpha- and beta-thalassemia. All subjects included in this study were registered in 9 cities of Fujian province. A complete blood count and high performance liquid chromatography (HPLC) were performed in all samples, with microcytosis (MCV≤ 79 f1 and MCH≤ 27 pg) or HPLC positive cases further studied by DNA analysis. alpha- and beta-thalassemia were determined by using gap-PCR and reverse dot blot (RDB) assays. Unknown positive samples were analyzed directly with DNA sequencing. RESULTS: Of all 11 234 cord blood samples, 356 were identified as from alpha-thalassemia gene carriers, 7 deletion genotypes were identified including 236 (--SEA/ α α) cases, 67 (α 3.7/ α α) cases, 24 (alpha 4.2/alpha alpha) cases, 3 (alpha 3.7/ SEA) cases, 1 (alpha 4.2/ SEA) cases, 1 (alpha 3.7/ alpha 3.7) cases, 1 (alpha 3.7/ alpha 4.2) cases; 3 non-deletion genotypes were detected, including 7 (alpha alpha QS/ alpha alpha) cases, 3 (α α CS/α α) cases, 2 (α α WS/ α α) cases, the most common mutation was SEA/α α, which accounted for 66.29%, 148 individuals were found to have beta-hemoglobin gene mutations. 12 different mutations were identified, namely 65 IVS-2 654 (C>T) cases, 40 CD41-42(-TCTT, 12 CD17(A>T) cases, 10 -28(A>G) cases,7 CD27-28(+C) cases, 5 start codon ATG>AGG cases, 2 CD26(G>A) cases, 1 CD71-72(+A) cases, 1 IVS-1-1(G>T) cases, 1 CD43(G>T) cases, 2 -29(A>G) cases, 2 Codon 36 (-C) cases, the most common mutation was IVS-2 654(C>T) and CD41-42(-TCTT), which accounted for 70.95%. A novel beta-globin gene mutation CD36 (-C) allele was also detected. The carrier rate of thalassemia in Fujian population is 4.41%. In addition, 9 beta-thalassemia carriers were found with alpha-thalassemia mutation. CONCLUSION: The research has revealed the type of gene mutations in alpha- and beta-talassemia in Fujian province. The beta-thalassemia mutations in Fujian province are complex, which were also obviously heterogeneous. This will significant value for screening the incidence, provide the valuable information for genetic counseling and prenatal diagnosis.

Pulmonary sarcomatoid carcinoma: a case report.

Sarcomatoid carcinoma (SC) is a rare primary malignant tumor in which both carcinomatous and sarcomatous elements occur. It can occur in many different organs and anatomical locations, such as the skin, thyroid gland, bone, urinary tract, breast, pancreas, liver and other areas. Of them, pulmonary sarcomatoid carcinoma (PSC) is a rare malignant cancer composed of sarcoma and sarcoma-like tumors with spindle or giant cell features. Here a case of a 75-year-old Chinese man with a six-month history of cough and hemoptysis is reported. Chest X-ray showed a tumor shadow in the left lung field. Chest computed tomography (CT) scan showed a lobulated mass in his left hilum and even the left pulmonary artery. Pleomorphic interstitial cells were found by bronchoscopic brushing. To establish a definitive diagnosis for PSC, a left pneumonectomy was performed. The pathological stage was IIB (pT2N1M0) based on the tumor node metastasis (TNM) staging system. The tumor's pathology, histology, immunohistochemistry and treatment methods are discussed.

Prenatal diagnosis of 15q11.2 microdeletion fetuses in Eastern China: 21 case series and literature review.

OBJECTIVE: 15q11.2 microdeletion can lead to syndromes affecting the nervous system. However, 15q11.2 microdeletion has large phenotypic differences and incomplete penetrance, which brings challenges to prenatal diagnosis. We reported 21 cases of 15q11.2 microdeletion fetuses in Eastern China and reviewed literature on the prenatal clinical characteristics related to the deletion variants to provide a basis for prenatal genetic counseling. METHODS: The clinical data of 21 cases of 15q11.2 microdeletion fetuses collected from June 2018 to September 2021 were retrospectively analyzed, and chromosomal microarray analysis was performed. The reported prenatal clinical features of 15q11.2 microdeletion fetuses were reviewed and summarized. A meta-analysis of 20 studies was performed to test heterogeneity, data integration, and sensitivity on the correlation between 15q11.2 microdeletion and neuropsychiatric diseases. RESULTS: The median age of the women was 29.5 years. The median gestational age at interventional examination was 24 weeks. All fetuses showed deletion variants of the 15q11.2 fragment, and the median deletion range was approximately 0.48 MB. Ultrasound of five cases showed no abnormalities; however, four of them showed a high risk of Down's syndrome (risk values were 1/184, 1/128, 1/47, and 1/54, respectively). The remaining 16 fetuses showed congenital heart disease (7/16), elevated nuchal translucency (5/16), abnormal brain structure (2/16) and renal disease (2/16). In a literature review of 82 prenatal cases, 44% (36/82) had abnormal ultrasound features, 31% (11/36) showed abnormal nuchal translucency, approximately 28% (10/36) showed abnormal cardiac structure, and 14% (5/36) had brain structural abnormalities. The meta-analysis revealed that the frequency of the 15q11.2 microdeletion mutation in patients with schizophrenia and epilepsy was significantly higher (odds ratio 2.04, 95% confidence interval: 1.78-2.33, p < 0.00001; odds ratio 5.23, 95% confidence interval: 2.83-9.67, p < 0.00001) than that in normal individuals. CONCLUSION: More than half of the 15q11.2 microdeletion cases presented no abnormalities in prenatal ultrasound examination. The cases with ultrasound features mainly showed isolated malformations such as elevated nuchal translucency, congenital heart disease, and brain structural abnormalities. Postpartum 15q11.2 microdeletion patients are at an increased risk of suffering from schizophrenia, epilepsy, and other neurological and mental diseases from 15q11.2 microdeletion. Therefore, prenatal diagnosis of 15q11.2 microdeletion not only depends on molecular diagnostic techniques but also requires cautious genetic counseling.

[Relationship between gene polymorphism of GABAA receptors gene and childhood autism].

OBJECTIVE: To explore the relationship between gene polymorphism of GABAA receptors and childhood autism by detecting rs140682, rs2081648 and rs140679 site of single nucleotide polymorphism (SNP) in GABAA receptors gene. METHODS: A total of 94 children with autism and 124 normal children were enrolled in a hospital from November 2010 to May 2011. Childhood autism rating scale (CARS) and autism behavior checklist (ABC) were used to evaluate or investigate the case group. After collecting venous blood and extracting the genome DNA, the allele and genotype of SNP rs140682, rs2081648 and rs140679 site in GABAA receptors gene were detected by PCR-RFLP. The allele and genotype of case group and control group were analyzed by χ(2) test, while the score of scales was analyzed by Spearman rank correlation analysis. RESULTS: The age of the case group was 5.12 ± 0.32, and it was 5.25 ± 0.27 in the control group (P < 0.05). In case group, the frequency of genotype CC, CT and TT of rs140682 site was 44, 41 and 9, while it was 48, 65, and 11 in control group (P > 0.05), respectively. The frequency of genotype AA, AG and GG of rs2081648 site was 8, 58 and 28 in case group, while it was 12, 49 and 63 in control group (P < 0.05), respectively. In case group, the frequency of genotype CC, CT and TT of rs140679 site was 15, 36 and 43, while it was 18, 59 and 47 in control group (P > 0.05), respectively. It was revealed by Spearman rank correlation analysis that of rs2081648 site, there was a positive correlation between genotype AG and sensation factor (S), social intercourse factor (R), and language factor (L) of autism behavior checklist (ABC) (r values were 0.149, 0.165 and 0.155, all P values < 0.05). A negative correlation between genotype GG and S, R, L and self-help factor (V) was proved (r values were -0.140, -0.173, -0.158 and -0.135, all P values < 0.05). There was a positive correlation between allele A and R and L factors (r values were 0.153 and 0.137, all P values < 0.05), while a negative correlation between allele G and R and L factors (r values were -0.153 and -0.137, all P values < 0.05). In case group, 42 children were diagnosed with mild-to-moderate autism, while 52 children were severe autism. There was no statistically significant correlation between allele or genotype of SNP rs140682 and rs140679 site and the degree of autism (P > 0.05). There was a positive correlation between allele A and genotype AG and the degree of autism (r values were 0.147 and 0.616, all P values < 0.05), while a negative correlation between allele G and genotype GG and the degree of autism (r values were -0.159 and -0.616, all P values < 0.05). CONCLUSION: The SNP rs2081648 site which located in GABAA receptors gene may be related to autism. No evidence for significant association between rs140682 and rs140679 site and autism was found.

Community Structure and Diversity of Hymenopteran Parasitoids of Chromatomyia horticola (Diptera: Agromyzidae) in Different Habitats in Changchun, China.

The community structure and diversity of hymenopteran parasitoids of the agromyzid leafminer Chromatomyia horticola (Diptera: Agromyzidae) were studied in agricultural, urban, and natural habitats in Changchun, Northeast China. In agricultural habitats, a total of 3,380 individuals and 19 species were collected, and the dominant species were Diglyphus isaea (Walker) (Hymenoptera: Eulophidae) (71.15%) and Chrysocharis pentheus (Walker) (Hymenoptera: Eulophidae) (12.10%). In urban habitats, a total of 5,996 individuals and 21 species were collected. There were three dominant species, C. pentheus (26.68%), Chrysocharis phryne (Walker) (Hymenoptera: Eulophidae) (22.18%), and D. isaea (22.13%). In natural habitats, a total of 1,566 individuals and 26 species were collected. There were three dominant species, C. pentheus (30.52%), D. isaea (15.52%), and Pediobius metallicus (Nees) (Hymenoptera: Eulophidae) (12.26%). The diversity indices of the parasitoid community in urban and natural habitats were higher than that in agricultural habitats, and the richness index in natural habitats was higher than that in agricultural and urban habitats. These results suggest that there are differences in the community composition and dynamics of parasitoids in different habitats. Hymenopteran parasitoids of C. horticola are less abundant in natural habitats; however, species richness is greater, and can be used as a species reserve for biological control.

Government Intervention Measures Effectively Control COVID-19 Epidemic in Wuhan, China.

The Coronavirus disease 2019 (COVID-19) outbreak has been brought under control through a nationwide effort, and now it has become a global pandemic and the situation seems grim. We summarized the measures taken in Wuhan and analyzed the effects to comprehensively describe the factors involved in controlling the COVID-19 in China. In China, several measures such as the lockdown of Wuhan, restriction of traffic and communities, increasing hospital beds, nationwide support from medical staff, epidemic prevention equipment and supplies, and establishment of makeshift shelter hospitals have been taken. The lockdown of Wuhan reduced the propagation of cases to other cities in Hubei province and throughout China, traffic and community restrictions reduced the flow of population and the spread of disease, increasing wards and beds and medical personnel reduced the incidence of severe cases and mortality, the establishment of the Fangcang shelter hospitals provided a good isolation and monitoring environment, and further reduced the spread and fatality of the disease. The fact that China was able to control the spread of COVID-19 within three months without a specific drug or vaccine suggests that these measures are more adequate and effective.

Construction and characterization of a full-length infectious clone from a fast-replicating, X4-tropic HIV-1 subtype B' isolate.

In HIV-1 epidemics in China, HIV-1 subtype B' is the most predominant subtype circulating in intravenous drug users. In this study, we constructed an HIV-1 full-length infectious molecular clone based on the primary virus LWJ, which was isolated from an HIV-infected patient in Fujian Province, China. Phylogenetic and bootscanning analysis of the viral sequence revealed that the isolate LWJ belonged to HIV-1 subtype B'. The infectious clone was designated as "pLWJ". The virus (LWJ-c) produced from this infectious clone by in vitro transfection of 293T cells could infect both human peripheral blood mononuclear cells (PBMCs) and human the T cell line MT4. Interestingly, the cloned LWJ-c virus utilized CXCR4 as its co-receptor and could replicate in vitro with similar efficiency and kinetics compared to its parental primary isolate LWJ as well as the clade B reference virus NL4-3. The LWJ-c virus could also cause cytopathic effects in both PBMCs and MT cells. Sequence analysis of the envelope glycoprotein of pLWJ showed that a conserved GPGR motif and an arginine at position 11 were present in the V3 loop, which was consistent with previous reports regarding CXCR4 co-receptor usage and syncytium-inducing (SI) phenotype. Thus, the infectious clone represents a fast-replicating, high-producing, CXCR4-tropic and syncytium-inducing isolate. Given the prevalence of HIV-1 subtype B' in China, this infectious clone can be a very useful tool to provide a versatile molecular model for research focusing on the biological properties of this subtype.

Effects of Substrates on the Production of Fruiting Bodies and the Bioactive Components by Different Cordyceps militaris Strains (Ascomycetes).

Cordyceps militaris is a type of food and medicinal species and is widely cultured in Asia. Substrate and strain are important factors for the production of fruiting bodies and bioactive components contents in fruiting bodies of C. militaris. This study aimed to select the excellent strains and suitable substrates by six strains of C. militaris cultivated on rice, wheat, and tussah (Antheraea pernyi) pupae. The results showed that the rice and wheat were suitable for fruiting body formation of strain CM3, with yields of 23.19 and 19.07 g per bottle, and biological efficiency of strain CM3 were 62.26% and 54.48%, respectively, which were significantly higher than other strains. Tussah pupae is suitable for fruiting body formation of strain CM9, with fruiting body length, yield, and biological efficiency of 5.57 cm, 6.80 g per each, and 291.70%, respectively, which were significantly higher than other strains. The content of adenosine in fruiting bodies of strain CM9 cultivated on tussah pupae was 2.62 mg g-1, followed by that of strain CM3 on rice of 2.51 mg g-1. The content of cordycepin in fruiting bodies of strain CM4 cultivated on wheat was 5.68 mg g-1, followed by that of strain CM9 on wheat of 5.41 mg g-1. To improve the product quality and the contents of bioactive components, C. militaris strains and substrates should both be considered, that is, different strains should be appropriate for different substrates.

Case Report: Compound Heterozygous Phosphatidylinositol-Glycan Biosynthesis Class N (PIGN) Mutations in a Chinese Fetus With Hypotonia-Seizures Syndrome 1.

Multiple congenital anomalies-hypotonia-seizures syndrome 1 (MCAHS1) caused by phosphatidylinositol-glycan biosynthesis class N (PIGN) mutations is an autosomal recessive disease involving many systems of the body, such as the urogenital, cardiovascular, gastrointestinal, and central nervous systems. Here, compound heterozygous variants NM_012327.6:c.2427-2A > G and c.963G > A in PIGN were identified in a Chinese proband with MCAHS1. The features of the MCAHS1 family proband were evaluated to understand the mechanism of the PIGN mutation leading to the occurrence of MCAHS1. Ultrasound was conducted to examine the fetus, and his clinical manifestations were evaluated. Genetic testing was performed by whole-exome sequencing and the results were verified by Sanger sequencing of the proband and his parents. Reverse transcription-polymerase chain reaction was performed, and the products were subjected to Sanger sequencing. Quantitative PCR (Q-PCR) was conducted to compare gene expression between the patient and wild-type subjects. The compound heterozygous mutation NM_012327.6:c.2427-2A > G and c.963G > A was identified by whole-exome sequencing and was confirmed by Sanger sequencing. The NM_012327.6:c.2427-2A > G mutation led to skipping of exon 26, which resulted in a low expression level of the gene, as measured by Q-PCR. These findings provided a basis for genetic counseling and reproduction guidance in this family. Phenotype-genotype correlations may be defined by an expanded array of mutations.

Molecular method for rapid detection of the red tide dinoflagellate Karenia mikimotoi in the coastal region of Xiangshan Bay, China.

The species Karenia mikimotoi is a common nearshore red tide alga that can secrete hemolytic exotoxin and ichthyotoxin, which can induce the death of fish and shellfish, causing severe economic losses. In this study, loop-mediated isothermal amplification (LAMP) was employed in combination with the lateral flow dipstick (LFD) visual detection method to establish the LAMP-LFD rapid detection method for K. mikimotoi. The internal transcribed spacer ITS1-5.8S-ITS2 of K. mikimotoi was used as the target sequence and was amplified with specific primers designed in this study. The results indicated that the amplification optimal reaction conditions for LAMP in this paper were for 20 min at 65 °C. Moreover, LAMP had excellent specificity, showing negative results for other common red tide causing algal species. In field samples, we successfully reduced the total time, with only 23 min needed from LAMP amplification to LFD result display, which was shorter than that of conventional PCR. Consequently, LAMP-LFD should be useful for rapid field detection of low-density K. mikimotoi and for the early prevention of red tide induced by such algae.

LZAP promotes the proliferation and invasiveness of cervical carcinoma cells by targeting AKT and EMT.

Objective: To explore the relationship and mechanism of LZAP in the occurrence and development of cervical cancer and to provide a new target and intervention method for the treatment of cervical cancer. Methods: Data mining and analysis of LZAP expression levels were performed using several online databases, including The Cancer Genome Atlas (TCGA). A cervical cancer cell line that stably overexpresses LZAP was established, and the effect of LZAP overexpression on cell proliferation, invasion, migration and tumor formation in vivo as well as its mechanism were explored. Results: Our study shows that the expression of LZAP is upregulated in cervical cancer. The overexpression of LZAP can significantly promote the proliferation, colony formation, and invasion and migration abilities of cervical cancer cells. The tumorigenesis test in nude mice showed that overexpression of LZAP could promote the tumorigenicity of cervical cancer cells in vivo. LZAP could also promote the phosphorylation of AKT at position 473 and the epithelial-mesenchymal transition (EMT). Conclusion: The expression of LAZP is increased in cervical cancer, which can enhance the invasion, metastasis, and EMT in cervical cancer cells by promoting AKT phosphorylation.

Genomic Characterization of Diverse Gyroviruses Identified in the Feces of Domestic Cats.

Gyroviruses (GyVs) are small, single-stranded, circular DNA viruses in the genus Gyrovirus, which consists of the chicken anemia virus (CAV) prototype and nine other viral species. These different GyV species have been reported in chickens, humans, mice, and companion animals. To date, CAV has been identified in the feces of domestic cats, while the circulation of other GyV species in cats is currently unknown. In the present study, 197 fecal samples were collected from pet cats in northeast China, and samples were screened for different GyV species by PCR. Twelve GyV strains were identified from the feces of pet cats. These included 4 positive for CAV, 3 for HGyV/AGV2, 3 for GyV3 and 2 positive for GyV6. The complete genome sequences of the 12 cat-sourced GyV strains showed 93.9-99.7% nucleotide identities to the homologous reference GyV strains. Phylogenetic analyses based on the complete genomes, VP1, VP2 and VP3 genes showed the identical classification of GyV species with previous reports. Moreover, one and four unique amino acid substitutions were identified in the VP1 protein of the cat-sourced HGyV/AGV2 and GyV6 strains, respectively, and one substitution was also observed in the VP2 protein of one GyV6 strain identified in this study. In conclusion, our investigation demonstrates that the diverse GyV species were circulating in domestic cats, and provides the first molecular evidence for the circulation of HGyV/AGV2, GyV3 and GyV6 in domestic cats. These cat-origin GyVs possessed considerable genetic diversity. This study also raises the possibility that domestic cats, as reservoirs for gyroviruses, may inadvertently disseminate viruses to other species, e.g., humans and chickens.

Distribution of Karlodinium veneficum in the coastal region of Xiangshan Bay in the East China Sea, as detected by a real-time quantitative PCR assay of ribosomal ITS sequence.

Athecate dinoflagellate Karlodinium veneficum is a universal toxic species possessing karlotoxins recognized especially as ichthyotoxic as well as cytotoxic and hemolytic. Blooms of K. veneficum, both single-species or accompanied with other species, occurred more frequently worldwide in recent years, including the coastal region of China. Normally, K. veneficum present in relatively low abundance in phytoplankton communities in estuary regions. Being small and difficult to identify with light microscopy, it has been ignored for a long time till its blooming and toxins being confirmed. How it presents in background level and what is its relationship with critical geological and hydrological environment factors are basically not clear. In this study, the paper reports the application of a real-time quantitative PCR (qPCR) method to investigate the abundance and distribution of K. veneficum in the coastal waters of Xiangshan Bay in the East China Sea (ECS), a typical bay area of harmful algae blooms and heavily affected by anthropogenic activities. The real-time qPCR assay came out being an efficient method at detecting even low cell densities of K. veneficum of different genotypes. A total of 38 field samples of surface (0.5 m) and bottom water (9-100 m in depth) were analyzed and 12 samples were found positive for K. veneficum. At least 3 genotypes of K. veneficum present in this region. Temperatures in sites of K. veneficum positive ranged from 21.7 to 23.4 °C, and salinity levels were between 21.1 and 26.3. The K. veneficum distributed quite extensively in the waters of Xiangshan Bay, cell abundance varied from a low of 4 cells/L to a maximum of 170 cells/L. Most of the samples containing K. veneficum were collected from bottom water in different sites. At three of the 19 sampling sites, K. veneficum was detected in both surface and bottom water samples. Especially at sampling site near Beilun port, where the water is typically muddy with low transparency, relative high cell numbers of K. veneficum were found in both surface and bottom waters. Mixotrophy and vertical migration of K. veneficum could be important eco-physiological factors to consider in terms of understanding these distribution characteristics. The ideal conditions for K. veneficum growth and aggregation in this area still needs further study.

Application of chromosomal microarray to investigate genetic causes of isolated fetal growth restriction.

BACKGROUND: Application of chromosomal microarray analysis (CMA) to investigate the genetic characteristics of fetal growth restriction (FGR) without ultrasonic structural anomalies at 18-32 weeks. METHODS: This study includes singleton fetuses with the estimated fetal weight (EFW) using the formula of Hadlock C below the 10th percentile for gestational age. FGRs without structural anomalies were selected, and the ones at high risk of noninvasive prenatal testing for trisomy 13, 18 and 21 would be excluded. The cases were divided into two groups: early-onset group (< 24+ 0 weeks) and late-onset group (24-33 weeks). All patients were offered invasive prenatal testing with CMA and karyotype analysis. RESULTS: CMA detected 10 pathogenic copy number variants and 2 variant of uncertain significance case. CMA has a 5.5% (7/127) incremental yield of pathogenic chromosomal abnormalities over karyotyping. The positive detected rate was 9.6% (5/52) in early-onset group and 9.3% (7/75) in late-onset group respectively. CONCLUSIONS: When FGR without structural anomaly is diagnosed before 33 weeks, an invasive prenatal procedure is strongly recommended. CMA can identify a 5.5% (7/127) incremental detection rate of pathogenic chromosomal abnormalities, which would impact clinical management for FGR.

High-level expression of recombinant dengue virus type 2 envelope domain III protein and induction of neutralizing antibodies in BALB/C mice.

Dengue fever is a growing public health problem in many countries since so far no effective vaccines are available. In this study, the domain III of dengue virus type 2 envelope was expressed in Escherichia coli without fusion of any carrier protein. The recombinant protein was detected in the form of inclusion bodies, which were solubilized in 8M urea and could be purified subsequently by high-performance liquid chromatography (HPLC) on an ion exchange column. After refolding, the recombinant protein inhibited the DEN-2 plaque formation on C6/36 cells, demonstrated its function of receptor-interaction was retained. The recombinant protein was inoculated into BALB/c mice to test its immunogenicity and ability to induce neutralizing antibodies. The mice immunized with the purified protein developed high antibody titers. A neutralizing titer of 1:64 was also obtained by a cytopathogenic effect (CPE) inhibition assay in C6/36 cells. Mice challenged with lethal dose of DEN-2 in combination with sera from immunized mice were protected completely. The results suggested that these expression and purification strategies have the potential for development of an inexpensive vaccine.

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Karlodinium veneficum.

The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) combined with a chromatographic lateral flow dipstick (LFD) assay to rapidly and specifically detect the Karlodinium veneficum ITS gene. Four groups of LAMP primers were specially designed to target the K. veneficum ITS gene. The LAMP-LFD detection limit was 7.4pg/µL (approximately 6.5cells/mL) of K. veneficum genomic DNA and was 10 times more sensitive than standard PCR. The LAMP-LFD method exhibited high specificity and accurately identified K. veneficum algal isolates, but not other algal isolates. To test the assay's accuracy, samples from positive results were further analyzed by sequencing and phylogenetic analysis, all of which were identified as K. veneficum. Over all, the LAMP-LFD assay established in this paper can be used as a reliable and simple method to detect the K. veneficum.